Preparedness Against Bioterrorism And Re-Emerging Infectious Diseases (Nato Science Series I:Life and Behavioural Sciences)

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PREPAREDNESS AGAINST BIOTERRORISM AND RE-EMERGING INFECTIOUS DISEASES

NATO Science Series A series presenting the results of scientific meetings supported under the NATO Science Programme. The series is published by IOS Press and Kluwer Academic Publishers in conjunction with the NATO Scientific Affairs Division. Sub-Series I. II. III. IV. V.

Life and Behavioural Sciences Mathematics, Physics and Chemistry Computer and Systems Sciences Earth and Environmental Sciences Science and Technology Policy

IOS Press Kluwer Academic Publishers IOS Press Kluwer Academic Publishers IOS Press

The NATO Science Series continues the series of books published formerly as the NATO ASI Series. The NATO Science Programme offers support for collaboration in civil science between scientists of countries of the Euro-Atlantic Partnership Council. The types of scientific meeting generally supported are "Advanced Study Institutes" and "Advanced Research Workshops", although other types of meeting are supported from time to time. The NATO Science Series collects together the results of these meetings. The meetings are co-organized by scientists from NATO countries and scientists from NATO's Partner countries - countries of the CIS and Central and Eastern Europe. Advanced Study Institutes are high-level tutorial courses offering in-depth study of latest advances in a field. Advanced Research Workshops are expert meetings aimed at critical assessment of a field, and identification of directions for future action. As a consequence of the restructuring of the NATO Science Programme in 1999, the NATO Science Series has been re-organized and there are currently five sub-series as noted above. Please consult the following web sites for information on previous volumes published in the series, as well as details of earlier sub-series: http://www.nato.int/science http://www.wkap.nl http://www.iospress.nl http://www.wtv-books.de/nato_pco.htm

Series I: Life and Behavioural Sciences - Vol. 357

ISSN: 1566-7693

Preparedness Against Bioterrorism and Re-Emerging Infectious Diseases Edited by Janusz Kocik Military Institute of Hygiene and Epidemiology, Warsaw, Poland

Marek K. Janiak Military Institute of Hygiene and Epidemiology, Warsaw, Poland

and Marian Negut Cantacuzzino Institute, Bucarest, Romania

IOS

Press Amsterdam • Berlin • Oxford • Tokyo • Washington, DC Published in cooperation with NATO Scientific Affairs Division

Proceedings of the NATO Advanced Research Workshop on Preparedness Against Bioterrorism and Re-Emerging Infectious Diseases 15-18 January 2003 Warsaw, Poland

© 2004, IOS Press All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without prior written permission from the publisher. ISBN 1 58603 4170 Library of Congress Control Number: 2004103094

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LEGAL NOTICE The publisher is not responsible for the use which might be made of the following information. PRINTED IN THE NETHERLANDS

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Editorial: Building Integrated Preparedness against Bio-Terrorism 1

Janusz KOCIK1, Marek K. JANIAK1, Marian NEGUT2 Military Institute of Hygiene and Epidemiology, Warsaw, Poland Cantacuzzino Institute, Bucarest, Romania

NATO Advanced Research Workshop (ARW) entitled "Preparedness against BioTerrorism and Re-Emerging Infectious Diseases - Regional Capabilities, Needs, and Expectations in Central and Eastern European Countries" was held on 15-18 January 2003 in Warsaw, Poland. The meeting was organized by the Military Institute of Hygiene and Epidemiology, Warsaw Poland, in co-operation with the Cantacuzzino Institute, Bucarest, Romania. The primary objective of the ARW was to provide countries in which bio-defence systems are currently under development with the experience and expertise of those that are more advanced and/or have already been exposed and responded to a bio-terrorist attack. Such an approach was taken to allow avoiding mistakes and to properly allocate limited resources in building preparedness against intentional and natural outbreaks of infectious diseases. The ARW also aimed at increasing awareness of the problem at the political level in the Region. Yet another objective was to consolidate the community of experts in the Region and to reinforce connections with NATO and other international organisations in this particular area. This forum allowed for integration of thinking, elaboration of common approach to the problem, and exchange of the experience. One of the main lessons learned from the ARW is that, in most of the countries, the existing systems of the management of biological threats are convoluted and fragmented. There is also no single international system that would provide all the information needed to rapidly assess each particular situation and help bring a biological crisis under control. Several international bodies only fragmentally deal with parts of the problem that are within their competence. Some of these efforts were presented during the Workshop (Cosivi, Niedrig, Kyncl). Projects for the informal experts' networks have been demonstrated (Price et al, Woodal). Also, national approaches to the bio-defense and disease surveillance were presented (Plochev, Faludi, Niedrig et al., Asokliene, Chomiczewski, Kapustiri). Moreover, as a result of the attendance of specialists from such relevant fields as epidemiology, infectious diseases, NBC threats, medical planning, vaccinology, detection and identification of biological agents, physical protection and decontamination it was possible to examine most of the avenues a country should ideally explore in order to build its preparedness and capacity to respond to a biological attack. Development of the integrated system of response against bio-terrorism is a very complex endeavor requiring integration of the activities of the relevant institutions at the national level. In this regard, a multidisciplinary approach is crucial. Physicians should be trained to entertain suspicions facing unusual profiles of the disease (Pavlin). Other medical personnel and first responders should be also familiar and adequately trained to cope with signs and symptoms presented by victims of a biological attack (Stopa). New laboratory systems and approaches can revolutionize disease surveillance. Integration of the real time epidemiological data collection, analysis, and dissemination with the timely laboratory

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support based on the advanced technology platforms (Morse, Hanson et al, Niemeyer) will accelerate diagnosis and increase awareness of the situation. Even though rapid progress is being made in the field of the remote sensing (Niemeyer), biosensors (Donlori), proteomics (delVecchio), and other relevant methodologies (Bartoszcze), there is currently no "silver bullet" for application in the real time biological detection and identification. Since a variety of approaches and technologies are evolving in the biological threat detection, prophylaxis, and therapeutics, a flexible and rapidly responsive bio-manufacturing infrastructure is an essential part of the effective overall strategy for bio-terrorism preparedness (O'Connell). Consequence management should be planned in advance (Ahrens), preferably based on the effective risk assessment? (Bellenkes). Innovations in medical countermeasures and prophylaxis (Oyston) as well as in the decontamination procedures (Pivovarov et al., Kartel et al.) are still necessary and being introduced. In view of the fact that infectious diseases do not respect boundaries preparedness against outbreaks should be built from the scratch using the "thinking outside-of-the-box" approach with consideration for the international interdependence. This requires unification of the epidemiological and laboratory procedures as well as of the public health planning in countries of the Region. Taking into account the differences and gaps in the existing health systems of these countries, the very complicated goal of leveling the approaches certainly requires firm and sustained regional co-operation. We hope to witness it in the nearest future. Hopefully, the present NATO ARW will help to pave the way for such co-operation. The Editors: Dr. Janusz Kocik, M.D., Ph.D. Dr. MarekK. Janiak, M.D., Ph.D. Prof. Marian Negut, M.D., D.Sc.

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Acknowledgements The meeting was sponsored by the NATO Scientific and Environmental Affairs Division, the U.S. Army Research Office and the U.S. Army Soldier, Biological and Chemical Command; the US. Naval Research International Field Office, as well as Baxter Vaccines. We take this opportunity to express our thanks to the sponsors. We also are grateful to Dr. Deborah Niemeyer, Kevin O'Connel, Mildred Donlon, and Adam Wilczynski for their efforts to spread the news about the ARW in the scientific community and help attract the outstanding key speakers to the event. Finally, the hard and dedicated work of Mrs. Magdalena Baranowska, Dr. Dr. Emil Lisiak, Marek Brytan, and Mr. Hubert Radziejewski of the Military Institute of Hygiene and Epidemiology is highly appreciated. Indeed, without their organizational and technical support this meeting would not have gone as smoothly as it did. Dr. Marek K. Janiak, M.D.,Ph.D. - Co-Director Prof. Marian Negut, M.D., D.Sc. - Co-Director Dr. Janusz Kocik, M.D., Ph.D. - Head of the Organizing Committee

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KEY SPEAKERS Theodore Ahrens US European Command Feldbergstrasse 102 71134Aidlingen, GERMANY e-mail: [email protected] LTC Gerard P. Andrews, Ph.D. Chief, Bacteriology Division USAMRIID, Ft. Detrick, MD, 21701, USA Dr. Loreta Asokliene Public Health Specialist Centre for Communicable Diseases Prevention and Control Kalvariju str. 153, LT-2042 Vilnius, LITHUANIA e-mail: [email protected] tel: +370 5 277 86 61 fax: +370 5 277 87 61 Dr hab. n. med. Michal Bartoszcze Military Institute of Hygiene & Epidemiology 2 Lubelska St., 24-100Pulawy, POLAND CDR Dr. Andrew H. Bellenkes MSC USN, School of Aviation Safety Naval Postgraduate School (Code 10) 1588 Cunningham Rd., Monterey, California 93943-5002, USA tel. US (country code) + 831.656.2581 e-mail: [email protected] Prof. Krzysztof Chomiczewski Military Institute of Hygiene & Epidemiology 4 Kozielska St., 01-163 Warsaw, POLAND

Ditta Ciganikowa Bio Weapons Prevention Project C/o PSIS 132, Rue de Lausanne 1211, Geneva 21, SWITZERLAND tel: +41 22 908 57 34 fax: + 41 22 738 35 82 e-mail: [email protected], www.bwpp.org Dr. Ottorino Cosivi World Health Organization Department of Communicable Disease Surveillance and Response 20, Avenue Appia CH-1211 Geneva 27, SWITZERLAND tel:+41 22 791 25 31 fax: +41 22 791 48 93 e-mail: [email protected] Helene van Cuyck Expert in Biology Weapons of Mass Destruction Centre, AB 310 NATO Bd. Leopold III BE. IIIo. Brussels, BELGIUM tel: 00322 707 1905 e-mail: [email protected] Prof. Vito DelVecchio Institute of Molecular Biology and Medicine University of Scranton Scranton, PA 18510-4625, USA Dr. Mildred Donlon DARPA/SPO 3701 North Fairfax Drive Arlington, VA 22203-1714, USA

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Dr. Phil Elzer Louisiana State University Dept. of Veterinary Science 111 Dalrymple Building Baton Rouge, LA 70803, USA Col. Dr. Gabor Faludi Hungarian Defence Forces Institute of Health Protection General Director 1555 Budapest Pf. 68, HUNGARY e-mail: [email protected] Major Eric Hanson Office of the Air Force Surgeon General Directorate, Expeditionary Medical Operations, Science & Technology 5201 Leesburg Pike, Suite 1401 Falls Church, VA, 22041, USA tel:(703)9243172wk tel: (703) 216 0839 cell e-mail: [email protected] Nick Kanellopoulos Membranes & Materials for Environmental Applications Laboratory National Center for Scientific Research "Demokritos" 15310 Agia Paraskevi, Athens, GREECE tel: +30 210 65 35 294 fax:+30 210 65 11766 e-mail: [email protected] Dr. Alexander Kapustin Chemical and Biological Weapons Conventional Problems Sector Ministry of Foreign Affairs of Ukraine 01018, Mykhaylivska sqr.l, Kyiv, UKRAINE tel: (380) 44 238 17 26 fax:(380)442381653 e-mail: [email protected]

Nikolai T. Kartel, Prof, Dr. Sci. (Chem.) Institute for Sorption & Problems of Endoecology National Academy of Sciences of Ukraine 13 General Naumov str. Kiev 03164, UKRAINE tel: +380 44 452 93 25 fax: +380 44 452 93 27 e-mail: [email protected] [email protected] Dr. Janusz Kocik Military Institute of Hygiene & Epidemiology 4 Kozielska St., 01-163 Warsaw, POLAND tel: +48 22 681 61 06, mobile (preferred) + 48 607 461 970 e-mail: [email protected] Dr. Mzia Kutateladze G. Eliava Institute of Bacteriophages, Microbiology and Virology, Georgian Academy of Sciences Tbilisi.3, Gotua str., Tbilisi 380060, GEORGIA tel/fax.: + 99 532 91 1836 e-mail: [email protected] Dr. Jan Kyncl Dept. of Epidemiology Srobarova 48, 100 42 Prague 10, CZECH REPUBLIC tel: +420 267 082 891 fax: +420 272 741 433 e-mail: [email protected] Dr. Christian Loucq Acambis plc Peterhouse Technology Park 100, Fulbourn Road Cambridge CB1 9PT, UK tel: +44 1223 275 300 mobile:+44 7881 81 75 41 fax: +44 1223 416 300 e-mail: [email protected]

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Dr. Stephen Morse Associate Director for Science Bioterrorism Preparedness and Response Program, Centers for Disease Control and Prevention 1600 Clifton Rd., Atlanta, GA 30333, USA Prof. Marian Negut MD, PhD Cantacuzzino Institute 103, Splaiul Independentei sect. 5 Bucharest 70 100, ROMANIA tel: +402 14 10 73 30 fax:+402 14 11 56 72 e-mail: [email protected] Dr. Mattias Niedrig Secretary of the European Network for Diagnosis of "Imported" Viral Disease (ENIVD) Robert-Koch-Institut Nordufer 20 13353 Berlin, GERMANY tel: +49 30 45 47 23 70 fax: +49 30 45 47 26 25 e-mail: [email protected] Dr. Kevin O'Connell AMSSB-RRT-BMResearch and Technology Diorectorate Edgewood Chemical Biological Center Aberdeen Proving Ground, MD 210105424, USA Dr. Petra Oyston Microbiology B07A, CBS Porton Down Salisbury, Wiltshire SP4 OJQ, UK

LTC Julie Pavlin DoD Global Emerging Infections System 503 Robert Grant Ave. Silver Spring, MD 20910-7500 USA tel: 301-319-9346 fax: 301-319-9104 [email protected] Prof. Alexander Pivovarov Department of Equipment and Technology of Food Industry Ukrainian State University of Chemical Engineering 8 Gagarina Av., Dnepropetrovsk 49005, UKRAINE tel: +38 0562 47 05 55 fax: +38 0562 36 68 37 e-mail: [email protected] a.pi vo varov@ua. fm home tel:+38 056 772 1050 Dr. Kamen Plochev Military Medical Academy G. Sofiiski 3 1606 Sofia, BULGARIA Home address: 97' Erlogi Georgiev Blvd. Sofia 1000, BULGARIA tel: +35 92 922 53 (i) 952 53 50 mobile: +359 98 48 98 27 Barbara B.S. Price, PhD Senior Scientist Biodefense Systems Battelle Eastern Regional Technology Center (BERTC) 1204 Technology Drive Aberdeen, MD 21001 USA tel (main): 410-306-8500 fax: 410-306-8420 direct: 410-306-8580 cell: 207-831-3398 [email protected]

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Col. Richard M. Price, USAF Ret. Applied Science and Analysis, Inc. PO Box 1144, Aberdeen, MD 21001 USA Tel 410-638-9480 Fax 410-638-9481 Web: http://www.asanltr.com Dr. Peter J. Stopa 5183Blackhawk Rd BldgE3549 AMSSB-REN-E-MC US Army Edgewood Chemical Biological Center Aberdeen Proving Ground, MD 210105424, USA Tel: 410 343 22 78 Fax: 410 612 50 83 e-mail: [email protected] private email: [email protected] Marek Tomaszewski Chief Sanitary Inspectorate (GIS) Epidemic Dept. 38/40 Dluga str., Warsaw, POLAND tel: +48 22 635 45 81 (i) 107 e-mail: [email protected] Dr. David Trudil New Horizons Diagnostic Corporation 9110 Red Branch Road Columbia, MD 21045-2014, USA tel: +410 992 93 57 ext. 222 fax: +410 992 03 28 e-mail: [email protected]

Prof. Tomasz Twardowski Kierownik Zespolu Biosyntezy Bialka Instytut Chemii Bioorganicznej PAN Noskowskiego 12/14, 61-704Poznan, POLAND Dr. Adam Wilczynski Permanent Mission of Poland to the UN Office at Geneva 15 1'Ancienne Route 1218 Grand Saconnex (CH), SWITZERLAND tel: +41 22 7109 710 fax: +41 22 7109 799 e-mail: [email protected] Dr. Jack Woodall Director, Nucleus for Investigating Emerging Infectious Diseases, Dept. of Medical Biochemistry, Institute of Biomedical Sciences, Federal University of Rio de Janeiro Cidade Universitaria Rio de Janeiro - RJ - 21941-590, BRAZIL tel: +5524 2225 1395 e-mail: [email protected] Doc. dr hab. Andrzej Zielinski Zaklad Epidemiologii Panstwowy Zaklad Higieny ul. Chocimska 24 00-791 Warszawa, POLAND

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OTHER PARTICIPANTS AND OBSERVERS Dr. Pawel Abramczyk Department of Defence Affairs / Ministry of Health Team of Reaction on Emergency Situations Miodowa str. 15, 04-950 Warsaw, POLAND e-mail: [email protected] fax: +48 22 634 94 24 Dr. Vladimir Bundin Russian Agency for Munitions Department for BTWC Shepkina 42, 107996 Moscow, RUSSIA tel: 007 095 206 43 64 fax: 007 095 206 35 93 Eric Bush-Petersen Baxter Vaccines Industriestrasse 67 A-1220 Vienna, AUSTRIA [email protected] Prof. Dumitru Carstina Infectious Diseases Clinic 3400 Cluj-Napoca Str.Iuliu Moldovan, 23, ROMANIA tel/fax:+40264193 105 e-mail: [email protected] Agnieszka Chycak-Kryczka Glowny Inspektorat Sanitarny, Departament Przeciwepidemiczny 38/40 Dtuga str., Warsaw, POLAND tel: +48 22 635 45 81 w. 109/107

Zbigniew Ciolek Dept. of International Cooperation Polish Ministry of Defense 1 Krolewska Str. 00-909 Warsaw, POLAND tel: +48 22 68 73 106 fax:+48 22 68 73 182 e-mail: [email protected] Flavio Del Ponte Humanitarian Aid / SDC Federal Department of Foreign Affairs 130 Freiburgstrasse 3003 Bern, SWITZERLAND tel:+4122 73162 04 e-mail: [email protected] Dr. Antonio della Guardia Presidenca Del Consiquo Dei Ministri Via XX Settembre, 8 00100 Roma, ITALY tel:+39 06 61174 612 mob: +39 347 039 63 66 e-mail: [email protected] Dr. Marek K. Janiak Military Institute of Hygiene & Epidemiology Kozielska 4 01-163 Warszawa, POLAND tel:+48 22 685 3101 m.j [email protected]

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Dr. Tomasz Szkoda Respiratory Virus Laboratory Department of Virology National Institute of Hygiene Chocimska 24, 00-791 Warszawa, POLAND tel. +48 (22) 54 21 230 tel./fax+48(22)5421385 NIH oper.: tel +48 (22) 54 21 400 e-mail: [email protected] http://www.pzh.gov.pl/ Major Dr. Corina Taubner Chief laboratory Central Military Emmergency Clinic Hospital in Bucharest 134, Calea Plevnei, 77103 Bucharest 1, ROMANIA tel:+40 21224 94 06/160 fax:+40 2141156 72 Prof. Sadi Osman Yenen General Manager KANSAS AS Blood and Health Service Co. Ataturk Bulvary, 219/14 Kavaklydere06680- Ankara, TURKEY

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Contents Editorial: Building Integrated Preparedness against Bio-Terrorism Janusz Kocik, Marek K. Janiak and Marian Negut Key Speakers Other Participants and Observers

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Part 1. Contemporary Epidemiology With Laboratory Support as a Biological Attack Identification Tool Epidemiology of Bioterrorism J.A. Pavlin

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Health Preparedness for the Deliberate Use of Biological Agents to Cause Harm: WHO's Activities 9 O. Cosivi Epidemic Outbreak Systems (EOS) - Microarray Incorporation for Pathogen Identification E. Hanson, R. Roweley, B. Agan, C. Tibbetts and D. Niemeyer

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Preventing Emerging Infectious Diseases: Epidemiology and Laboratory Capacity Support S.A. Morse

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The Laboratory Response Network S.A. Morse, R. Kellogg, S.R. Perry, R.F. Meyer, D. Bray, D. Nichelson and M.J. Miller

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Imported Viral Diseases, Surveillance and Control in Europe - European Network for the Diagnostics of "Imported" Viral Diseases (ENIVD) M. Niedrig

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Surveillance of Acute Respiratory Infections in the Czech Republic and in Europe Example of an Early Warning System J. Kyncl and B. Kriz

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Part 2. National Approaches to Biodefence in Central and Eastern Europe Countries Organization of Military Medical Response to Bioterroristic Attacks K. Plochev and E. Penkov

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Georgian Approach to Biodefense M. Kutateladze

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National Approach of Germany to Bioterrorism and Bio-Warfare M. Niedrig, R. Fock and E. Finke

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National Approach of Hungary to Bioterrorism and Biowarfare G. Faludi, A. Csohan and G.Berencsi

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Emerging Infectious Diseases and Their Surveillance in Lithuania L. Asokliene

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Biological Recognition Teams in the Polish Army K. Chomiczewski

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National Approach of Ukraine to Bioterrorism and Biowarfare A. Kapustin

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Part 3. Risk Assessment, Crisis Management and NBC Training Effective Risk Management in the Human Factors Assessment of Chemical/Biological Threats A.H. Bellenkes

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Crisis Management: Expediting Information and Resource Flow via the Global Incident Analysis and Alerting System (GIAAS) for CBRN R. Price, J. Woodall and S. Netesov

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Civilian Relief after Release of Weapons of Mass Destruction: Need for a New Task Force 'Scorpio' J. Woodall

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Consequence Management of a Bioterrorist Incident (CMBI) J. T. Ahrens

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GM Modified Food/Feed as Biowarfare T. Twardowski

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Antibiotic Resistance Bacteria - A Potential Threat M. Negut

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Effective Training for First Responders P.J. Stopa, K. Quinn-Doggett and R.A. Vigus

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Part 4. Applied Research Brucellosis-A Biowarfare Threat and Public Health Concern P.H. Elzer

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Medical Countermeasures Research at Dstl Porton Down, UK P.C.F. Oyston

164

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Current Problems Regarding Detection and Identification of Biological Threats M. Bartoszcze

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Rediscovering Remote Sensing: Improving Infectious Disease Surveillance D.M. Niemeyer

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The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Debra M. Niemeyer

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Biosensors - The Tool for Fast Detection M.A. Donlon

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A Flexible Approach to Biomanufacturing K.P. O'Connell, P.E. Anderson, D.C. Lukens, M.H. Kim, A.S. Khan, R.G. Thompson, J.T. Park and J.J. Valdes

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Development of Rapid Fingerprinting and Detection Assays for Biological Agents of Mass Destruction V.G. DelVecchio

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Decontamination of Drinking Water and Liquid Media by Cold Plasma in the Special Periods A.A. Pivovarov

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Antibacterial Adsorption-Filtering Materials for Individual Protection Means of Organism - Antibacterial Action of Metal-Containing Carbon Adsorbents N. Kartel, A. Grigoriev, D. Shvets and V. Strelko

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Antibacterial Adsorption-Filtering Materials for Individual Protection Means of Organism - Fine Filtering Materials on the Base of Polypropylene Microfibres and Their Antibacterial Property M. Tsebrenko, V. Rezanova, I. Tsebrenko, M. Mayboroda and N. Kartel

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Antibacterial Adsorption-Filtering Materials for Individual Protection Means of Organism - Composite Materials Based on Polyurethanes and Active Carbon Yu. Savelyev, L. Robota, O. Savelyeva, N. Kartel and V. Strelko

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List of Tables and Figures Author Index

251 255

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Part 1

Contemporary Epidemiology With Laboratory Support as a Biological Attack Identification Tool

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Preparedness Against Bioterrorism and Re-Emerging Infectious Diseases J. Kocik et al. (Eds.) IOS Press, 2004

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Epidemiology of Bioterrorism* Julie A. PAVLIN Walter Reed Army Institute of Research, Silver Spring, MD USA * Adapted from Emerg Inf Dis 1999 Jul-Aug;5(4):528-30. Abstract. The risk of a bioterrorist attack occurring anywhere in the world has increased with the increased willingness of terrorists to inflict mass casualties. A sound epidemiologic investigation of a disease outbreak, whether natural or humanengineered, will assist medical personnel in identifying the pathogen, as well as instituting the appropriate medical interventions. Documenting who is affected, possible routes of exposure, signs and symptoms of disease, and the rapid identification of the causative agents will greatly increase the ability to plan an appropriate medical and public health response. Good epidemiologic information will allow the appropriate follow-up of those potentially exposed, as well as assist in risk communication and responses to the media. To minimize the effects of a biological terrorist attack, health care professionals and public health authorities must be aware of the threat of biological warfare and terrorism and have an increased index of suspicion that such an attack can occur. They must have some understanding of the classes of agents that have been and can be weaponized and their effects after inhalation and other dissemination methods. They need to be trained to recognize and treat casualties of biological warfare or terrorism and they must be able to apply appropriate preventive measures rationally and without unnecessary panic or alarm. Well before any event, public health authorities must implement surveillance systems so they can recognize patterns of nonspecific syndromes that could indicate the early manifestations of a biological warfare attack. The system must be timely, sensitive, specific, and practical. To recognize any unusual changes in disease occurrence, surveillance of background disease activity should be ongoing, and any variation should be promptly followed up with a directed examination of the facts regarding the change.

The use of Bacillus anthracis spores as an agent of terror in the United States in October 2001 has demonstrated the need for increased vigilance and preparedness for attacks using biological agents. It is impossible to monitor every nation, group and individual who may be considering the use of biological terrorism, so in addition to increased law enforcement vigilance, we must improve our public health monitoring. Even as this will improve our ability to detect and react to biological attacks, it will improve basic public health capacity to watch for new naturally occurring disease trends and outbreaks and to institute appropriate preventive measures. Today more than ever we must be on the alert for new emerging infections and bioterrorism, and we need to be able to rapidly investigate them and prevent further cases. The awareness of the threat of biological agents as weapons is not new. Since the discovery of Iraq's biological weapons program, today's military leaders and policy makers have had concerns regarding the threat of biological warfare [1]. However, the increase in terrorist acts both domestically and internationally demonstrates the need to protect civilian populations as well as military forces against this possibility. Increases in the acquisition of, and alleged, or actual use of biological organisms as weapons of terrorism had been claiming

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J.A. Pavlin /Epidemiology ofBioterrorism

front-page status in our newspapers even before the anthrax letter attacks. The threat of bioterrorism is a reality, and to effectively counter the potentially devastating effects of an attack, we need to first understand basic epidemiologic principles of biological agents used as weapons. Best-selling fiction novels about bioterrorism attacks in the United States tend to portray a biological agent as highly contagious, genetically engineered organisms resistant to all known vaccines. While this scenario is not impossible, experiences to date have not proven it to be true. Besides the attacks with non-communicable anthrax that was susceptible to commonly used antibiotics, alleged attacks by the Aum Shinrikyo did not result in a single illness from a biological agent [2], and the successful 1984 attack contaminating salad bars in The Dalles, Oregon by a religious cult utilized a common salmonella strain that was not lethal, not contagious, and susceptible to antibiotics [3]. These examples are not intended to imply that a bioterrorism attack could not have a devastating impact, but only to point out that our level of suspicion and diligence in identifying and reacting to a biological attack must remain high, since it will likely not appear as popular literature describes. Furthermore, a small outbreak of illness could be an early warning of a more serious attack, and recognition and prompt institution of preventive measures such as effective vaccines and antibiotics could save thousands of lives. To facilitate the rapid identification of a bioterrorist attack, all health care providers and public health personnel should have basic epidemiologic skills and knowledge of what to expect in such a setting.

1. Differential Diagnosis Any outbreak of disease, in small or large numbers, should be evaluated as a potential bioterrorist attack. This initial investigation does not have to be time consuming or involve law enforcement. A simple look at the facts surrounding the outbreak to determine if anything seems unusual or indicative of a man-made epidemic should suffice. Since a disease outbreak can be of natural or unnatural origin, the differential diagnosis of an outbreak should first be considered. The possibilities include a spontaneous outbreak of a known endemic disease, a spontaneous outbreak of a new or reemerging disease in that area, a laboratory accident, or an intentional attack with a biological agent. Epidemiological tools can assist in differentiating between these possibilities. It may be very difficult to determine the cause of a disease cluster, or even that something unusual is occurring, especially if the initial cases are small in number. Not only should unusually high rates of illness trigger an investigation, but also any unusual disease event should signal a warning. For example, even one case of inhalation anthrax should cause immediate concern and action. To further complicate awareness of an attack, unlike chemical terrorism, biological terrorism will not be immediately obvious, but will most likely appear insidiously, with primary care providers witnessing the first waves of casualties. However, it may not even be emergency room personnel who first realize there is a problem. The first to notice could be a hospital laboratory seeing unusual strains of organisms, or the county epidemiologist keeping track of hospital admissions, or even pharmacists distributing more antibiotics than usual, 911 operators noticing an increase in respiratory distress calls, or funeral directors with increased business. All potential avenues of epidemiologic data need to be tracked and aggressively followed to ensure the most rapid recognition and response.

J.A. Pavlin /Epidemiology of Bioterrorism

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2. Epidemiologic Approach The basic epidemiologic approach in the evaluation of a potential bioterrorist or biowarfare attack is not different from any standard epidemiologic investigation. The first step is to confirm that an alleged disease or event actually has occurred using laboratory and clinical findings. A case definition should be constructed and used to determine the actual number of cases, and consequently the approximate attack rate. The use of objective criteria in the development of a case definition is very important in determining an accurate case number, as both additional cases may be found, and some may be excluded, especially as the potential exists for hysteria to be confused with actual disease. Once an estimated rate of illness is calculated, a comparison of rates during previous years should be performed to determine if this event constitutes a deviation from the norm. Once the case definition and attack rate have been determined, one can now characterize the outbreak via the conventional time, place and person. This data will provide crucial information in determining the potential source of the outbreak, and consequently, whether it appears to be of natural or man-made origin.

3. Epidemic Curve Using data gathered on cases over time, an epidemic curve can be calculated. The disease pattern is an important factor in differentiating between a natural outbreak and an intentional attack. In most naturally occurring outbreaks, numbers of cases gradually increase as a progressively larger number of people come in contact with other patients, fomites and vectors that can spread disease. Eventually, most of the population has been exposed, and is immune to further disease, and the number of cases, or epidemic curve, gradually decreases. Conversely, a bioterrorism attack is most likely to be caused by a point source, with everyone coming in contact with the agent at approximately the same time. The epidemic curve in this case would be compressed, with a peak in a matter of days or even hours, even with physiological and exposure differences. If the biological agent is contagious, it is possible to see a second curve peak after the first, as original cases expose originally unexposed people to the agent. The steep epidemic curve expected in a bioterrorism attack is similar to what would be seen with other point source exposures, such as food-borne outbreaks. Therefore, the compressed epidemic curve is still not pathognomonic for an intentional bioterrorism attack. If a specific group of people have become victims, the epidemic curve may give some suggestion on when they were exposed. From this information, a possible incubation period can be calculated. The incubation period can assist in determining the potential cause of illness, as well as suggesting a possible intentional attack if the incubation period is shorter than usual due to an unusually high inoculum or more effective exposure route than what is seen normally. Calculating the incubation period may also help in determining if the disease is contagious via person-to-person spread. The implications of a contagious disease are obviously of extreme importance to effective disease control measures.

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4. Epidemiologic Clues As steep epidemic curves can be seen in natural point-source exposures, additional characteristics of the outbreak should be investigated in determining the potential for it being man-made [4,5]. None of the following clues alone constitute proof of intentional use of a biological agent, but together can assist greatly in determining if further investigation is warranted. 1 - The presence of a large epidemic, with greater case loads than expected, especially in a discrete population. 2 - More severe disease than would normally be expected for a given pathogen, as well as unusual routes of exposure, such as a preponderance of inhalational disease as was seen in Sverdlovsk after the accidental release of aerosolized B. anthracis spores [6]. 3 - A disease that is unusual for a given geographic area or presents outside of the normal transmission season, or is impossible to transmit naturally in the absence of the normal vector for transmission. 4 - Multiple simultaneous epidemics of different diseases. 5 - A disease outbreak with zoonotic as well as human consequences, as many of the potential threat agents are pathogenic to animals. 6 - Unusual strains or variants of organisms, or antimicrobial resistance patterns disparate from those currently circulating. 7 - Higher attack rates in those exposed in certain areas; such as inside of a building if it was released indoors, or lower rates in those inside a sealed building if an aerosol was released outdoors. 8 - Intelligence that an adversary has access to a particular agent or agents. 9 - Claims by a terrorist of the release of a biologic agent. 10 - Direct evidence of the release of an agent, with findings of equipment, munitions or tampering. Even with the presence of more than one of the above indicators, it may not be easy to determine that an attack occurred through nefarious means. For example, the outbreak of salmonellosis in Oregon took months to determine that it was caused by an intentional contamination of salad bars [3]. Other naturally occurring incidents have been suspected of being caused by unnatural means, such as the hantavirus outbreak in the Four Corners area of the United States [7] and the West Nile virus epidemic in New York City [8]. There are no definite answers to this dilemma, but most importantly, even if no conclusive answer can be derived quickly, the means employed in reaching for that answer will still provide medical personnel with information that will help them prevent further morbidity and mortality. 5. Anthrax Example The anthrax letter attacks demonstrated many of the epidemiologic characteristics previously described. The cases were of a disease unusual for the geographic location, of exceptional severity and an unusual exposure route (inhalational) and warning was given by the perpetrator through the letters. In other ways, this outbreak would have been very difficult to detect or confirm as bioterrorism should another agent have been used. Only 22 cases occurred, they were spread out over a six week time frame, the cases were geographically widespread, and the strain of bacteria was not unusual and had normal antibiotic susceptibilities [9]. This attack has also provided more insight into how a bioterrorist attack might present. The presentation of an attack very much depends on the population targeted, the characteristics of the agent and the mode of transmission [10]. That a terrorist might use an unsuspected dissemination device (i.e., a letter) must be factored into any investigation. The attack may even have consequences unexpected by the perpetrator as it was unknown at that time the ability of anthrax spores to disseminate from letters through the mechanical action of mail

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sorters. The infinite number of variables present in an attack with a biological agent (target, dissemination method and device, differences in population immunity and susceptibility, type of agent, wind speed and direction if an aerosol, etc.) means that the medical and response community must always stay vigilant in detecting and responding to a bioterrorist attack. It is impossible to accurately predict all the epidemiologic features for the next occurrence. 6. Recommendations for Preparedness Recommendations to improve awareness and readiness should a bioterrorism attack occur include education of all medical personnel, especially those primary care providers and emergency personnel who will probably be the first to see victims of a biological attack. Training should include the basic epidemiologic principles outlined here as well as clinical information on diagnosing and treating the highest threat agents. Training should be refreshed periodically to ensure that skills remain current. Improved surveillance efforts should be instituted with as close to real time data gathering as possible. Robust surveillance systems are essential to detect any emerging or reemerging disease, whether natural or man-made. Quick recognition of any change in disease patterns will facilitate determining the source and preventing further exposure, which should be the key driving force behind any epidemiologic investigation. Many new types of disease surveillance systems have been developed for the purpose of rapid detection of disease outbreaks and bioterrorism [11-17]. Some of the systems use medical data sources that are routinely collected for other purposes, (e.g., emergency room logs), some collect new data at the point of patient encounter, and some use non-clinical data (e.g., pharmacy sales, school absenteeism) to trigger an alert. Many use new statistical methods to detect aberrations. Since the terrorist attacks of September 2001 and the subsequent anthrax mailings, there has been a proliferation of these systems developed and in use by city, county and state public health personnel, as well as by academia and the military [18,19]. Public health departments can use the expertise of those working in this area to assist in setting up additional real-time surveillance. Through strong epidemiologic training, a close attention to disease patterns, and a healthy respect for the threat of biological terrorism, potential problems can be discovered rapidly, and actions can be taken to decrease the impact of disease, regardless of its origin. References [1] [2] [3] [4] [5] [6] [7] [8]

Proliferation: threat and response. Office of the Secretary of Defense; November 1997. Broad WJ, Miller J. The threat of germ weapons is rising. Fear, too. New York Times 1998 Dec 27. Torok TJ, Tauxe RV, Wise RP, Livengood JR, Sokolow R, Mauvais S, et al. A large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars. JAMA 1997;278:389-95. Weiner SL. Strategies of biowarfare defense. Milit Med 1987; 152:25-28. Noah DL, Sobel AL, Ostroff SM, Kildew JA. Biological warfare training: infectious disease outbreak differentiation criteria. Milit Med 1998; 163:198-201. Meselson M, Guillemin J, Hugh-Jones M, Langmuir A, Popova I, Shelokov A, et al. The Sverdlovsk anthrax outbreak of 1979. Science 1994;266:1202-1208. Horgan J. Were four corners victims biowar casualties? Sci Am 1993;269(5):16. Fine A, Layton M. Lessons from the West Nile viral encephalitis outbreak in New York City, 1999: implications for bioterrorism preparedness. Clin Infect Dis 2001;32:277-82.

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[9] [10] [11] [12] [ 13] [ 14] [15] [16] [17] [18] [19]

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Centers for Disease Control and Prevention. Update: investigation of anthrax associated with intentional exposure and interim public health guidelines, October 2001. MMWR 2001;50:889-93. Jernigan DB, Raghunathan PL, Bell BP, et al. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiologic findings. Emerg Inf Dis 2002;8:1019-28. Miller JR, Mikol Y. Surveillance for diarrheal disease in New York City. J Urban Health 1999;76:38890. Pavlin JA, Kelley PW, Mostashari F, et al. Innovative surveillance methods for monitoring dangerous pathogens. In: Institute of Medicine (US). Biological threats and terrorism: assessing the science and response capabilities. Washington, DC: National Academy of Sciences; 2002. p. 185-196. Espino JU, Tsui F-C, Wagner M. Realtime outbreak detection system (RODS). Available from https://www.health.pitt.edu/rods/rods.htm. Accessed on 11 Feb 2002. Lazarus R, Kleinman KP, Dashevsky I, DeMaria A, Platt R. Using automated medical records for rapid identification of illness syndromes (syndromic surveillance); the example of lower respiratory infection. BMC Public Health 2001; 1:9. New Mexico Department of Health. Rapid Syndrome Validation Project (RSVP) Project Description. Available from http://epi.health.state.nm.us/rsvpdesc/default.asp. Accessed on 11 Feb 2002. Lober WB, Karras BT, Wagner MM, et al. Roundtable on bioterrorism detection: information systembased surveillance. J Am Med Inform Assoc 2002 Mar-Apr;9(2):105-15. Green MS, Kaufman Z. Surveillance for early detection and monitoring of infectious disease outbreaks associated with bioterrorism. Isr Med Assoc J 2002 Jul;4(7):503-6. Centers for Disease Control and Prevention. Syndromic surveillance for bioterrorism following the attacks on the World Trade Center - New York City, 2001. MMWR 2002;51:13-5. Lewis MD, Pavlin JA, Mansfield, JL, et al. Disease outbreak detection system using syndromic data in the greater Washington, DC area. Am J Prev Med 2002;23(3): 180-6.

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Health preparedness for the deliberate use of biological agents to cause harm: WHO'S activities Dr Ottorino COSIVI Project Leader, Preparedness for Deliberate Epidemics, Global Alert and Response Team Department of Communicable Disease Surveillance and Response World Health Organization, Geneva In most of the world, the public health infrastructure is already stretched to its limits in coping with natural health hazards. Against such a background, the additional threat to public health services posed by the deliberate use of biological or chemical agents could be considered as little more than a slight addition to the existing burden. The deliberate use of such agents, however, could be of such a scale or of such a nature that health-care systems would be unable to cope. For deliberate releases or threats of release of such agents, a spectrum of outcomes can be envisaged that ranges between two extremes: relative insignificance at one end, and mass destruction of life or mass casualties at the other. Widespread panic and fear are expected to follow any use or threat of use such agents, whatever the actual number of casualties it may provoke, leading to increased demand for medical and other emergency services. Remedies or countermeasures may be beyond the resources of many countries and therefore only available, if at all, through international cooperation. In 1970, technical guidance was made available to Member States by WHO in the publication Health aspects of biological and chemical -weapons. The WHO report was instrumental in achieving international consensus on the Biological and Toxins Weapons Convention [1] and the Chemical Weapons Convention [2]. The events of 11 September 2001 in New York and Washington in the United States of America and the dissemination of anthrax spores though the United States Postal Service in the fall of 2001 highlighted the need for public health preparedness for the possible use of biological and chemical agents to cause harm. Health organizations in both developing and industrialized countries were overwhelmed by requests for information and guidance on various aspects of chemical and biological weapons (CBW). Through the World Health Assembly resolution WHA55.16 of 18 May 2002 [3], WHO Member States requested the Director General to strengthen activities on global public health preparedness and response to deliberate use of biological and chemical agents or radionuclear material that affect health. WHO focuses exclusively on the public health aspects of preparedness and response of such threats and its actions are implemented through the existing framework provided by the Global Health Security: epidemic alert and response strategy as per WHA resolution WHA54.14 of 21 May 2001. WHO activities relevant to resolution WHA55.16 include four main areas of work: (a) international preparedness; (b) national capacity strengthening on preparedness and response; (c) public health preparedness for diseases associated with biological warfare, and (d) global outbreak alert and response. Ongoing activities within these four areas of work summarized below. In addition, a WHO

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Headquarters working group on Biological, Chemical Radiological Threats (BCR Working Group) has been established to exchange information and co-ordinate activities within the Organization. International preparedness The objective of this area of work is to provide international guidance and to monitor closely international developments on public health aspects of biological weapons, to avoid duplication and explore possibilities for new collaborations. Activities in this area of work include the fmalization of the second edition of the Public health response to biological and chemical weapons: WHO guidance, which is expected to be published by June 2003. Plans have been developed to publish further technical guidance material in the form of Supplements to the second edition. A WHO CBW Advisory Group is being established as a permanent resource for WHO and its Member States. WHO also continues monitoring international developments on various aspects of biological weapons to explore further possibilities for collaboration. Such activities particularly include the follow-up process of the Biological Weapons Convention, as agreed during its Fifth Review Conference in November 2002.

National capacity strengthening on preparedness and response The objective of this area of work is to respond to the increased number of requests for technical assistance by Member States for assessment of their national CBW preparedness and response programmes and training of staff. Field missions to advise Ministries of Health have been conducted in 2002 and 2003. WHO is developing and field testing guidelines to assess national health CBW preparedness and response programmes as these remain a high priority for reinforcing country capacities. WHO is strengthening laboratory and epidemiology country capacities for epidemic prone diseases, including possible deliberate diseases, through a programme targeting microbiologists and epidemiologists from several countries of the African, Eastern Mediterranean and European regions. This includes, in-depth review of surveillance system leading to national plan of actions for strengthening surveillance and early warning systems for both deliberate and naturally occurring epidemic diseases [4]. An Intercountry Meeting on Emergency Preparedness Strategies, was held in Bangkok, Thailand, 17— 20 March 2003, including countries of the WHO Regional Office for South-East Asia. WHO is also working with the United Nation Disaster Management Programme [5] (UN DTMP) on the development of a training module on the management of preparedness and response programmes on chemical, biological and radio-nuclear incidents for policy makers.

Public health preparedness for diseases associated with biological warfare selected diseases The objective of this area of work is to contribute to international preparedness on diseases associated with biological warfare, by (a) establishing global networks of experts and laboratories; (b) establishing standard and procedures, and disseminating information, and (c) setting up and implementing training. WHO is strengthening selected disease-specific networks, starting with anthrax. Activities on plague and smallpox are also being carried out.

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Other priority diseases — identified by a WHO risk assessment — include tularaemia, brucellosis, glanders, melioidosis, Q fever, typhus fever, coccidioidomycosis, and Venezuelan equine encephalomyelitis. Work in ongoing for the setting up a global database of anthrax experts and laboratories capabilities. Priorities of this project include the establishment of quality control mechanisms for laboratories of the anthrax laboratory network, the development of training materials as well as publication of the fourth edition of Guidelines for the surveillance and control of anthrax in humans and animals. In addition, the development of a plan of work for tularaemia —similar to that developed for anthrax — for implementation in late 2003 onwards is being considered.

Global alert and response The objective of this area of work is to provide the global public health community with timely information on "public health emergencies of international concern" and to provide support to Member States in their response activities, when required. Alert and response operations includes disease intelligence, verification, response and follow up. The Global Outbreak Alert and Response Network includes some 110 partners has proven its capacity to provide Member States with technical assistance in case of outbreaks of epidemic origin and intoxication. This mechanisms is being strengthen to address the challenge posed by CBW. The International Health Regulations of 1969 are being revised to provide the legal framework of the alert and response operations. The revised IHR will require Member States to report all "public health emergencies of international concern". The revised IHR would include provision for the provision of assistance to Member States for response and an option of confidential/provisional notification. Any "public health emergencies of international concern" would include events related to possible use or threat of use of CBW. However some key issues should be considered with regard the possible inclusion of deliberate epidemics in the revised IHR: (a) the need for WHO to maintain neutrality and focus its action on the public health component of response only; and (b) WHO has no mandate to assess the deliberate nature of a possible CBW use or threat of use. Should Biological Weapons be involved in such events, the responsibility to investigate such reports in order o ascertain the possible deliberate nature remain with the UN; and for Chemical Weapons with the Organization for the Prohibition of Chemical Weapons (OPCW). However, the UN may request WHO technical assistance in case of such investigations. References [1] [2] [3] [4] [5]

Convention on the prohibition of the development, production and stockpiling of bacteriological (biological) and toxin weapons and on their destruction, which came into force in 1975. Convention on the prohibition of the development, production, stockpiling and use of chemical weapons and on their destruction, which came into force in 1997. Global public health response to natural occurrence, accidental release or deliberate use of biological and chemical agents or radionuclear material that affect health, WHA55.16, 18 May 2002. http://www.who.int/cosr http://www.undmtp. rg/

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Preparedness Against Bioterrorism and Re-Emerging Infectious Diseases J. Kocik et al. (Eds.) IOS Press, 2004

Epidemic Outbreak Systems (EOS) Microarray Incorporation for Pathogen Identification* Eric HANSON1, Robb ROWELY1, Brian AGAN2, Clark TIBBETTS2, Debra NIEMEYER3 Expeditionary Operations, Science & Technology Directorate, Office of the Air Force Surgeon General, Falls Church, VA 22041, 2Infectious Disease Department, WilfordHall USAF Medical Center, Lackland AFB, TX78236, 3Joint Program Office for Chemical and Biological Defense, Falls Church, VA 22041 Originally published in the Society of Armed Forces Medical Laboratory Scientists (SAFMLS) Newsletter, Society Scope Fall 2002; 5(3): 1. Reprint coordinated through Editor; modified for the NATO Conference Series Book

Abstract. A model is proposed for validating the use of oligonucleotide arrays (microarrays) in the rapid detection and identification of biological agents. Random samples will be obtained from a study population of 76,000 active duty military personnel requiring approximately 30,000 evaluations for respiratory infections per year. This high incidence density of infectious disease in a controlled military setting creates a real-world opportunity for implementing and validating genomics technologies for the rapid detection of disease causing pathogens. Program processes include rapid biologic agent identification (target time for detection is less than two hours); diagnostic correlation with specific syndromic indicators for outbreak and surveillance purposes; longitudinal tracking of affected personnel; and utilization of hybrid information system technologies for relationship, association and predictive modeling. Program outcomes include assessments of 1) clinical accuracy of respiratory pathogen diagnosis and therapeutics utilizing genotypic-phenotypic information correlation; 2) cross platform comparison with existing polymerase chain reaction technologies and 3) predictive modeling techniques for outbreak investigations and disease surveillance. Future research applications based on this test program will include microarray assessments of other biologic, chemical and physical exposures (i.e., ecogenomics) and incorporating a genomics module into an electronic surveillance system, the Lightweight Epidemiology Advanced Detection and Emergency Response System (LEADERS) for nationwide surveillance information.

Disclaimer. The conclusions and opinions expressed in this document are those of the authors. They do not reflect the official position of the United States Government, Department of Defense, Joint Program Executive Office for Chemical & Biological Defense, United States Army or Air Force.

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1. Introduction The primary goal of the Epidemic Outbreak Systems (EOS) project is the rapid detection and identification of respiratory pathogens using genotypic specific information. The EOS project will serve as a model for microarray identification of organisms in an outbreak investigation. Success with this methodology will be a first step in revisiting the current methodology used in outbreak investigation protocols. This project also provides a real-world model for pathogen identification, which is required for early detection purposes in a scenario of a biologic agent release. Early pathogen detection and identification is key to disease prevention and maintenance of airpower projection. Case in point: 57% of personnel deployed in Desert Shield had diarrheal disease and 20% of the affected personnel were incapable of carrying out their duties [1]. Early detection of the diarrhea-associated pathogen can limit disease. This was demonstrated in December 1999 when a laboratory team deployed in Southwest Asia using new testing technology (real-time PCR) rapidly identified a food borne outbreak, thereby limiting the effects to only 3% of the base population with minimal mission impact [2,3]. Real-time PCR screening of a variety of environmental samples was key to alerting public health officials to improper food-handling practices. Subsequent molecular assessment of the food borne pathogen afforded determination of the contamination source4. The utility of PCR was again demonstrated when the technology was successfully applied to aid agent identification and clear sample backlogs following intentional release of anthrax October 2001 [5,6]. Worldwide, laboratories currently utilize culture for pathogen detection. This results in delays with specific pathogen identification, feedback to health care providers (HCPs) and proper implementation of public health interventions. Furthermore, this technology is difficult to use in the deployed environment. The end result is inadequate epidemiologic information for HCPs and other decision-makers to promptly stop disease transmission. Additional delays result from a combination of disjointed record-keeping often inherent in a mobile patient population, "shoe leather" epidemiologic techniques, inadequate real-time disease surveillance data and inability to access filtered or analyzed data. Advances in the fields of genomics and information systems have produced breakthroughs that will eliminate many of the current outbreak investigation problems.

2. The Approach To accomplish rapid pathogen identification, EOS will innovate current technology in microarrays. The EOS project will use low-density, high-throughput oligonucleotide-based microarrays for the rapid detection of respiratory pathogens in a controlled population. Basically, microarrays are miniature DNA-based testing platforms upon which a test sample is applied. Short pieces of single stranded DNA on the array corresponding to DNA of various pathogens will adhere to complementary DNA in the test sample. A visible signal is generated with DNA strand matching or hybridization (Figure 1) [7-9]. Innovations will include the combination of an advanced microarray substrate and matrix, sequence-specific identification of pathogens, and advanced signal detection methodologies. A project goal is the identification of a viral respiratory pathogen from clinical sample to identification in less than two hours. This represents outbreak investigation timesavings of over 500 hours based on current viral culture methods.

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Figure 1. Microarray Technology a. A High Throughput Oligonucleotide Microarray [9] Custom arrays offering up to 500,000 features

106 -107 copies of each DNA probe per feature

b. Sample Preparation and Testing [7,8]

Clinical and epidemiological surveillance for infectious disease outbreaks in a large training population is critical to military mission accomplishment. This has been true throughout the history of warfare and the threat of infectious disease remains an omnipresent threat for the Department of Defense (DoD) to confront. Controlled populations in the DoD, Veterans Administration (VA) and civilian communities are numerous [10]. They include special operations training, deployed sites, military academies, prisons, nursing homes, childcare and even the dining facilities at the Pentagon. Time savings in pathogen identification in any one of these settings will significantly decrease to mission accomplishment and save significant amounts of time, rescued productivity, money and resources by decreasing morbidity and mortality in the event of a biological disease outbreak. EOS will demonstrate rapid pathogen detection capabilities in a controlled population and realworld setting. Likelihood of technology transition to civilian industry and other governmental

E. Hanson et al. /Epidemic Outbreak Systems (EOS)

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agencies will be favorable when total cost-effectiveness evaluations are modeled for outbreak scenarios. Decreased microarray costs with increased production will follow the initial pilot studies proposed here. Data generated from the microarrays will be fed into a genomics module surveillance system to include the Lightweight Epidemiology Advanced Detection and Emergency Response System (LEADERS). LEADERS is currently being developed by the US Air Force and tested by DoD as a nationwide surveillance information system to provide early warning of aberrant events, particularly the presence of environmental hazards [11-13]. The goal of phase I is to standardize production of disease-specific diagnostic microarrays to improve the speed and sensitivity of respiratory pathogen detection in the Basic Military Training (BMT) population. Young recruits residing in close quarters are physically and mentally stressed during this training and this can increase their susceptible to a variety of communicable diseases. Close monitoring with improved pathogen identification will limit disease spread, diminishing morbidity and mortality. Phase II goals are performing comparisons with the current "gold standard" of viral cell cultures, and real-time polymerase chain reaction (PCR) technology. An additional application consideration for future development is a field-capable handheld microarray processing and reader unit. The assembled resources and team of USAF personnel partnering with other DoD institutions, civilian industry and academic leaders will provide the research setting and expertise to create this model system. This system will demonstrate the effectiveness of microarray technologies in pathogen identification. EOS will start with detection of viral respiratory pathogen-specific sequences, but the system proposed can be expanded to evaluate bacterial, fungal, parasitic and chimeric nucleotide sequences. Development efforts for EOS have emphasized future capabilities that allow not only direct hybridization and re-sequencing capabilities, but will evaluation for simultaneous assessment of biochemical reactions and proteomics capabilities.

3. Conclusion Challenges for the future microarray applications to overcome will be decreasing the expense of microarray production. It is not cost-effective to use microarrays to diagnose every respiratory pathogen seen in the HCPs office. However, this model will demonstrate utility of microarray use in a controlled population to rapidly diagnose militarily important disease processes in an operationally relevant model. Performing additional molecular assessments of biological, chemical, physical, and social exposures (i.e., ecogenomics) will be the logical progression in this developmental process. Continued collaborations within DoD, with other governmental agencies, industry and academia will be necessary to make this happen. References [1]

[2]

[3]

Hyms, KC, AL Bourgeois, BR Merrell, et.al. Diarrheal Disease During Desert Shield, NEJM, Vol 325:1423-1428, No. 20, November 1991; abstract: http://content.nejm.org/cgi/content/abstract/325/20/1423?maxtoshow=&HITS=10&hits=10&RESULTF ORMAT=&searchid=1015709380815 1301 l&stored search=&FIRSTINDEX=0&volume=325&firstpa ge= 1423&iournalcode=nejm. USAF Force Protection Battlelab News Release: Rapid Biological Agent Identification Initiative Enhances Force Protection through Early Foodborne Outbreak Detection, 20 June 2000: http://www.idahotec.com/rapid/success.htm. Niemeyer, D, M Corkern, W Mobley, M Eitutis, D Dubois, W Barnes, W Hamilton, K Lohman

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[4]

[5] [6] [7] [8] [9] [10] [11]

[12] [13]

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R. McBride. Real-time PCR Speeds Laboratory Diagnosis to Rapidly Identify a Salmonella Outbreak at a Deployed Location. 24th Annual Meeting of Society of Armed Forces Medical Laboratory Scientists (SAFMLS) Proceedings, p 34, March 2000. Niemeyer, D, R Watson, N Wertz, A Salmen, F McCleskey. Salmonella Surveillance Using Molecular Genotyping-Part of a Prevention Oriented Process Approach to Food Safety in the Deployed Environment. Presented at the International Conference on Emerging Infectious Diseases, Atlanta, GA, Jul 00. Jernigan, J, et. al. Bioterrorism-Related Inhalational Anthrax: The First 10 Cases reported in the United States. Emerging Infectious Diseases, Nov-Dec 01;Vol.7, No. 6, Nov 01, Center for Disease Control: http://www.cdc.gov/ncidod/eid/vol7no6/jernigan.htm. Air Force Link. Air Force sends teams to help anthrax assessment. 22 October 2001: http://www.af.mil/news/Oct2001/n20011022_1502.shtml. Brazma, A, H Parkinson, T Schlitt, M. Shojatalab. A quick introduction to elements of biology—cells, molecules, genes, functional genomics, microarrays. October 2001. EMBL European Bioinformatics Institute: http://www.ebi.ac.uk/microarrav/biologv intro.htm. Brown, PO, D Botstein. Exploring the new world of the genome with DNA microarrays. Nature Genetics, Vol 21, Supplemental pp 33-37, 1999: http://www.nature.com/cgi-taf/DvnaPage.taf?file=/ng/journal/v21/nls/ruU/ng0199supp 33.html. Agan, B, E Walter. Presentation entitled, "Genomics and Bioinformatics: Revolutionizing Medicine through Integrated Technologies." USAF Force Protection Battlelab Meeting, 11 December 2002. Hanson, E., B Agan, L Folio, R Rowley, D Niemeyer. Evaluation of Potential Methods for Rapid Identification of Central Nervous System Pathogens Using Microarray Technology. Submitted for publication, Mil Med April 2003. Niemeyer, D, E Hanson, R. Rowley, R Munson, K Schafer, Real-time Medical Surveillance for Early Warning and Mitigation of Environmental Hazards. Proceedings of the International Conference on Protection Against Biological Threats, Sponsored by DARPA and the General Karol Kaczkowski Military Institute of Hygiene and Epidemiology, Warsaw, Poland, June 2001. Jackson, J. On the edge. Washington Technology, Vol 16, No. 20, 21 January 2002: http://www.washingtontechnology.com/news/16 20/emergingtech/17706-1.html. Graham-Rowe, D. Hospital internet system could spot bioterrorist attack. 17 December 2001: http://web.mit.edu/hst.921/www/IEEE.htm.

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Preventing Emerging Infectious Diseases: Epidemiology and Laboratory Capacity Support Stephen A. MORSE Bioterrorism Preparedness and Response Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention Atlanta, Georgia 30333, USA Abstract. Epidemiology and laboratories have been vital components of the public health system. The realization that emerging infectious diseases were a major threat to the U.S. provided the stimulus for limited resources to enhance these capacities. However, the concern about the growing threat of bioterrorism has emphasized to need to further enhance the public health system including many of the same components and activities needed to respond to naturally occurring outbreaks of infectious diseases. A strengthened disease surveillance system will be important for detecting covert bioterrorist attacks. New laboratory systems and approaches can revolutionize disease surveillance by providing real-time data to epidemiologists.

1. Introduction By the middle of the twentieth century, it was widely believed that humans were winning the centuries-long war against infectious microorganisms [1]. However, this optimism was premature. Infectious diseases continue to be a menace to all people, regardless of age, gender, lifestyle, ethnic background, and socioeconomic status. They cause suffering and death, and impose an enormous financial burden on society. While some diseases have been conquered by improvements in urban sanitation and water quality, antibiotics and vaccines, new ones are constantly emerging (e.g., AIDS and hantavirus pulmonary syndrome), and others reemerge in drug-resistant forms (e.g., tuberculosis and malaria). In the early 1990s, the growing concern about the threat of emerging infectious diseases was highlighted in a report issued by the Institute of Medicine of the National Academy of Sciences [2]. The report emphasized the intimate links between U.S. health and international health and concluded that emerging infectious diseases were a major threat to U.S. health. There have been a number of events over the past few years that have focused attention on the growing threat of bioterrorism in the U.S. [3]. There is no guarantee that a terrorist will announce an attack. Therefore, without such an announcement, there will be no recognition that a biological attack is occurring until enough cases, perhaps including a number of fatalities, are observed and reported to allow recognition of an epidemic of an unusual disease. Many biological agents can cause illness in humans, but not all are capable of impacting public health and medical infrastructure on a large scale [4]. Historically, the military has been primarily concerned with an aerosol release of a biological agent on the battlefield [5], In

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contrast, public health preparedness activities have had to focus on a number of ways that a biological agent could be introduced into a civilian population. Thus, a bioterrorist may choose from among a number of different agents and dispersal mechanisms, e.g., aerosol, food, water, and infected animal vectors. Intentional release of pathogenic microorganisms could result in an outbreak similar to a naturally occurring one. Thus, with few exceptions (e.g., smallpox), an epidemiologic investigation would be needed to determine whether the outbreak was naturally occurring or due to the intentional release of an infectious microorganism. It has been recognized that there is also a need to improve the public health infrastructure to respond to bioterrorism [6], which would involve enhancing many of the same components and activities needed to respond to naturally occurring outbreaks of infectious diseases [7]. Public health has been a cornerstone of health protection and public safety, yet it has often lacked resources commensurate with its role, resulting in erosion in the capability of public health agencies to do their job [8].

1.1 U.S. Public Health Infrastructure In 1994, the Centers for Disease Control and Prevention (CDC) launched the first phase of a nationwide effort to revitalize national capacity to protect the public from infectious diseases [9]. This effort focused on four goals: improving disease surveillance and outbreak response; supporting research to understand and combat emerging infectious disease threats; preventing infectious diseases by implementing disease control programs communicating public health information; and rebuilding the infectious disease-control component of the public health infrastructure. An updated plan, which was organized under the same four goals, but in a different order, was published in 1998 [10]. The new document describes the second phase of CDC's strategy, taking into account new discoveries and challenges, and building on the experience, success, and knowledge gained from implementing the 1994 plan. In Goal I: Surveillance and Response, the objectives called for strengthening infectious disease surveillance and response in the U.S. and internationally, as well as improving methods for gathering and evaluating surveillance data. In Goal II: Applied Research, the objectives included improving tools for identifying and understanding emerging infectious diseases; determining risk factors for infectious diseases; and conducting research to develop and evaluate prevention and control strategies. The public health infrastructure is the underlying foundation that supports the response to emerging infections. In Goal III: Infrastructure and Training, the objectives and activities focused on enhancing epidemiologic and laboratory capacity in the U.S. as well as internationally. In the U.S., this required improving CDC's ability to communicate electronically with its partners as well as strengthening it's capacity to serve as a reference center for infectious diseases and drug-resistance testing. The objectives and activities of Goal III also addressed the need to enhance the nation's capacity to respond to outbreaks, including those caused by bioterrorism. All of the efforts described in the plan [10] are ultimately directed at the national and international implementation of Goal IV: Prevention and Control. 1.2 Global Infectious Disease Strategy It has been recognized that it is not possible to adequately protect the health of the U.S. without addressing infectious disease problems that occur elsewhere in the world [2]. In an age of

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expanding air travel and international trade, infectious microorganisms are transported across borders, carried by infected people, animals and insects, and contained within commercial shipments of contaminated food. Diseases can emerge in one region and spread throughout the world and foreign governments have come to rely on CDC to provide outbreak assistance and public health information whenever a new or reemerging disease threat is detected anywhere on the globe. This outbreak assistance would also be required if an intentionally caused outbreak occurred in the U.S. or abroad. CDC in consultation with public and private partners, both domestic and international, developed a document to further define it's global mission and to improve the capacity to detect, control, and prevent infectious diseases [11]. CDC's ongoing efforts to strengthen the U.S. public health infrastructure are critical to the success of its international efforts. The global infectious disease strategy defines priorities in six areas: international outbreak assistance; a global approach to disease surveillance; applied research on diseases of global importance; the application of proven public health tools; global initiatives for disease control; and public health training and capacity building. The priorities are described in Table 1. Table 1. Priorities for CDC's global infectious disease strategy, 2001-2002 (modified from [11]).

Area

Priority

International outbreak assistance

Dedicate specific resources (e.g., epidemiologic, diagnostic, and logistic) to international outbreak investigations.

Global approach to disease surveillance

Work with WHO and other partners to provide technical assistance to regional networks in Africa, Asia, and Latin America that can fill gaps in global disease surveillance and become components of a global network of networks.

Applied research on diseases of global Importance

Establish two or more long-term, on-site research collaborations in developing countries to test new strategies for disease control and prevention.

Application of proven public health tools

Work with developing country partner to launch a demonstration project that employs three or more proven public health tools to prevent and control infectious diseases, depending on local priorities.

Global initiatives for disease control

Work with foreign ministries and WHO on specific programs (e.g., eradication of polio and guinea worm disease, HIV/AIDS control).

Public health training and capacity building

Establish the first International Emerging Infections Program as a partnership among a ministry of health, CDC, a Field Epidemiology training Program, and one or more local universities or medical research institutes.

This article will address the enhancement of epidemiology and laboratory capacity and it's role in identifying and responding to a bioterrorism attack.

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2. Epidemiology and laboratory capacity (ELC) program The purpose of the Epidemiology and Laboratory Capacity for Infectious Diseases (ELC) program was to implement the CDC plan [9,10] by assisting state and eligible local public health agencies in strengthening their basic epidemiologic and laboratory capacity to address infectious disease threats. The ELC program has focused on notifiable diseases [10], food-, water-, and vector-borne diseases, vaccine-preventable diseases, and drug-resistant infections. The ELC program has supported activities that enhance the ability of a program to: identify and monitor the occurrence of infectious diseases of public health importance in a community; characterize disease determinants; identify and respond to disease outbreaks and other infectious disease emergencies; use public health data for priority setting and policy development, and; assess the effectiveness of activities. Funding for the ELC program began in fiscal year 1995 with ten awards and has grown steadily (Figure 2). By the end of fiscal year 2002, all states, six local health departments, and Puerto Rico had been funded for the ELC program. The average award per grantee was just over $900,000. Selected examples of how ELC program resources were used by states and large public health agencies to improve infectious disease surveillance and outbreak response are shown in Figure 3.

Figure 2. Epidemiology and Laboratory Capacity (ELC) program, fiscal years 1995 through 2000 (Courtesy of Debbie Deppe, CDC).

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Figure 3. Selected examples of how ELC program resources were used by states and large public health agencies to improve infectious disease surveillance and outbreak response (Courtesy of Debbie Deppe, CDC).

Strengthening collaboration between laboratory and epidemiology practice has been a crucial component of this plan. This collaboration is exemplified by the role of PulseNetTM, which was initially funded through the ELC program, in food-borne disease surveillance. Food has been used as a vehicle to intentionally spread infectious microorganisms among civilians [12,13]. Thus, PulseNet™, or a similar system, may be useful in detecting a bioterrorism event involving food. PulseNet™ is a national laboratory network, which includes state, local and federal public health laboratories. The laboratories perform standardized molecular typing of foodborne, disease-causing bacteria by pulsed-field gel electrophoresis (PFGE) using a rapid one-day protocol [14]. The PFGE patterns are shared electronically by participants via the Internet and are compared to a dynamic database of PFGE patterns maintained at CDC. PulseNet™ functions as a "cluster" detection tool. Clusters, which are identified by PulseNet™ are investigated by epidemiologists. If epidemiologic links are found between cases, the "cluster" is classified as an outbreak. Thus, PulseNet™ can both facilitate the early identification of common source outbreaks as well as assist in rapidly identifying the source of the microorganism. PulseNetTM began in 1996 with a standardized PFGE protocol for Escherichia coli O157:H7 and 10 participating laboratories. By 2002, it had grown to 65 participating laboratories with standardized protocols for additional important food-borne pathogens including Salmonella serotypes, Shigella spp., Listeria monocytogenes, and Clostridium perfringens. A similar network has been developed in Canada. Participating laboratories receive training and are certified. Laboratories also participate in proficiency testing. These

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networks have revolutionized food-borne disease surveillance in North America by providing real-time data to epidemiologists. An example of the impact that PulseNetTM on an outbreak of E. coli O157:H7 due to contaminated meat is shown in Figure 4. It has been estimated that the outbreak would have been detected about a week earlier and that the number of cases would have been reduced by 68%.

Figure 4.

Estimated impact of PulseNetTM if the 1993 multistate food-borne outbreak of Escherichia coli O157:H7 had occurred in 1998 (Courtesy of Balasubra Swaminathan, CDC).

3. Epidemiology and laboratory capacity for detecting a bioterrorism event An evaluation of the vulnerability of the U.S. to an attack on civilians with biological agents has focused on the role public health would have in detecting and managing a covert bioterrorist incident [15]. It was earlier noted that the local, state, and federal public health infrastructure was already stretched thin as a result of other public health problems and lack of resources. Laboratories are a vital component of the public health system. Local, state, and federal public health laboratories have been protecting the health of the U. S. population for more than a century [16]. They detect and report infectious diseases and work in concert with epidemiologists to control outbreaks, including those resulting from a covert bioterrorist incident. Laboratory reports form the basis of active surveillance systems [17], which will be needed for early detection and to determine the extent of a covert incident. The epidemiological skills, surveillance methods, diagnostic techniques, and resources required to identify and respond to an attack with a biological agent are similar to those required to detect and investigate unusual or unknown diseases. Although additional resources were needed to rebuild the public health infrastructure and reduce U. S. vulnerability to biological terrorism, the new resources would also strengthen U. S. capacity to respond to any outbreak of infectious disease. CDC's strategic plan for biological and chemical terrorism preparedness and response

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[15] was implemented in 1999 through funding of cooperative agreements to states and several large municipalities. Awards were based on up to five focus areas, with each area integrating training and research. These focus areas were: preparedness and planning; detection and surveillance; diagnosis and characterization of biological and chemical agents; response; and communication. After three years of funding, there were substantial improvements (see article on the Laboratory Response Network); however, additional preparedness efforts were needed. In the aftermath of the incidents involving the World Trade Center disaster and the dissemination of spores of Bacillus anthracis via the mail [18,19], a supplemental allocation of approximately $915 million went to all state health departments and select major metropolitan cities and territories for bioterrorism preparedness. In addition to the original focus areas, two new focus areas were added: risk communication and health information dissemination; and, education and training. Of these funds, $183 million went toward epidemiology and surveillance activities and $119 million went for laboratory capacity. There was also awareness that the issue is broader than bioterrorism. These funds support state and local health departments as they greatly enhance their surveillance, epidemiology, and laboratory capacity for bioterrorism, other infectious disease outbreaks, and other public health threats and emergencies. CDC is working to integrate surveillance for illness resulting from biological terrorism into a strengthened U.S. disease surveillance system. As part of this program, there is increased accountability on the part of the awardees to ensure that specific goals are met, such as increasing the number of epidemiologists in a given population area and strengthening ties with the public health laboratory.

4. Surveillance Systems for Emerging Infections There are a number of surveillance systems that have been developed that are useful in detecting outbreaks of emerging infectious diseases or illness due to the covert release of infectious microorganisms. Some examples are described below. The Emerging Infections Network (EIN), which functions as a sentinel system to monitor new or resurgent infectious diseases, was developed by the Infectious Diseases Society of America (IDSA) in cooperation with CDC [20]. Members of the EIN include adult and pediatric infectious disease consultants. Its membership represents a ready source of infectious disease expertise for CDC and state health departments to draw on during outbreaks or when unusual illnesses occur. EIN is a bridge between the infectious diseases and public health communities. GeoSentinel is a global surveillance network consisting of 22 travel/tropical medicine clinics located in the U.S. and elsewhere. It was begun in 1995 by the International Society of Travel Medicine and is based on the concept that these clinics are ideally situated to effectively detect geographic and temporal trends in morbidity (specific etiology or as syndromes) in travelers. The U.S.-Mexico Border Infectious Diseases Surveillance (BIDS) Project was designed to coordinate syndromic surveillance for infectious diseases (e.g., syndromes consistent with hepatitis and febrile-rash illness) along the 2,000-mile U.S.-Mexican border. EMERGEncy ID NET is a network of academically affiliated emergency medicine centers that operate emergency departments at 11 hospitals in large U.S. cities [21]. The network monitors a number of syndromes, including bloody diarrhea, illnesses that follow exposure to animals, and illnesses in immigrants. Laboratory-based surveillance for emerging antimicrobial resistance is also a critical element in preventing emerging infectious diseases. A recent example is the emergence of vancomycin-resistant Staphylococcus aureus in the U.S. and the necessity for laboratory testing [22,23].

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5. Summary Preparedness for bioterrorism will require certain specialized programs and policies; however, many aspects of bioterrorism preparedness will use strategies that are built on existing systems that are used routinely for naturally occurring infectious disease threats. Epidemiology and laboratories have been vital components of public health. Strengthening the public health infrastructure for surveillance and outbreak response, including on-the-ground epidemiologic expertise and enhanced laboratory capability will have a dual use in protecting the U.S. against emerging infectious diseases and covert bioterrorism attacks. References [1] [2] [3] [4] [5] [6] [7]

[8] [9] [10] [11] [12] [13] [14] [15]

Burnet, M., and D. O. White. 1972. Natural History of Infectious Diseases. Cambridge University Press, London, U. K. Institute of Medicine, National Academy of Sciences. 1992. Emerging Infections. Microbial Threats to Health in the United States. National Academy Press, Washington, D. C. Tucker, J. B. 1999. Historical trends related to bioterrorism: an empirical analyis. Emerg. Infect. Dis. 5:498-504. Rotz, L. D., A. S. Khan, S. R. Lillibridge, S. M. Ostroff, and J. M. Hughes. 2002. Public health assessment of potential bioterrorism agents. Emerg. Infect. Dis. 8:225-230. Eitzen, E. M., Jr. 1997. Use of biological weapons, p. 437-450. In Sidell, F. R., E. T. Takafuji, and D. R. Franz (ed.), Medical Aspects of Chemical and Biological Warfare, Textbook of Military Medicine, Part 1. Office of the Surgeon General, Washington, D. C. Institute of Medicine, National Academy of Sciences. 1999. Chemical and Biological Terrorism, National Academy Press, Washington, D. C. LeDuc, J. W., S. M. Ostroff, J. E. McDade, S. Lillibridge, and J. M. Hughes. 1999. The role of the public health community in detecting and responding to domestic terrorism involving infectious agents, p. 219230. In W. M. Scheld, W. A. Craig, and J. M. Hughes (ed.), Emerging Infections 3. ASM Press, Washington, D. C. Hamburg, M. A. 2002. Public health preparedness. Science 295:1425. Centers for Disease Control and Prevention. 1994. Addressing emerging infectious disease threats: a prevention strategy for the United States. Public Health Service, U. S. Department of Health and Human Services, Atlanta, Ga. Centers for Disease Control and Prevention. 1998. Preventing emerging infectious diseases: a strategy for the 21st century. Public Health Service, U. S. Department of Health and Human Services, Atlanta, Ga. Centers for Disease Control and Prevention. 2002. Protecting the nation's health in an era of globilization: CDC's global infectious disease strategy. Public Health Service, U. S. Department of Health and Human Services, Atlanta, Ga. Torok, T. J. R. V. Tauxe, R. P. Wise, et al. 1997. A large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars. J. A. M. A. 278:389-395. Kolavic, S. A., A. Kimura, S. L. Simons, L. Slutsker, S. Barth, and C. E. Haley. 1997. An outbreak of Shigella dysenteriae type 2 among laboratory workers due to intentional food contamination. J. A. M. A. 278:396-398. T. J. Barrett, H. Lior, J. H. Green, et al. 1994. Laboratory investigation of a multistate food-borne outbreak of Escherichia coli O157:H7 by using pulsed-field gel electrophoresis and phage typing. J. Clin. Microbiol. 32:3033-3037. Centers for Disease Control and Prevention. 2000. Biological and chemical terrorism: strategic planning, preparedness and response. Recommendations of the CDC strategic planning workgroup. Morbid. Mortal. Wkly. Rep. 49(RR04):1-14.

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[16] [17] [ 18] [19] [20] [21] [22] [23]

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Anderson, G., A. DiSalvo, W. Hausler, J. Liddle, J. McDade, and J. Sampson. 1993. Task force report on the public health laboratory: a critical national resource. Association of Public Health Laboratories, Washington, D. C. Roush, S., G. Birkhead, D. Koo, A. Cobb, and D. Flemming. 1999. Mandatory reporting of diseases and conditions by health care professionals and laboratories J. A. M. A. 282:164-170. Centers for Disease Control and Prevention. 2001. Recognition of illness associated with the intentional release of a biologic agent. Morbid. Mortal. Wkly. Rep. 50:893-897. Jernigan, J. J., P. L. Raghunathan, B. B. Bell, et al. 2002. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiological findings. Emerg. Infect. Dis. 8:1019-1028. Executive Committee of the IDSA Emerg. Infect. Network. 1997. The emerging infections network: a new venture for the Infectious Diseases Society of America. Clin. Infect. Dis. 25:34-36. Talan, D. A., G. J. Moran, W. R. Mower, et al. 1998. EMERGEncy ID NET: an emergency departmentbased emerging infections sentinel network. Ann. Emerg. Med. 32:703-711. Centers for Disease Control and Prevention. 2002. Staphylococcus aureus resistant to vancomycin— United States, 2002. Morbid. Mortal. Wkly. Rep. 51:565-567. Centers for Disease Control and Prevention. 2002. Public health dispatch: vancomycin-resistant Staphylococcus aureus—Pennsylvania, 2002. Morbid. Mortal. Wkly. Rep. 51:902.

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Preparedness Against Bioterrorism and Re-Emerging Infectious Diseases J. Kocik et al. (Eds.) IOS Press, 2004

The Laboratory Response Network Stephen A. MORSE, Richard KELLOGG, Samuel R. PERRY, Richard F. MEYER, David BRAY, David NICHELSON, Michael J. MILLER Bioterrorism Preparedness and Response Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention Atlanta, Georgia 30333, USA Abstract. The Laboratory Response Network is an international, unified consortium of laboratories that was established in response to the threat of bio-terrorism. The Laboratory Response Network is fully capable of responding quickly and appropriately to national or local public health emergencies. It has the capacity to respond to both bioterrorism events and naturally occurring outbreaks of infectious disease.

1. Introduction There have been a number of events over the past few years that have focused attention on the growing threat of bioterrorism in the United States [1]. From the public health perspective, bioterrorism is defined as the deliberate release of pathogens or their toxins into a population for the purpose of causing illness or death. Although some authorities had initially felt that the threat of bioterrorism was exaggerated [2], the recent incident using spores of Bacillus anthracis has made bioterrorism a reality in the United States [3], and has focused attention on national preparedness should another crisis occur. The Centers for Disease Control and Prevention (CDC) was designated by the Department of Health and Human Services to prepare the nation's public health system to respond to a bioterrorism event [4]. To enhance state and local preparedness, CDC funded cooperative agreements with every state and several large municipalities that focused on preparedness efforts [5]. Five critical areas were emphasized during the first three years of this program that began in 1999: preparedness planning and readiness assessment; surveillance and epidemiology capacity; biologic laboratory capacity; chemical laboratory capacity; and health alert network and information technology [4]. Based on lessons learned from the recent anthrax attack [3], additional resources and focus areas have been added: communicating health risks and health information dissemination; and, education and training. Bioterrorist attacks can occur as one of two scenarios, i.e., covert and overt. Because we currently lack the ability to conduct real-time monitoring for the release of a biological agent in U. S. cities, an unannounced (i.e., covert) release of a biological agent would likely go unnoticed for some time, with those exposed leaving the area long before the act of terrorism becomes evident. Due to an incubation period, the first signs that a biological agent has been released may not become apparent until days or weeks later, when individuals become ill and seek medical care. Thus, the "first responders" to a covert bioterrorism attack will likely be the astute clinician, laboratorian, or public health worker who recognizes the index case or identifies the responsible agent. Because of their terrorism training, traditional "first

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responders" (e.g., firefighters, hazmat or law enforcement personnel) are the most likely to respond to an announced (i.e., overt) release of a biological agent or, more likely, to a hoax. Thus, the initial recognition of a bioterrorism event (or hoax) in the U. S., whether announced or unannounced, would be at the local and state level. A comprehensive public health response to bioterrorism (or for that matter, to any outbreak of infectious disease) will involve epidemiologic investigation, medical treatment and prophylaxis for affected persons, and the initiation of disease prevention activities. The success of these activities is dependent, to a large extent, upon a rapid and accurate identification of the threat agent. 2. Agents Many biological agents can cause illness in humans, but not all are capable of impacting public health and medical infrastructures on a large scale [6]. In order to bring focus to public health preparedness activities, CDC convened a meeting of national experts in 1999 to review the criteria for selecting the biological agents that posed the greatest threat to civilians and to help develop a prioritized list of agents [6]. This list of "Critical Agents" (Table 2) was prioritized based on considerations such as: the ability of the agent to cause mass casualties; the ability of the agent to be widely disseminated either by aerosol or other means; the ability of the agent to be transmitted from person to person; the public's perception, correctly or incorrectly, associated with the intentional release of the agent; and special public health preparedness needs (e.g., vaccines, therapeutics, enhanced surveillance, or diagnostics). Table 2. Critical biological agents for public health preparedness (modified from [5]) Agent

Disease

Category A Variola major Filoviruses (e.g., Ebola and Marburg) Arenaviruses (e.g., Lassa and Junin) Bacillus anthracis Yersinia pestis Francisella tularensis Clostridium botulinum neurotoxin Category B

Smallpox Hemorrhagic fever Lassa fever, Argentine hemorrhagic fever Anthrax Plague Tularemia Botulism

Alphaviruses (e.g., Venezuelan, Eastern, and Encephalomyelitis Western encephalomyelitis viruses) Q fever Coxiella burnetti Brucella spp. Brucellosis Burkholderia mallei Glanders Staphylococcal enterotoxin B Staphylococcal food poisoning Ricin from Ricinus communis Ricin intoxication Clostridium perfringens epsilon toxin Food- and water-borne agents including (but not limited to): Salmonellosis Salmonella species Bacillary dysentery Shigella dysenteriae Escherichia coli O1 57: H7 Hemolytic uremic syndrome

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S.A. Morse et al. / The Laboratory Response Network Vibrio cholerae Cryptosporidium parvum Category C

Cholera Cryptosporidiosis

Multidrug-resistant Mycobacterium tuberculosis Yellow fever virus Tickborne encephalitis complex (flavi) viruses Tickborne hemorrhagic fever viruses Nipah and Hendra Complex viruses Hantaviruses

Tuberculosis Yellow fever Encephalitis Hemorrhagic fever Hantavirus pulmonary syndrome

As currently defined, Category A, which includes some of the classic biowarfare agents, are high priority agents that are most likely to cause mass casualties if deliberately disseminated and require broad-based public health preparedness efforts. Natural infections caused by agents in Category A are uncommon in the U. S. or nonexistent. For example, prior to the bioterrorist attack with B. anthracis in 2001, the last case of inhalational anthrax in the U. S. was in 1976 [7]. Furthermore, the World Health Organization declared smallpox eradicated in 1977 [8]. Category B agents also have some potential for large-scale dissemination, but generally cause less illness and death than those in Category A. Many of these agents have been weaponized in the past, or are being considered as weapons by some state-sponsored programs [9]. Some of the Category B agents could be used to contaminate food or water sources. In addition, many of these agents are relatively easy to obtain, and are thus more likely to be used in the setting of a biocrime [10]. Biological agents that are not currently believed to present a high bioterrorism risk to public health, but which could emerge as future threats were placed in Category C. Some of these agents are associated with emerging infections or have characteristics that, in the future, could be exploited for deliberate dissemination. The Critical Agent list has been used by the National Institutes of Health (NIH) in establishing priorities for bioterrorism-related research [11]. In the U. S., both clinical and laboratory experience is limited for the recognition and confirmation of the Category A agents and for many of the agents in Category B. The low numbers of human infections in the U. S. caused by Category A agents (as well as for many of those in Category B) has been given as a reason why there has been a general lack of interest by the commercial sector in spending money for the development, manufacture, and FDA approval of diagnostic tests. This situation has created the need for the development and restricted distribution of biodetection assays and specialized reagents, which would not otherwise be available to support the public health infrastructure and national security interests of the U. S. [12].

3. Laboratory Response Network Some individuals had suggested that a single high-throughput laboratory or perhaps a few regional laboratories would be sufficient to detect and respond to a bioterrorist attack in the U.S. However, an effective public health response would have to be rapid since there is only a small window of opportunity during which prophylaxis or other control measures could be implemented to reduce the morbidity and mortality associated with a bioterrorism event [13]. In order to facilitate the rapid identification of threat agents, the Laboratory Response Network (LRN) was created. The LRN was initially established as a national system designed to link

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state and local public health laboratories with advanced capacity clinical, military, veterinary, agricultural, water- and food-testing laboratories. It is a critical component of CDC's mission to lead the effort in strengthening the public health infrastructure, and consequently enhancing readiness to detect and respond to bioterrorism at the federal, state and local levels. Operational in August 1999, the LRN was established in recognition that the existing national infrastructure of diagnostic testing laboratories competent to deal with biological (or chemical) terrorism was extremely limited. The LRN was developed by the CDC in concert with the Association of Public Health Laboratories (APHL) and with collaboration from the Federal Bureau of Investigation (FBI) and the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). The LRN is the first example of a public health-law enforcement partnership. It was conceived to build on the existing interaction of nationwide public health laboratories, which participate in routine disease surveillance activities [14]. The LRN has a dual function in that it has the ability to detect and respond to agents released by a bioterrorist as well as to those that occur naturally. This capacity is particularly important, since it will generally not be known at the time of detection whether the outbreak was intentional or natural. The LRN collaborative partnership operates as a national network of laboratories designated level A through level D. Because there were marked differences in capabilities and infrastructure, each member laboratory initially provided an agent-specific designation of level A, B, or C. Each of these levels represented progressively stringent levels of safety, containment, and technical proficiency necessary to perform the essential tests to rule-out, rulein, as well as perform the referral functions that are required for agent identification and confirmation (Figure 5A). Thus, a laboratory could be level C for botulinum toxin testing and level B for anthrax.

Figure 5. Conceptual diagram of the Laboratory Response Network as it was conceived in (A) 1999; and, a proposed diagram to reflect the expansion of the Laboratory Response Network to include other types of sentinel laboratories (2002)

Level A laboratories are, for the most part, hospital and other community clinical laboratories. The reasoning behind this decision is that in the aftermath of a covert bioterrorism

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attack, patients will seek care at many widely dispersed hospitals where these laboratories exist [14]. These laboratories participate in the LRN by ruling out or referring critical agents (Table 2) that they encounter in their routine work to the nearest LRN level B or C laboratory. To make this process as rapid as possible, protocols and algorithms have been developed in collaboration with the American Society for Microbiology (ASM) that can be implemented by the clinical laboratory in its routine day-to-day operation. The algorithm for Bacillus anthracis is shown in Figure 6.

Bacillus anthracls: Level A laboratory flowchart Morphology: Large aerobic, gram positive rods (1 to 1.5 by 3 to 5 (in.) Smears/blood/CSF: Short chains of 2-4 Cells that appearencapsulated Sheep blood agar (amble nt atmosphere): Oval, central-to-sub terminal spores which do not cause significant swelling of cell; often In long chains Growth on sheep blood agar. 2-5 mm, tenacious, nenhemolytic colonies after 15-24 h (flat/slightly convex, Irregularly round colonies with Irregular/wavy border and ground glass appearance) Perform all additional work In biosafety cabinet Hemolysis: Negative Catalase: Positive Motility: Nonmotile

NO

(features net present) Report:Bacillusspecles, NOT B. anthracls; continue Identification per laboratory procedures

Yes (features present) Report Bidllus spedes, sent to reference laboratory to rule out a anthracis

Figure 6. Algorithm developed for the rapid rule-out of Bacillus anthracis by clinical laboratories

This algorithm could be used to rapidly rule-out B. anthracis from blood cultures growing Gram positive rods. The algorithms for B. anthracis, Yersinia pestis, Francisella tularensis, Clostridium botulinum neurotoxin, and Brucella spp. can be accessed on the internet at either www.asmusa.org or www.bt.cdc.gov. In order to make the referral process work effectively, level A (i.e., clinical) laboratories must know the location of the nearest LRN level B or C laboratory. This has been facilitated through an educational process carried out at the state and local levels. At the beginning of the program, Level B laboratories were primarily state and local public health laboratories, with biosafety level 2 facilities where biosafety level 3 practices are observed. Level C laboratories were primarily public health laboratories with biosafety level 3 facilities, or those with certified animal facilities, which are necessary for performing the mouse toxicity assay for botulinum toxin. Level C laboratories could perform all of the Level B tests as well as additional tests that required biosafety level 3 containment (e.g., handling powders suspected of containing anthrax spores). Many Level B/C laboratories work closely with local FBI agents. This partnership was especially evident during the recent anthrax attack when FBI agents helped LRN laboratories triage specimens for analysis by performing threat assessments. There are currently 110 LRN level B and C laboratories in the U. S. and Canada,

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with new members being added quickly. All 50 states now have the capacity to perform presumptive and confirmatory laboratory identification methods for B. anthracis, Y. pestis, and F. tularensis as well as access to specialized level C laboratories for confirmation of Brucella spp. and C. botulinum neurotoxin. With the increased threat of smallpox and the plans to begin smallpox vaccination, all LRN level B and C laboratories have been given the capacity to test for Vaccinia virus and Varicella zoster; selected laboratories will have the capacity to test for Variola major. There are two Level D federal laboratories (CDC and USAMRJID) with biosafety level 4 facilities that can handle agents such as Ebola and Variola major, for which other laboratories have insufficient safety facilities or unvaccinated staff. Level D laboratories also have the capacity to perform all of the Level B and C procedures. In addition, they identify agents in specimens (i.e., human and animal) and samples (i.e., environmental, food, etc.) that have been referred by the Level B and C laboratories, identify recombinant microorganisms (e.g., chimeras) that may not be recognizable by conventional isolation and identification methods or by those used in the Level B/C laboratories. Level D laboratories also maintain extensive culture collections of critical agents against which the isolate(s) from a bioterrorist event may be compared using molecular methods to determine its likely origin. Over the past four years, the LRN has matured and clear distinctions between Level B and Level C have all but vanished because of the improvements that were made in every state as a result of the funds provided by the U. S. Congress to strengthen the public health infrastructure. Among the improvements were the construction of a number of biosafety 3 laboratories, hiring of additional personnel, and the purchase of state-of-the-art equipment for performing real-time nucleic acid amplification and time-resolved fluorescence antigen detection assays. As a result, a new description of the structure is currently being developed. The LRN is now more accurately described as a unified network of integrated laboratories functioning through a single operational plan and represented by sentinel laboratories of laboratory 1st responders (formerly Level A) and by confirmatory testing laboratories (formerly Levels B, C, and D) representing several disciplines (Figure 5B). The maturity of the LRN structure is commensurate with a parallel increase in technical capacity and response ability of the public health system. The basis of admission into the LRN is predicated by: (1) the laboratory's contribution to the public health needs of the state; (2) the ability of the laboratory to meet safety requirements for handling select agents [15]; (3) the ability of the laboratory to meet physical and personnel security requirements [16,17]; (4) successful completion of a checklist evaluating these and other items; and (5) participation in the secure communication of test results. For non-public health laboratories, such as clinical laboratories, to become members of the LRN in a confirmatory role, requires documentation from the state public health laboratory director that there is a need for the additional laboratory capacity and that the invited laboratory meets all requirements. CDC's role through the LRN is to support the public health infrastructure, which is defined by public health laboratory work. In this regard, CDC currently develops, produces, validates, packages, and ships all reagents used in the rapid screening tests developed by CDC laboratories and its federal partners for these agents. State funding for the LRN has come from the Federal cooperative agreements, which began in 1999. In the first year of this program, 43 project areas (41 states, New York City and Los Angeles County) received $8.7 million (range of award was $58,755-$566,906, with an average award of $203,000) to enhance public health laboratory capacity to respond to a bioterrorism event. In order to extend laboratory coverage throughout the country, states that were not initially funded were invited to join the LRN and were thus able to receive protocols,

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reagents and training. By the beginning of the third year, and prior to 9/11/2001, the number of funded project areas increased to 54 (50 states, New York City, Los Angeles County, and Guam). During the first three years of this program, a total of $23.1 million was awarded to enhance laboratory capacity to respond not only to bioterrorism, but to any outbreak of infectious disease. In the aftermath of the anthrax attack, project areas received supplemental awards totaling $118 million for biologic laboratory capacity. The LRN has been strengthened through collaborations with the Department of Defense (Army, navy, Air Force), Department of Justice (FBI), Department of Energy (Lawrence Livermore and Los Alamos national Laboratories), Food and Drug Administration (Centers for Food Safety and Devices and Radiological Health), U. S. Department of Agriculture (National Veterinary Service Laboratory and Food Safety Inspection Service), Environmental Protection Agency (Office of Research and Development), and National Institutes of Health (Office of Research Service), as well as professional societies (American Society for Microbiology), and public health organizations (Association of Public Health Laboratories), thus leveraging not only highly developed expertise, but also galvanizing larger networks for emergency response and overall continuity of operations. Many of these collaborations have been incorporated into the new proposed LRN structure (Figure 5B). The LRN has cautiously begun to expand internationally. In 2000, two laboratories in Canada were added to the LRN. More recently, laboratories in the United Kingdom and Australia have become members. 3.1 Protocols, Reagents and Assays Confirmatory laboratories use standard protocols and reagents for the identification and confirmation of threat agents. Bioterrorism is a criminal act and specimens or cultures will be evidence in a criminal investigation. Thus, the protocols also have information concerning chain of custody requirements. Protocols for the Category A and B agents were written by subject matter experts at CDC, USAMRIID and the FBI and reviewed for accuracy and ease of use by laboratorians representing the LRN. The protocols, which are available to LRN members on a secure web site currently managed by CDC, contain the information for ordering the necessary reagents and control strains for performing the tests. Technologically, many U. S. public health laboratories have lagged behind those in the private sector. Thus, the original protocols relied on techniques (e.g., culture and staining with fluorescein-labeled antibodies [DFA]) that were already familiar to all public health laboratories to identify many of the critical Category A agents. Due to the lack of commercially available DFA reagents, the LRN relied on reagents produced at CDC or purchased from USAMRIID. Subsequently, protocols were updated as new, rapid assays were developed and validated [18]. The focal point for the advances in technology within the LRN was the Rapid Response and Advanced Technology (RRAT) laboratory at CDC, which was created in 1999. The RRAT laboratory is a vital component of CDC's bioterrorism preparedness effort and a partner to the LRN where it serves as a source of test methods, validation data, training, and proficiency testing for the member laboratories. The RRAT laboratory develops novel approaches to molecular screening for the Category A and B agents using real-time polymerase chain reaction (RT-PCR) assays, time-resolved fluorescence (TRF) immunoassays, and other technologies. LRN laboratories were given the option of choosing which commercially available thermocycler capable of performing real-time PCR was best suited for its laboratory. With the availability of supplemental funds, many laboratories

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purchased additional instruments. The instruments for which RT-PCR assays were optimized by the RRAT laboratory were: LightCycler (Roche Diagnostics Corporation, Indianapolis, IN); SmartCycler (Cepheid, Sunnyvale, CA); and the GeneAmp 5700, Prism 7000 and 7700 (Applied Biosciences, Foster City, CA). Protocols for RT-PCR assays for the detection of nucleic acid from a number of Category A agents have been added to the LRN web site together with protocols for TRF assays for antigen or antibody detection. The Victor2 (Perkin Elmer-Wallach) instrument was selected for the TRF assays because many state public health laboratories were using a similar instrument for newborn screening assays. The new rapid assays have markedly reduced the amount of time it takes to make an identification of a critical agent. For example, culture may take 24 to 72 hours to grow and identify the agent; with DFA staining, some agents could be identified in 3 to 4 hours. Today, using technology available in LRN laboratories, an answer can be obtained in approximately 1 hour. 3.2 Training Training has always been considered critical in the overall effort to prepare the nation to respond to a bioterrorism attack. Initial efforts centered on increasing the awareness of the clinical laboratories to the threat of bioterrorism, the agents of concern, and the LRN. A 1-day course, cosponsored by CDC and the National Laboratory Training Network (NLTN), was presented during the summer of 1999 in several cities in the U. S. (Atlanta, GA; Boston, MA; Philadelphia, PA; Denver, CO; Chicago, IL; Dallas, TX; Phoenix, AZ; Seattle, WA). More than 600 individuals attended these courses. Since 1999, the NLTN has put on more than 115 awareness courses for clinical microbiologists involving 4,915 students. In an attempt to reach additional individuals who worked in clinical laboratories, talks were presented at national and regional meetings of professional societies (e.g., ASM, American Society for Clinical Pathology). The presentations at these meetings were publicly available on CDC's bioterrorism web site. The results of a survey of state public health laboratory capacity conducted by APHL during the summer of 1998, indicated that most respondents had limited experience in identifying the bacterial agents in Category A and B. In order to address this need, a one-week course consisting of lectures and laboratory training was developed and offered four times in the fall of 2000 at the Georgia State Public Health Laboratory. This course addressed the Level B protocols for B. anthracis, Y. pestis, F. tularensis, and Brucella spp. Additional topics included the role of the RRAT laboratory, handling hazardous evidence, and automated microbial identification systems. One individual representing each of the cooperative agreement sites as well as individuals from states that did not receive funding were invited to attend. The intent of the course was to train additional trainers. After completion of the course, these individuals were expected to return to their respective public health laboratories and train additional laboratorians in their respective jurisdictions. However, the results of a recent survey suggested that some of these "trainers" have not trained additional laboratorians [19]. Rapid nucleic acid amplification and antigen detection assays and protocols were developed and validated by the RRAT laboratory and placed on the LRN web site in October 2001. The bioterrorism-related anthrax attack caused a delay in the training necessary for performing these assays. Six one-week training courses for these RT-PCR and TRF were completed by the spring of 2002. Again, one individual representing each of the cooperative

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agreement sites received training and was expected to return to their respective laboratory and train additional laboratorians.

3.3 LRN Response The LRN played a critical and successful role in the nation's response to the bioterrorismrelated anthrax incidents during October through November 2001. The LRN concept was validated when on October 3, 2001 the LRN laboratory in Jacksonville, FL rapidly confirmed that the Gram positive rod isolated from the cerebrospinal fluid and blood of the index case in Palm Beach County was B. anthracis [20]. Although the bioterrorism-related anthrax cases were limited to four states (Florida, New Jersey, New York, Connecticut) and the District of Columbia [21], the impact was felt nationwide. During this period, LRN laboratories tested over 125,000 clinical specimens and environmental samples involving approximately 1 million assays: 69% of the specimens were processed by public health LRN laboratories, 25% by Department of Defense LRN laboratories, and 6% by the CDC. Of particular interest was the observation that the majority of the environmental specimens were from states where there was no anthrax (Figure 7). Most of these were either hoaxes or specimens provided by frightened or concerned individuals.

Figure 7.

Environmental and clinical specimens processed by LRN laboratories, October - November 2001.The black portion of the graph represents specimens processed by laboratories in states where there were anthrax cases; the gray portion of the graph represents specimens processed in states where there were no anthrax cases.

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3.4 Data Management and the LRN Coordination of the LRN's daily operations is entrusted to a dedicated help desk resource that acts as a clearinghouse and funnels tasks to the appropriate individuals. This eliminates confusion by providing a single point of triage for all ongoing membership activities from which the needs of laboratories and laboratorians can be reviewed and tracked. An added benefit has been the positive feedback from LRN member laboratories, who have been reassured by the presence of a committed help desk and point of contact with whom they could develop a relationship. A Data Management Team was established to address the IT needs of the LRN. For example, to support electronic reporting of proficiency testing results through the LRN website, a reporting database was designed and built, which permitted LRN member laboratories to compare the results they had submitted to the correct results along with a grade of each laboratory's performance. A database of LRN members eligible for the licensed anthrax vaccine was also developed. To track the number of laboratorians desiring the vaccine, each laboratory was contacted, information was collected, and subsequently conveyed upon request to a contractor who was administering the vaccine. Currently, near term and long term solutions are being developed for the secure, real-time reporting of laboratory data as well as the sharing of information that is critical to the public health response to bioterrorism.

4. Summary The LRN is an international, unified laboratory consortium that was established in response to the threat of bioterrorism that is fully capable of responding quickly and appropriately to national or local public health emergencies. Funding for the LRN has helped rebuild the country's public health laboratory capacity to respond to a bioterrorism event as well as to naturally occurring outbreaks .of infectious disease. By providing funding for infrastructure, state-of-the-art equipment, and personnel, LRN laboratories are in a better position to respond to infectious disease threats. A well-trained laboratory workforce, including American Board of Medical Microbiology (ABMM) or equivalently certified specialists, is essential for national security. However, this program was not designed to address the current nationwide shortage of laboratorians. This cannot be accomplished through short-term funding, but requires a longterm national strategy.

References [1] [2] [3] [4]

[5]

Tucker, J. B. 1999. Historical trends related to bioterrorism: an empirical analysis. Emerg. Infect. Dis. 5:498-504. Cohen, H. W., R. M. Gould, and V. W. Sidel. 1999. Bioterrorism initiatives: public health in reverse. Am. J. Pub. Health 89:1629-1631. Centers for Disease Control and Prevention. 2001. Update: investigation of anthrax associated with intentional exposure and interim public health guidelines, October 2001. MMWR, Morbid. Mortal. Wkly. Rep. 50: 889-893. Centers for Disease Control and Prevention. 2000. Biological and chemical terrorism: strategic plan for preparedness and response. Recommendations of the CDC Strategic Planning Workgroup. MMWR, Recomm. Rep. 49(RR-4):1-14. Khan, A. S., S. Morse, and S. Lillibridge. 2000. Public-health preparedness for biological terrorism in the USA. Lancet 356:1179-1182.

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[6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]

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Rotz, L. D., A. S. Khan, S. R. Lillibridge, S. M. Ostroff, and J. M. Hughes. 2002. Public health assessment of potential bioterrorism agents. Emerg. Infect. Dis. 8:225-230. Centers for Disease Control. 1976. Anthrax-California. MMWR, Morbid. Mortal. Wkly. Rep. 25:33-34. World Health Organization. 1980. The global eradication of smallpox: final report of the global commission for the certification of smallpox eradication. World Health Organization, Geneva, Switzerland. Miller, J., S. Engelberg, and W. Broad. 2001. Germs. Biological weapons and America's secret war. Simon & Schuster, New York. Kolavic, S. A., A. Kimura, S. L. Simons, L. Slutsker, S. Barth, and C. E. Haley. 1997. An outbreak of Shigella dysenteriae type 2 among laboratory workers due to intentional food contamination. JAMA 278:396-398. National Institute of Allergy and Infectious Diseases. 2002. The counter-bioterrorism research agenda of the National Institute of Allergy and Infectious Diseases (NIAID) for CDC Category A agents. National Institutes of Health, Bethesda, MD Meyer, R. F., and S. A. Morse. 2002. Bioterrorism preparedness for the public health and medical communities. Mayo Clin. Proc.77:619-621. Kauiman, A. F., M. I. Meltzer, and G. P. Schmid. 1997. The economic impact of a bioterrorist attack: are prevention and post-attack interventions justifiable? Emerg. Infect. Dis. 3:83-94. Gilchrist, M. J. R. 2000. A national laboratory network for bioterrorism: evolution from a prototype network of laboratories performing routine surveillance. Mil. Med. 165(suppl. 2):28-31. Department of Health and Human Services. 2002. 42 CFR Part 1003. Possession, use, and transfer of select agents and toxins. Fed. Reg. 240:76886-76905. Centers for Disease Control and Prevention, National Institutes of Health. 1999. Biosafety in microbiological and biomedical laboratories, 4th ed. U. S. Govt. Printing Office, Washington, D. C. Pub. L. No. 107-56. Uniting and Strengthening America by Providing Appropriate Tools Required to Intercept and Obstruct Terrorism ("USA PATRIOT") Act of 2001. Hoffmaster, A. R., R. F. Meyer, M. P. Bowen, et al. 2002. Evaluation and validation of a real-time polymerase chain reaction assay for rapid identification of Bacillus anthracis. Emerg. Infect. Dis. 8:1178-1181. Association of Public Health Laboratories. 2002. State public health laboratory bioterrorism capacity. Public health laboratory issues in brief, October 2002. Washington, D. C. Jernigan, J. J., D. S. Stephens, D. A. Ashford, et al. 2001. Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States. Emerg. Infect. Dis. 7:933-944. Jernigan, J. J., P. L. Raghunathan, B. B. Bell, et al. 2002. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiological findings. Emerg. Infect. Dis. 8:1019-1028.

Preparedness Against Bioterrorism and Re-Emerging Infectious Diseases J. Kocik et al. (Eds.) IOS Press, 2004

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Imported viral diseases, surveillance and control in Europe - European Network for the Diagnostics of "Imported" Viral Diseases (ENIVD) Mattias NIEDRIG Robert Koch-Institut, Berlin, Germany

In the last decades we became aware of the emergence of several viral disease outbreaks in humans which have their origin in non-human species. The most important of these, AIDS caused by HIV-1 and -2 virus probably entered the human population from the simian host more than 60 years ago and became pandemic since the 1980s. Viruses like Hendra and Nipah passed the species barrier just recently and cause fatal diseases in humans with close contact to infected horses or pigs. Others are known for many years as zoonotic viruses like Hanta and human Influenza and are a constant threat for the developed world, and yet others are only endemic in tropical areas but cause increasingly problems like Yellow Fever, Lassa and Dengue. There are several reasons for the emergence of viral threats: Increase of migration by tourism and of refugees; Increase of world population; Spread of vectors by microbial adaptation & climate change; Breakdown of public health systems. Through the imported Yellow Fever and Lassa cases to Europe we all became aware that these dangerous infections could be imported to Europe from endemic regions in a very short time. The surveillance of such diseases very much depends on good and reliable case definitions and good diagnostic tools. Just recently the EC has decided for setting up a network for epidemiological surveillance and control of communicable diseases the European community. There are also several viral diseases like Yellow Fever and all viral haemorrhagic fevers (Crimean Congo HF, Ebola, Lassa fever, etc.) on the list. To improve the diagnostic capacity and quality for "imported" viral infections scientists from nearly all European countries from laboratories working in the field of diagnostics of "imported" viral diseases have started to build up a network and have worked out the objectives to be addressed in this collaboration. In eleven meetings scientists from laboratories working in the field of diagnostics of "imported" viral diseases in the United Kingdom, Sweden, France, Greece, Spain, Denmark, Netherlands, Belgium, Portugal, Finland, Italy, Ireland, Slovenia, Lithuania, Switzerland, Bosnia-Herzegovina, Czech Republic, Slovakia, and Germany have started to build up a network to improve the diagnostics of "imported" viral infections and have worked out objectives to be addressed in this collaboration: 1. Build a network of European laboratories working on diagnostics of "imported", rare and emerging viral infections. Provide mutual help in the exchange of diagnostic samples, i.e. sera, viruses, methods, and information in order to improve diagnostics

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2.

3. 4. 5. 6. 7. 8.

Identify those viral infections more likely to be imported and co-ordinate the objectives and identify laboratories, capable and willing to perform the rapid diagnostics (<24h) of an acute case, suspected to be infected with a viral haemorrhagic fever. Work out recommendations for standardisation and quality control in diagnostics laboratories involved in the diagnostics of such diseases. Identify and operate standard assays according to defined quality control criteria. Optimise limited resources by exchanging reagents, methodologies, and expertise. Encourage regular contact within the network through meetings, exchange and training of laboratory personnel. Open the network for members of other European laboratories. Organise and co-ordinate international activities with the "Surveillance network group", the "Task force on vaccines and viral diseases", or other national or international organisations like CDC and WHO . Time

lmported from

to

Viral pathogen

Kenya S Susp VHF1 Ivory Coast CH Ebola Brazil CH YF Nigeria ? D Susp VHF1 Zimbabwe GB CCHF Gambia B Susp VHF2 YF Ivory Coast D Ivory Coast D Lassa Sierra Leone GB Lassa Nigeria D Lassa Sierra Leone NL Lassa Kenya D Susp VHF3 D Susp VHF2 Sierra Leone Chile/ F Hanta Argentine (Andes) 03/01 D Susp VHF2 Sierra Leone 08/01 D CCHF Bulgaria 11/01 B YF The Gambia no final diagnosis,2 final diagnosis: Malaria, 3 final diagnosis: generalised HSV-1 01/90 11/94 04/96 09/97 02/98 11/98 08/99 01/00 02/00 03/00 06/00 12/00 03/01 03/01

No of cases / fatalities

Business / Tourist

1/0 1/1

Tourist Business

1/1 \l \l \l 1/1 \l 1/1 1/1 1/1 1/1 1/0 1/0 1/0 1/0

1/1

Refugee Tourist Business Tourist Business Repatriation Business Tourist Business Tourist Business Tourist Tourist

The table above gives a brief overview on the recent imported cases of suspected or real Viral Haemorrhagic Fevers (VHFs) to Europe. Most of these cases had a severe outcome. According malaria cases imported to Europe most experts feel that several cases are not detected due to the missing knowledge of medical staff on VHF disease symptoms. For better management of VHFs the advisory board of the ENIVD developed the first European recommendation for "Management and Control of VHFs" which is like other useful information available on the website: www.enivd.org For further development and the integration of other European members, the project needs European funding in order to maintain the European network and to improve and standardise diagnostic techniques. This will offer an opportunity for technology transfer within the European Community and result in better and more efficient diagnostics in the individual

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countries. In the long run, this initiative offers a chance of increasing the awareness of responsible medical institutions in each country of the EU regarding the problem of emerging and re-emerging viral diseases. By creating these surveillance activities, we will be able to better recognise and respond to threatening emerging and re-emerging diseases in the future. In supporting this group of experts, it will be possible to initiate a co-ordinated international effort to deal with these problems. This offers the chance for future interventions and an effective and close co-operation with the WHO, and also for the future implementation of proposed activities. The ENIVD provides it's members with continuous information on world outbreak situations and shares information on outbreak investigations and diagnostic methods and expertise. This is also achieved by regular meeting which provide an excellent opportunity to gather information on informal basis. In the last two years we performed some quality assurance evaluations. Two for serology of Hanta and Dengue diagnostics and one for Dengue PCR diagnostic. A further QA for evaluation of PCR diagnostics for Ebola, Lassa and Orthopox is performed at the moment. The ENIVD is presently funded by the DG SANCO (SPC.2002396) under the program AIDS and other communicable diseases.

References [1]

Biel S., O. Donoso Mantke., K. Lemmer, A. Vaheri, A. Lundkvist, P. Emmerich, M. Niedrig (2003) Quality control measures for the serology-diagnosis of hantavirus infections. J. of Clinical Virology, in press

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Preparedness Against Bioterrorism and Re-Emerging Infectious Diseases J.Kociketal. (Eds.) IOS Press, 2004

Surveillance of Acute Respiratory Infections in the Czech Republic and in Europe - Example of an Early Warning System Jan KYNCL and Bohumir KRIZ Centre of Epidemiology and Microbiology National Institute of Public Health, Czech Republic Abstract. Any existing infectious diseases reporting system would also be a valuable part of an early warning system of terrorism-related illness outbreaks. Acute respiratory infection is one of the potential syndromes to be monitored if biological agents are misused. Respiratory virus activity is detected in Europe each winter, yet the precise timing and magnitude of this activity remain highly unpredictable. The impact of acute respiratory infection and/or influenza infection in European countries is continuously monitored through a variety of surveillance indices. All these sources of information are used to assess the nature and extent of activity of influenza and other respiratory viruses, and to offer guidance on the prevention and control of morbidity and mortality due to influenza at local, national and international levels. The early warning system of a forthcoming influenza epidemic is mainly based on use of a set of thresholds. In the Czech Republic, the acute respiratory infection (ARI) reporting system with automated data processing uses a statistical model for early detection of unusual increased rates of the indicators monitored. The entered data consists of numbers of ARI, numbers of complications of ARI and population registered with the general practitioners and paediatricians that reported, all separately in four monitored age groups. The European Influenza Surveillance Scheme (EISS) is a modern and efficient European surveillance system that collects data from the national networks on a regular weekly basis, combining both clinical and virological information. The EISS facilitates exchanging timely information on influenza and other acute respiratory virus activity. It includes 28 reference laboratories in 19 European countries and not less than 10,500 sentinel physicians and covers a total population of 438 million. Since using an Internet-based platform, it is easily accessible and provides timely information.

1. Introduction Information on the occurrence of infectious diseases is of great significance for maintaining public health. Every European country has its own national notification and surveillance systems and legislation [1,2]. Furthermore, their laboratories participate in many international surveillance programmes organized by the EU, WHO and other organizations. Plenty of diseases caused by biological agents may manifest themselves by a wide variety of non-specific symptoms frequently including acute respiratory infection or flu-like syndrome. It is very difficult to distinguish between the natural and other outbreaks. Therefore

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any existing infectious diseases reporting system would also be a valuable part of an early warning system of terrorism-related illness outbreaks.

2. Acute Respiratory Infections and Influenza Acute viral rhinitis, pharyngitis, laryngotracheitis, tracheobronchitis, bronchitis, bronchiolitis or pneumonia are associated with a large number of viruses, each of which is capable of producing a wide spectrum of acute respiratory illness, differing in etiology between children and adults [3]. Viral diseases of the respiratory tract may be characterized by fever and one or more systemic reactions, such as chills, headache, general aching, malaise and anorexia. Morbidity from acute respiratory diseases is particularly significant in children. In adults, the relatively high incidence and resulting disability, with consequent economic loss, make acute respiratory diseases a major health problem worldwide [4, 5]. As a group, acute respiratory diseases are one of the leading causes of death from any infectious disease. Influenza virus activity is detected in Europe each winter, yet the precise timing and magnitude of this activity remain highly unpredictable. The age groups of the population affected and the severity of illness that they experience are dependent on a number of factors including the virus types and subtypes that circulate during a given season. Epidemics of influenza are reported almost every year. Influenza pandemics occur at irregular intervals (approximately once per 1 0 - 3 0 years) and are associated with unpredictable recombination of human and swine or avian antigens.

3. Surveillance of Acute Respiratory Infections and Influenza Surveillance of acute respiratory infections and influenza is based above all on clinical surveillance (morbidity reports and mortality statistics of influenza and respiratory infections as well as of all causes) and virological surveillance from the community and hospitals. All sources are used to assess the nature and extent of activity of influenza and other respiratory viruses, and to offer guidance on the prevention and control of morbidity and mortality due to influenza at local, national and international levels. 3.1. Surveillance in the Czech Republic In 1951, the influenza morbidity monitoring program started in the Czech Republic. The age specific incidence of acute respiratory infections (ARI) and total incidence of complications have been monitored weekly since 1968. The system now includes approximately 2230 general practitioners (GP) and 1240 paediatricians. Virological surveillance is performed by the National Reference Laboratory (NRL) for influenza and NRL for non-influenza respiratory viruses. The data on morbidity from epidemiological surveillance are integrated with those from virological surveillance. After validation and advanced assessment, the results are presented weekly [6]. Comprehensive outputs for international organizations such as the European Influenza Surveillance Scheme (EISS) or WHO-FluNet are provided by the NRL for influenza.

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In an attempt to improve the health care information systems, substantial changes were made to the ARI reporting system from 2000 to 2002 [7]. Starting from the season 2001/2002, each District Public Health Service enters the data from collaborating general practitioners and paediatricians into the central database, using an encrypted Web transfer with name and password controlled access. The entered data consists of numbers of ARI, numbers of complications of ARI and the population registered with the GPs and paediatricians that reported, all separately in monitored age groups (0-5, 6-14, 15-59, 60+ years). There is also space for comments. Pneumonia only is newly considered as a complication of the infection. The basic data processing is automated and uses a statistical model for early detection of unusual increased rates of the indicators monitored, based on a general linear model for leftcensored data. Usual weekly ARI incidence is modelled and this rate can only increase if a possible epidemic occurs. A threshold was established by averaging non-epidemic ARI incidences in the past years and applying an upper tolerance limit (covering 90% observations with 95% probability). The thresholds are available for the whole Czech Republic and also for each region. Direct standardization and weighting for the size of the monitored population is also used to enable comparison of ARI morbidity among regions. The virological surveillance program consists in weekly assessment of the results of routine laboratory testing of paired sera and nasopharyngeal swabs, provided by the collaborating virological laboratories. The methods commonly used for this purpose are the complement fixation reaction (CFR), direct antigen detection from clinical specimens (ELISA) and isolation of the causative agent from a suitable cell culture. Lately, rapid diagnosis of the major causative agents of acute respiratory virus infections such as influenza virus of types A and B, respiratory syncytial virus, adenoviruses and parainfluenza viruses has been used within this program [8]. Although a small part only of all clinical cases are analyzed virologically each year, the results obtained are equally as significant as epidemiological reporting. Substantially more data are available for the specimen analyzed, e.g. patient's age, clinical diagnosis, sampling date and that of the disease onset. First isolations of influenza virus and particularly an increase in their incidence may be predictive of the very beginning of an epidemic even before any change can be detected in the morbidity development. Routine detection of other viral respiratory pathogens yields complementary data helpful in monitoring general trends in morbidity. Summary data are informative enough of the circulation of different agents in the population throughout the year.

3.2. Surveillance in Europe National networks for the clinical and virological surveillance of influenza have existed in Europe since the 1950s. In the late 1980s efforts were made to improve the clinical reports from sentinel physicians by integrating virological surveillance systems and by collecting data on a European level (Eurosentinel scheme, 1987-1991, followed by the ENS-CARE Influenza Early Warning Scheme, 1992-1995 and the European Influenza Surveillance Scheme, since 1996) [9]. The objectives of the European Influenza Surveillance Scheme (EISS) are as follows: a. To facilitate the rapid exchange of information on influenza activity in Europe; b. To combine clinical and virological data on the same population; c. To identify causal viruses in the population and to recognise virological changes; d. To provide standardised information of high quality.

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The EISS presents clinical and virological data on influenza in 19 European countries: Belgium, the Czech Republic, Denmark, France, Germany, Ireland, Italy, Lithuania, the Netherlands, Norway, Poland, Portugal, Romania, the Slovak Republic, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. The EISS has received funding from the European Commission since November 1999. There does also exist the EuroGROG (Groupes Regionaux d'Observation de la Grippe), a complementary influenza surveillance system that includes countries in Europe not covered by the EISS. The EISS clinical surveillance of influenza is generally based on reports made by 10,500 sentinel physicians, mostly general practitioners. Some of the sentinel surveillance systems also include paediatricians and physicians with other specialisations. The participating physicians usually represent 1-5% of physicians working in the country or region [9, 10]. The case definitions and denominator populations used for the clinical surveillance of influenza vary with the networks. Most sentinel surveillance systems report data on the number of new cases of influenza-like illness, whilst others report the number of new cases of acute respiratory infection. Some sentinel surveillance systems have denominator population based on patient lists (the results are presented as the number of cases per 100,000 population) and others on the total number of consultations (the results are presented as the number of cases per 100 consultations). In an effort to standardise and improve the presentation of the EISS data, a method of calculating population denominators was devised for the networks reporting the number of cases per 100 consultations. This method has allowed all national networks within the EISS to report influenza rates per 100,000 population starting from the 2001-2002 season [10]. The sentinel physicians are asked to take nose and/or throat swabs from patients with influenza-like illness or acute respiratory infection. Some national surveillance systems also collect blood samples [9]. The specimens are sent to the national reference laboratory and are screened for influenza and other respiratory viruses by virological tests that vary with the networks; most frequently used are ELISA, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR) and virus cultures. The virological results are used to validate the clinical reports. The EISS web site (www.eiss.org) is divided into two parts. The first part is only accessible to authorised persons to enable entering the data into the EISS database and working with the database. There are also public pages that provide background information, useful links, news items and the Weekly Electronic Bulletin. The Bulletin provides a weekly update of influenza activity in Europe in the form of a map, a table, graphs and expert commentary. The Bulletin appears every Friday at 12:00 (GET) and presents the data collected during the previous week. 4. Conclusions Infectious diseases continue to be an important cause of suffering and death worldwide. Acute respiratory infections and influenza are still one of the main health problems. Their occurrence and impact are continuously monitored through a variety of surveillance indices. It is necessary to continue the surveillance at local, national and international levels. Global surveillance provides necessary information for monitoring and evaluating disease control measures as well as an early warning system for epidemics [11]. The EISS helps to reduce the burden of disease associated with influenza in Europe by collecting and exchanging information on influenza activity, providing relevant information

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about influenza to health specialists and the public, contributing to the annual determination of the influenza vaccine composition and contributing to European influenza pandemic preparedness activities. For efficient information at all levels, high quality local and national surveillance is vitally necessary. Since using an Internet-based platform, the reporting systems are easily accessible and provide timely information. Acknowledgement Thanks are given to Dr. Marie Otavova and Dr. Martina Havlickova from the National Reference Laboratory for influenza, National Institute of Public Health in Prague for stimulating discussions. References [1] [2]

[3] [4] [5] [6] [7] [8] [9] [10]

[11]

Van Dalen, P.: Outbreaks of disease: Current European reporting system. In: Dando M. et al., eds. Maximizing the Security and Development Benefits from the Biological and Toxin Weapons Convention. Kluwer Academic Publishers, Dordrecht, the Netherlands, 2002, p. 97-119. Kriz, B., Kyncl, J.: Natural Outbreaks of Disease: Communicable Disease Surveillance in the Czech Republic. In: Dando M., Pearson G., Kriz B., eds. Scientific and Technical Means of Distinguishing Between Natural and Other Outbreaks of Disease. Kluwer Academic Publishers, Dordrecht, the Netherlands, 2001, p. 35-39. Chin, J., ed.: Control of Communicable Diseases Manual, 17th ed., American Public Health Association, Washington, 2000. Fleming, D. M.: The contribution of influenza to combined acute respiratory infections, hospital admissions, and deaths in winter. Communicable Disease and Public Health, 3, 2000, No. 1, p. 32-38. Monto, A. S.: Viral respiratory infections in the community: Epidemiology, agents, and interventions. American Journal of Medicine, 99, 1995, suppl. 6B, p. 24S-27S. Acute respiratory infections in the Czech Republic. Available at http://www.szu.cz/cema/aro/aro.htm Kyncl, J. et al.: Improving the acute respiratory infection notification system in the Czech Republic. The First European Influenza Conference, St.-Julians, Malta, 20-23 October 2002, poster No. P-W1-11, abstract book p. 54. Roubalova, K. et al.: Doporucene metody ve virologicke diagnostice (Standard operational procedures in virological diagnostics), in Czech. Acta hygienica, epidemiological et microbiologica, 2000, No. 1. The European Influenza Surveillance Scheme. Available at http://www.eiss.org Paget, W. J., Meerhoff, T. J., Goddard, N. L. On behalf of EISS: Mild to moderate influenza activity in Europe and the detection of novel A(H1N2) and B viruses during the winter of 2001-02. Eurosurveillance, 7, 2002, No. 11, p. 147-157. Cosivi, O.: WHO contribution to global surveillance of microbial threats. In: Dando M. et al., eds. Maximizing the Security and Development Benefits from the Biological and Toxin Weapons Convention. Kluwer Academic Publishers, Dordrecht, the Netherlands, 2002, p. 167-175.

Part 2

National Approaches to Biodefence in Central and Eastern Europe Countries

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Organization of Military Medical Response to Bioterroristic Attacks Kamen PLOCHEV, Emil PENKOV MMA - Sofia, Bulgaria, Clinic of Infectious, Parasitic and Tropical Diseases MMA - Sofia, Bulgaria, Center of Military Epidemiology and Hygiene Abstract. This report describes the regularity of infectious diseases in Bulgaria for the recent five years; the regional localization, force and means of the national health network for diagnosis, treatment and prophylaxis of these diseases. There is data about the morbidity of infectious diseases in the army and the ability of the Medical service of the Bulgarian Army to control and treat the infectious diseases. Based on this data and following evaluation of the possibilities, there is a draft project on Strategy for Protection of the Population, and in particular, protection of the army from bio-terrorism. The underlying principles of the Strategy are mobility, modularity, anti-epidemic regime, interaction and coordination. Development and introduction of a biological module is proposed, including one expert in infectious diseases, one epidemiologist, one expert in viral diseases, one microbiologist and one pathologist. There are two stages defined - preliminary and operative and the relevant activities and tasks are described. Special attention is paid to the elements of observation and quarantine. A problem, which has to be solved by the Bulgarian Army is the treatment-evacuation support and this issue is described in details.

1. Introduction Nowadays the threat of biological weapon use has been increased significantly. World changed political situation, available data about circulation of strains outside labs or infectious agents "home" preparation and their hiding in private properties, and the possible use of infected vectors are part of the challenges we face. Anthrax distribution in the US showed how fragile the existing health and security systems are and how easy the normal way of life could be disturbed. The responsible institutions should keep in mind the possible danger of combined use of chemical and biological weapons. They should consider that civilians without experience and focused thinking about the possible use of weapons of mass destruction will register the first cases. The panic distribution amongst civilian population and municipal health workers should not be ignored. As a part of modern society the military poses vast capabilities to counter any threat community would face. Troops are better protected than civilians are. Their training allows high quality performance under immense pressure. The newly developed situation must be handled by newly designed manners. The main goal of this work is to propose an unified organization of preparation, reaction, and collaboration in case of biological attacks from terrorists with designated goals and

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responsibilities of all participants both military and civilians. We believe cooperation between military and civilian institutions will be the society best interest. 2. Materials and methods We used the data published in standards of NATO medical service activities during usage of weapons of mass destruction and immunizations (Amed p-7, STANAG 2122 and 2037). Data from the National Institute of Medical Statistics about the morbidity levels and levels of resources available and papers, prepared by the Bulgarian Army Medical Service about troops medical support during use of biological weapon were used too. In the Bulgarian Army we accept 22 different infectious agents, which can be used against troops and civil population, separated in 3 groups according to the risk they bring, (see table 3) Table 3. List of infectious agents, adopted by Bulgarian Army Medical Service as possible biological agents Group Bacterial agents

Viral agents

Ricketsial agents

Biological agent Yersinia pestis Vibrio cholerae Brucella species Francisella tularensis Pseudomonas melitensis Bacillus anthracis Smallpox virus Hemorrhagic fever Ebola virus Hemorrhagic fever Marburg virus Hemorrhagic fever Lassa virus Bolivian hemorrhagic fever virus Argentinian Hemorrhagic fever virus Congo Crimean Hemorrhagic fever virus Rift valley fever virus Dengue virus Yellow fever virus Venecuellian equine encephalitis Western equine encephalitis Eastern equine encephalitis Hanta viruses Coxiella burnetti Chlamydia psittaci

Before starting any kind of work we must clarify the basic terms we are going to use. According to our understanding on the discussed subject we propose the following definitions. (see table 4) Table 4. Basic terms definitions Biological weapon Biological agent Bioterrorism Infected Influenced Identification Decontamination

biological agents and the means of their carrying and dissemination microorganism or toxin with biological nature capable of causing diseases within people, animals and/or plants. applying biological agents for terror goals. The main targets are civilian citizens. all human beings located in the affected area during the use and removal of the agent. all human beings with clinical and laboratory data for a current infection determination of the species and/or characteristic of the used as a biological weapon microorganism or toxin. elimination and/or deactivation of biological agents overlaying the body, materiel and/or environment by using physical or chemical methods.

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3. Discussion The three necessary steps to achieve the goals of the military medical response against bioterrorism are: 1. Preparatory step - before the moment of attack. 2. Operational step - after the registration of the attack. 3. Conclusive step - elimination of the consequences. The preparatory step includes: • evaluation of the situation in the world and in the country, • constant analysis of the normal infectious diseases morbidity in the respective geographic area, • evaluation of the resources (men, materiel, finance, time, etc), • personnel education, • development of a computer model for military medical response management, and • applying of specific prophylaxis The evaluation of the situation in the world and in the country means to assure effective biological reconnaissance. The goals of this recon are: 1. Collection of preliminary information about possible biological weapon use. 2. Constant observation of objects supposed to be targets of attacks for accidents maximum rapid detection. 3. Clarification of the regional medical and veterinary units capabilities which could be used for situation management. The constant analysis of the normal infectious diseases morbidity in the country or in the respective geographic area should be made with co-operation with the national medical statistics specialists. The team must monitor the general morbidity data, but more important task is to trace unusual diseases increasing in certain areas. The main topics to follow should be established on the start of the preparatory step. They must be declared very carefully because any change during follow on stages could compromise the final results. General data about infectious diseases morbidity in Bulgaria for the last several years are shown on table 5. Table 5. Infectious diseases morbidity in the country Year 1994 1995 1996 1997 1998 1999 2000 2001

Infected/number 88523 88388 114183 71843 86078 77394 81769 52637

Morbidity/100000 1043.30 1044.81 1349.73 856.73 1026.60 923.04 993.50 657.96

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A declining of infectious diseases morbidity is observed. We consider it as a result of incorrect information. The most common infectious diseases in the country (in particular in the Bulgarian Armed Forces) are Viral Hepatitis, Intestinal and Respiratory Infections. Peculiar characteristic is the presence of diseases, which are seldom diagnosed in some of the NATO members, such as Q-fever (150-300 cases per year), Marseille fever (more than 1500 cases in 2001), Tularemia (179 cases since 1997 and more than 50 cases since the beginning of 2003). There is a trend of natural foci dissemination and spreading over new territories. During the last 10 years diseases, which are not common or endemic for the country, have not been registered. The evaluation of the resources (men, materiel, finance, time, etc.) must begin simultaneously with all other activities basic for the preparatory step. The process is constant with weekly or monthly changing circumstances. It requires fully dedicated workers both military and civilian who posses access to classified information. At the moment 127 infectious diseases wards in 105 cities in 27 regions and 1 specialized hospital are established in Bulgaria. There is 1667 infectious beds, 9150 therapeutic beds, 2618 pediatric beds within them. 223 infectious diseases specialists, 788 pediatricians, 2013 internal medicine specialists and 16527 nurses work in these hospitals. The hospitals have all necessary equipment and medicines. Additionally 32 centers of emergency medicine came out. 1515 doctors and 2308 nurses are attached to these centers. 970 vehicles (ambulances, cars, vans) are assigned, but 90% of them are in use. The centers of emergency medicine are well equipped with the necessary life-saving items and medicines. In every administrative region a unit for hygiene & epidemiology is established (28 at all). The staff includes epidemiologists, hygienists, disinfection specialists, etc. Microbiological, viral and parasitic laboratories are created in every unit. A modern laboratory with biosafety level 3 is under construction. The microbiological and the parasitic labs have the capacity to do all spectrum of study. 5 of the viral labs have capability to diagnose Influenza, Hepatitis, Poliomyelitis, AIDS, Rubella, Measles, Mumps, Q-fever, Ornithosis, Marseille fever, Mycoplasmosis, Lyme borelliosis, Cytomegaloviral, Herpal and Enteroviral Infections, EBV Infections, Acute Respiratory Infections, but 23 of them do AIDS and Hepatitis only. 5 hospitals, 3 rehabilitation bases and a chain of recreational units are established in the Bulgarian Army, that are a convenient place to isolate infected people. A military medical unit for rapid reaction is created in MMA - Sofia. An equipped infectious disease module is made within it. Bulgarian Army medical service has equipped an infectious diseases clinic in the capital (40 beds) and an infectious diseases ward in the town of Sliven (15 beds). 9 infectious disease specialists and 2 internal medicine specialists work in these units. They have field experience from Cambodia and Macedonia. There are 4 hygiene-epidemiological inspectorates with capacity of sampling and expressing tentative microbiological, viral and parasitic diagnosis. After careful evaluation of the resources available several problems were find out. Part of them is linked with the national heath system resource support but the rest could create troubles in case of using military units to counter terrorist attack. These narrow places within national health resources support system includes: 1. Lack of sampling and transportation resources. 2. Infectious disease wards are not fitted to deal with patients in case of biological weapon use or presence of unknown or genetically modified microorganisms. 3. Insufficient protection suits and materiel to support treatment and isolation procedures. 4. Lack of technical items and experience to evacuate infected and influenced.

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5. Lack of updated national information system for daily control and analysis of contagious and other infectious diseases. 6. Insufficient means to perform decontamination, disinfection, and disinfestation; 7. The medical staff is insufficiently trained. Problems within Bulgarian Army resource support includes: 1. Extremely old and unfit for use and isolation bed stock. 2. Insufficient medical staff for diagnosis, treatment, nursing and isolation of infected and influenced soldiers. 3. Insufficient materiel supplies for performing decontamination, disinfection, and disinfestation. 4. The resources of evacuation and isolation are not synchronized to the NATO standards. 5. Lack of mobile means for sampling and transportation. 6. Supply for vaccine prophylaxis is limited to mission support. 7. Lack of computer model for management of resources available and situation analysis. 8. Insufficient evacuation means. 9. Lack of synchronized with the NATO standards liaison technical devises within military medical service, including satellite communication. 10. Lack of well synchronized cooperation system between military medical service on national level with national security services and other government institutions or on local level with regional government units. During the preparatory step the government institutions should be obliged to work in the following directions: 1. Diagnostic reagents delivery to local and national laboratories. 2. Preparation of communicational programs for receiving actual information in real time. 3. Improving the population education about the bioterror problems by using educational materials and the mass information media. 4. Delivery of serums, vaccines, toxoids, and specific immunoglobulins supplies. 5. Reassurance of antiviral medicines and vaccines researches. 6. Creation the necessary organization by committing the government institutions capabilities to assure that the persons taking decisions will receive all political, operational and specialized information they need. 7. Performing military medical exercises with aim to improve the bioterror response groups personnel training. 8. Medical personnel education. The preparatory step switches to the operational step in case of convincing data about imminent application of biological weapon. The following measures must be perform immediately. • Determination of the affected area and designing its borders. • Location of the staff under the attack. • Specifying the area objects, capable of transmitting the used agent. • Epidemiological investigation amongst the population in the affected area. • Collection any other information linked with the attack.

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We propose the first action on this level to be a declaration of emergency situation in the accident region in order to prevent panic and mass movement. It's important to underline that all these activities are local - they will be implemented in the designated area only. • According to our opinion the operational activity includes: • Evaluation of the situation - clinical and epidemiological; • Infected people's triage and evaluation under 4-level scale; • Sampling and organization of diagnosis; • Full sanitary handling of infected; • Admission in the nearest medical hospital; • Organization of autopsy and casualties burial. We propose the bioattack response action to be taken on two stages - stage one - prior the causative agent identification and stage two - after its identification. Prior the causative agent identification has two level organization. Level one is performed by the Military Medical Unit for Rapid Reaction. It is its responsibility to locate the area, to evaluate the number of the infected, to perform life-saving procedures on the spot, to organize evacuation to the nearest hospital according to pre-prepared files in order to isolate, diagnose, and treat. Operational group for consequences elimination will do level two. The group organizes the diagnostic process in national reference labs and the treatment in specialized hospitals. The group is responsible for the personnel, vehicles, and the designated territory decontamination. By using computer model and the situation data the group prognosticates medical casualties and plans the needed means to coup with the situation. The group makes epidemiological analysis of the bioterroristic attack. Considering the capabilities on one side and the difficulties on the other for the medical service activities after biological attack detection we believe that its activity must follow some basic principles: • mobility, • module type of work, • cooperation on the spot, • range of action, appropriate with the used microorganism type, its way of dissemination, and the degree of its influence. After the used biological agent identification the first job to be done is to make clear whether this organism is replicable or not. The agents must be evaluated according to the following additional indices: toxin-production capability, stability, invasiveness, and virullence. The action after receiving the preliminary or the definitive identification result is two types - observation or quarantine. Observation - it implements in case of biological agent group B or biological toxins. Observation should include the following activities: limitation of coming in and out of the area, limitation of the contact between infected, health promotion amongst soldiers and local citizens, constant medical observation over infected (daily anamneses about imminent symptoms, daily check of skin, mucous, and organs, temperature, labs data); application of non-specific immunoprophylaxis, and if it is necessary - antibiotic prophylaxis, implementation of specific immunoprophylaxis, introduction of special hygienic regime, and if it is needed performing disinfestation.

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Quarantine - it implements in case of use of biological agents group A, group C, unknown or unidentified organism or simultaneous use of biological agents (organisms + toxins). Quarantine must include the following: introduction of full area border control (all coming in and out of the area is strictly forbidden). The guards are military police staff. If it is necessary additional police, gendarmerie or unaffected military unit are involved, admission-transfer points will be organized (All supply needed to perform medical activities will be delivered through these points), dissemination of quarantined in individual modules with local lavatories, full sanitary handling, epidemic regime of work - protective suits, implementation of immunoand chemo-prophylaxis, performance of focused medical observation, strict hygienic control, obligatory disifestation, treatment on the point. One of the most important components of the operational step is the organization of the co-operation. Military Medical Academy organizes Medical Coordination Center (MCC) for planning and organization of the affected contingents (both military and civilians) medical support should function until the crisis situation end. The MCC regularly sends information to the Bulgarian Army Military Situation Center (BAMSC). The MCC organizes the cooperation with other health institutions on the topics of informational support and samples collection, analyses, diagnoses, and treatment practice as well. With the help of BAMSC coordinates the work with National intelligence service and military counter intelligence. After the end of biological attack consequences elimination an observation upon the infected and the territory should be made. During the observation epidemiological, epizootic, clinical, immunological, ecological, and laboratory data should be taken into consideration. The process of observation starts the final conclusive step of the attack response. During this step we think some additional activities should be done. They includes the foci data weekly update, military medical expertise, conclusive disinfection and disinfestation, prophylaxis amongst soldiers and civilians in the bordering areas, preparation of reports and prognoses for potential dangers of epidemic (epizootic) process activation by using the pre-prepared software, summarization of information about bioattack detection, reaction, and consequences elimination activity and preparing of report to the Command.

4. Comments The Bulgarian Army has partially readiness to neutralize a biological wartime attack, but there is a lack of experience and resources to support medical response in case of bioterrorism. A preparation and planning for action in peace- and wartime crisis situations, linked with biological weapon use is needed to coordinate and synchronize with the NATO-standard. It should be national priority to support all range of activities aimed to bioterroristic act neutralization.

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Georgian approach to Biodefense Mzia KUTATELADZE Georgian Academy of Sciences Tbilisi.3, Gotuastr. Tbilisi 380060, Georgia, kutateladze(a)pha.ge Abstract. The difficulties connected with the political changes due to the breakdown of Soviet system caused the problems in biosafety and biosecurity in FSU countries. The problem of protection of the collection of pathogenic bacterial strains became the serious issue. Insuring of physical security of the Institutions keeping the collection of dangerous pathogens is very important for Georgia, especially now, in case of increasing threat of biological attacks. Observance of standard rules of working on pathogens needs to be improved by technical means of security, as well as by the appropriate laboratory equipment. Open discussion about the former Soviet scientists' experience would help the expansion of biodefence research worldwide. G.Eliava Institute of Bacteriophages, Microbiology and Virology, Tbilisi, Georgia was one of the strongest centers in former Soviet Union working on dangerous pathogens. During many years, the pathogenic and conditionally pathogenic bacterial strains were being sent to the Institute from entire Soviet Union. G.Eliava Institute became the leading center of Bacteriophage research. The phage preparations are being used for the therapy and prophylaxis of different infectious diseases, especially caused by multi-drag resistant bacterial strains. Original schemes of phage typing of different bacterial strains have been elaborated at the Institute that could be used as an additional test for epidemiological surveillance and for the analyses of the acute epidemiological outbreak.

The difficulties connected with the political changes due to the breakdown of Soviet System caused problems in biosafety and security in FSU countries. During last 10-12 years, the lack of the state financing caused destruction of the systems of biosafety at the relevant Institutions. Connections between the different organizations throughout entire former Soviet Union has been destroyed; shortage in electricity, water supply system, destruction of the sewage (water purification) systems at the institutions caused the damage of the entire system of biological industry, especially the difficulties led to lost of the some part of the bacterial strain collections and/or non-systemic spread of the strains. The lost of the general means of security and control mechanism, lack of the finances for quarantine stations on customs create a threat coming from the different countries. It is extremely difficult to detect and trace the movement of dangerous biological or chemical substances by current means of security in our country. Current state programs for the sanitary inspection or epidemiologic surveillance cannot cover the entire country. The Government cannot provide enough finances for such activity. Huge facilities for the production of bio-preparations, vaccines, and sera in a large scale at the post-Soviet Institutions are idling; they need some reconstruction and redirect to gain the new functions. Tbilisi G.Eliava Institute of Bacteriophages, Microbiology and Virology was one of the strongest centers in the former Soviet Union working on dangerous pathogens and preparations

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against these pathogens. The Institute was under the responsibility of Soviet Ministry of Biological Preparations Industry. During many years, the pathogenic and conditionally pathogenic bacterial strains were being sent to the Institute from the entire Soviet Union. The large collection of bacterial strains has been constructed at the Institute. Some part of the collection is destroyed due to the shortage of electricity or other problems, but the most part of it does exist now. During the Soviet time, Eliava Institute was producing about 50 different types of biopreparations, including anti-diphtheria, anti-tetanus, gangrene, botulinus, scarlet fever, antimeningococcal and streptococcus sera, diphtheria antitoxin, anti-tuberculoses vaccine, smallpox vaccine, different type of intestinal vaccines. At the same time, G.Eliava Institute became the leading center of Bacteriophage research. The main direction of the Institute was studying bacteriophages isolated on aerobic and anaerobic bacterial strains. The Institute produced the phage preparations against the provocative of intestinal and purulent infections. Dysenterial, typhoid phages, staphylococcal, streptococcal, Clostridium perfringens phages preparations, the cocktails containing different type of phages were produced. The phage preparations were successfully used for therapy and prophylaxis in entire Soviet Union. Staphylococcal phage preparation should be especially mentioned. Highly purified and concentrated phage was used intravenously in cases of acute and chronic sepsis, septic staphylodermia, peritonitis, osteomielitis, purulent arthritis, acute and chronic lung abscesses, chronic pneumonia etc. Part of the phage preparations was produced by special Government order. Approximately 80% of the phage preparations has been sent for use in military - for prophylaxis as well as for the civilians - in kindergarten, schools etc. So, the Institute had the status of strategic organization. The Institute began the production of anti-anthrax vaccine (STI) in 1951, being first in the Soviet Union. The different methods for the vaccine production, including the production of dried preparation, the method for the continuous cultivation of the preparation, vaccine in aerosol has been elaborated at the Institute. During many years, the Institute was the only place in the Soviet Union producing the anti-anthrax vaccine. The case of Sverdlovsk in 1979 is vivid example of Georgian potential against bioterrorism. The outbreak of anthrax was contained and eliminated_by the preparation produced by our Institute. In addition to the vaccine, the Institute produced anti-anthrax globulin and diagnosticum for determination of immunologic changes during vaccination or re-vaccination of human and horse-producents. (In 1988, the Institute produced 670.000 flasks of Anthrax vaccine, 194 thousand flasks of Anthrax gamma-globulin, 17 thousand flasks of diagnosticum for anthrax. In 1989, 525 thousand flasks of anthrax vaccine, 2091 of anthrax gamma-globulin). The bacteriophage active against Brucella was isolated and studied at Eliava Institute. By the recommendation of WHO TB phage was considered as a reference for the interspecies differentiation of Brucella bacterial strains. The scientists from the different countries used widely this phage for the diagnostics. Epidemiological studying of the different infections has been carried out at the Institute. The central scientific-research laboratory of phage typing of pathogenic microbes had been working at the Institute since 1964. The leading laboratory had 50 regional laboratories in the different cities of the Soviet Union. Original phage-typing patterns (or lyso-typing schemes) designed for inter-specific differentiation of pathogens such as Salmonella paratyphi, Salm. typhimurium, Shigella flexneri, Shigella sonnei, Yersinia enterocolitica, Clostridium perfrigens and others have been elaborated. Application of the highly specific lyso-typing phage sets had

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been used for epidemiological surveillance and determination of the sources and ways of transmission of infections, as well as for rapid diagnosis of bacterial pathogens. Today, every country is facing the threat of bio-terrorism, especially the less developed, or transition countries. The leading countries having the best prepared, well-organized safety and healthcare systems, could avoid the results of the attacks in the shortest period. It is difficult to imagine the results of bio-terrorism in our country. We hope that NATO member countries and international organizations will provide some assistance and advice to improve our national preparedness, more so as we are very much open for collaboration. On the other hand, we think that open discussion on former Soviet scientists' experience would enhance biodefense research worldwide. It is necessary to re-build and restructure national systems of bio-defense in the country. Together with modern methods and techniques for strengthening capacity in different fields, it is crucial to revive the existing equipment and technologies. G.Eliava Institute of Bacteriophages, Microbiology and Virology has been one of the Institutes that worked and produced the vaccines and sera against the most dangerous infections. People who used to work on these biological agents in the Soviet period still work at the Institute. It is important to use their experience that could help global security as well. In the recent decades uncontrolled use of antibiotic therapy caused widespread of multidrug resistant bacterial strains. These kinds of microorganisms could be constructed artificially and be used for the bioterrorism. A renewed interest in the possibilities of bacteriophage use an alternative way of therapy, prophylaxis and diagnostic of the infections - becomes evident. In this direction, the interest about our Institute has been increased during last years. The Institute could provide the phage preparations against various bacteria. Experts consider that frequent and widespread infections, such as the provocatives of intestinal infectious diseases that could be included in nutrient chain and spread in the environment easily, will be used for bio-terrorist attacks. To handle with such scenarios it might be appropriate to build the phage "bank" including the bacteriophage in balk amounts. Such phage preparations could be sent immediately directly to the place of epidemics and used against different type of bacterial strains. The bacteriophage can be used as an indicators - for the epidemiological marking of microbial contamination pathways of natural and artificial water reservoirs, for identification of zones of sanitary control, as well as determination of the effectiveness of water purification, sewage systems etc. Original schemes of phage typing of different bacterial strains elaborated at the Institute could be used as an additional test for epidemiological surveillance, marking of microbial contamination pathways and analyses of the acute epidemiological outbreak. Current researches and experiments at the Eliava Institute are carried out with help of international organizations providing funds for specific scientific projects. But the research connected with the infections that could be used for the potential terrorist attacks is being carried out in small scale and only by the enthusiasm of the scientists. Therefore, ongoing economic problems coupled with the possibility of getting of some funds from the foreign or local "benevolent" people can cause the use of their experience in a wrong way. There were many cases of the efforts from the Middle East countries to contact the scientists from our Institute. That is why the conservation of the bacterial strain collection by modern technical means of security is so important. Observance of standard rules of working on pathogens needs to be improved by special up-to-date laboratory equipment.

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We are open and ready for cooperation and hope that these kinds of meetings will help us build up our preparedness and response capacity that will be conducive to build the systems of collective security.

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National Approach of Germany to Bioterrorism and Bio-Warfare M. NIEDRIG1, R. FOCK1, E. FINKE2 'Robert Koch-Institut, Berlin, 2Institut fur Mikrobiologie der Bundeswehr, Munich, Germany In 1998 the "Senior Defence Group on Proliferation" of the NATO considered the biological weapons presently and in the future as the biggest threats for the military forces and the population in NATO countries. The long list of biological threats makes it necessary to develop a clear strategy for analysis of biological risks, identification of agents, samples collection, transport and diagnosis of diseases, including a fast and efficient information structure. The way of exposure (aerogenic, alimentary, parenteral) has to be analysed as well as the probability of an naturally or artificial cause of an emerging disease. Therefore the analysis of unusual death, diseases and outbreaks is an important part for the identification of suspected or real biological threats. The surveillance of such diseases very much depends on good and reliable case definitions and good diagnostic tools. Just recently the EC has decided for setting up a network for epidemiological surveillance and control of communicable diseases for the European community. This is considered in combining the different expertise in an outbreak investigation team (epidemiologist, microbiologist, infectious disease specialist, public health specialist, etc.). Depending on the situation and the course of disease this team has to be expanded by other experts. Also the back up by logistic support for communication, protection measures and desinfection is a major issue which has to be addressed. Depending on the outbreak scenario within the country or in a military mission outside the preparedness is the most effective measure reacting properly in such situation. The different centres of expertise on the military and civilian sites have to work close together regarding information exchange and protection measures. In Germany since many years a civilian military working group develops a co-operative strategy for handling such outbreak situations. Based on the existing expertise we are trying to organise the most effective response in a suspected or real outbreak and /or biological threat situation. In this content it is very important to integrate all potential participants involved in a biological hazard response task forces, fire brigade, local public health officials, emergency services, etc. The integration and discussion with these partners will allow the best acceptance and implementation of recommendations and other instructions in anoutbreak situations. After discussion of the final draft the countries specific recommendations should be compared with other European countries recommendations to avoid great differences in measures which will create confusion in the population due to the different measures recommended by different experts in the field. Differences of the procedures in neighbouring countries must be explained seriously to avoid fear and panic in the population and must be communicated as quick as possible before a bad information policy will create a disastrous situation as we have seen in many real or suspected outbreak situations. This situation can only

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be improved by a good and serious preparedness planning for the response to bioterrorism and bio-warfare threats Reference [1]

Wirtz, A. Niedrig, M. and Fock, R., (2002) Management of patients with suspected viral haemorrhagic fever and other potentially lethal contagious infections in Germany. Eurosurveillance, 7, N°3, 36-42.

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National Approach of Hungary to Bioterrorism and Biowarfare Gabor FALUDI(l), A.CSOHAN (2), G.BERENCSI (2) (l)Institute of Health Protection of Hungarian Defense Forces (2) "Bela Johan " National Center of Epidemiology Theoretical changes in the assessment of BW in the past The problems of biological weapons have became suddenly important after some decades long deeply sleeping period - it was generated in our region perhaps intentionally because of several non medical factors. At first the long term effect of development and spreading of biotechnology, the new scientific and technical achievements and the illegal activity of several states (SU, Iraq) has been realized all around the world. The second problem was the so called - atomic blindness - when the nuclear weapons and the consequences of a possible war determined the specialists thinking and oriented the main direction of interest from the BW. The next one was a psychological factor of thinking which characterized the post '89 years. After the Cold War era as the bipolar world dramatically collapsed -subjective and euphoric opinions became dominant everywhere among the decision-makers, media members and also scientists in Hungary, that the weapons of mass destruction (WMD) lost their former importance because the probability of the eruption of a New World War drastically diminished if not totally terminated at all. This understandable but unfortunately false optimism caused more radical diminishing of defense funds, reduced the size of experienced expert staff in armed forces and turned out the focus of interest of civil medical society from the NBC medical defense questions for other more important problems from the point of view of the economical crisis management together with the establishment of the free market health care providing system. This tendency of modest indifference was occasionally enforced by the special adversary effect of successes of different international treaties as Atomic stop, CWC, BTWC. The results of long lasting effective diplomatic efforts were able to fulfill their roles to prevent or restrict the proliferation of WMD for a long time - but in the same time nearly paralyzed the professional interest of scientists for the problems of biodefence in Hungary. Consequently among such general conditions - it was not surprising - that only a very small group of experts could realize the silently emerging accumulation of negative tendencies. It is a very sad situation for everybody, that this success story of international diplomacy has been interrupted by Pakistan and India, or by the anthrax letter-bombs or perhaps the fentanyl usage. To break out from this isolated situation of topic helped us a lot the international scientific cooperation with NATO, the gradually opened sources of information on offensive and defensive sides of biological warfare and the slowly but efficiently spreading new possibilities of electronic information exchange on net. Nowadays we are able to collect easily series of excellent articles and E-books about this topic from everywhere. The information influence the significance of possible roles of

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biological weapons is shocking but also are shocking those events which had been put forward the hypothetic application of BW predicted by some "paranoid" experts. In the light of facts the reevaluation of strategy of NBC is unavoidable especially BC medical defense integrated into the preventive medical volume of medical support plans. Different workshops in the NATO and many other places, as WHO strongly criticized the weakness of the principles and practices of old NBC strategy "the system of systems" involving the "Holy Trinity": the avoidance, protection and decontamination. For instance, the WHO in its bioterrorism guide offered a new approach as a guide of thinking: the risk management, preparedness and response. The picture has not been clarified yet, but under the pressure of history we are obliged to deal with every part of biomedical force protection. The implementation of new defensive principles is going ahead a little bit toughly, the systematic acceptance of many magnificent molecular diagnostic tools - were born in recent times - progressed more slowly into the military medical support system, than the production possibilities of the sophisticated B weapons developed. But one of our greatest challenge to enforce the primary data sources - the clinical diagnostic capabilities by education, the stationary and mobile laboratory systems, and the practice of rapid epidemiological field investigations and sampling.

Bioterrorism influence on Hungary Those tragic attacks committed by international terrorist on 11th of September 2001, that can be taken only with great indignation, shocked every part of the society and finally changed our world, because the terrorism became a part of our everyday life. Even before the ruins had been cleared away in New York we had to face a new wave of terror by appearance of bioterrorism: from 4th of October 2001, the intentionally released anthrax letter bombs were detected mostly but not only in the USA. The phenomenon of terrorism earlier was less known - only from the journals - in Hungary, and the appearance of WMD in the arsenal of terrorists indisputable opened a new dimension of deterrence. Formerly we were less convinced about the reality of bioterrorism, when we began to read at first about the Aum Shinrikyo, the cult responsible for release of nerve gas sarin in the Tokyo subway in '95, has also attempted to weaponize the anthrax, Ebola and other microbiological agents for their special purposes. Consequently we had to study the nature of terrorism by bioterrorism applied the principles of descriptive and analytical epidemiology. Unfortunately since that time - 95 - several other even more dangerous terrorist networks have been created by Osama bin Laden. The prophetical truth of many authors like Osterholm, was clearly proven, who told: The question was not where - but only when? Fortunately different very good websites could be found as data sources, for example at the homepage of US State Department: The Report on Global Terrorism, and I have to mention the two Monterey Institutes of International Studies reports published at first in Emerging Infections at 97. In the light of analyzed evidences it could be well seen, that the bioterrorism means a theoretically new, arrogant, diplomatically and legally uncontrollable field of proliferation. The global threat of bioterrorism is needed for a global response, and it is necessary to use all the coordinated efforts for cooperation and information exchange at global, continental regional and national levels.

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We suppose, however, that the bioterrorism is practically and theoretically not a clearly military problem, in spite of the army has some ready for use defensive methods in its armaments against hidden, sabotage B attacks. The defense against a bioterror attack means a wider general problem of national security. We personally believe that one has to work out a more efficient defensive system including a complex long term program instead of a campaign task which may determine our future activity associated with the local public health systems. So, we had to work to strengthen the surveillance and the control of infectious diseases in Hungary. This target could be achieved if military and civil national regional and international organizations will find the way of closest cooperation, because of the bioterrorist attacks threatening dominantly the civil mostly defenseless population, being practically unprotected and theoretically a large proportion of the civil population cannot be protected at all. According to the mentioned new principles Hungary accepted and signed a regional treaty of health ministers in 2002 February in Prague. The NATO Summit also in Prague some weeks ago also encouraged the work in this direction. Thus the responsibility of the experts of NBC medical and especially of preventive medicine became larger today in order to create more efficient response plans against different bioterrorism scenarios or events.

Experiences in Hungary Unfortunately our interest for bioterrorism could not conserve its profile as a theoretical problem after 4th of October. There were many events in Hungary closely connected with the wave of anthrax letter bombs in the USA. It sounds strangely but our country was also affected by the effects of aggressive manifestation of bioterrorism even in double sense. At first: The potential and direct possibility of a bioterror attack in Hungary too and the real application of the biological weapon has crept in the consciousness of Hungarian people resulting a mass psychosis with a slight but manageable fear or panic, what is regularly accompanying the occurrence at every WMD application according to the principles of the military medicine and therefore is well known before us. The second complication was that many Hungarians had no confidence in any consignments by mail. The fear resulted some difficulties in the work of mail-services, caused a sort of extra administrative efforts and control measurements. In the same time unfortunately there were some undesirable surprising incidents too. Many irresponsible malevolent or humorous persons abusing the situation and general feeling of the population mailed suspicious letters to individuals or institutions. There was an immediate need to arrange the safe handling of postal consignments, to organize the urgent local field investigation by the emergency defense groups. Several measures were put into the force by public health service both national and military level. Coordinative works started abruptly among different organizations and services. Special connections were kept up with the media to inform honestly the civilian society. A higher level bio-safety laboratory (BSL3) was suddenly designated for microbiological investigation of suspected materials in the National Center for Epidemiology, and a second reserve was planned to establish for military purposes, but the latter has not been necessary. In the National Center of Epidemiology 957 suspicious postal consignments were investigated between 16 October and 31 December 2001. The daily number of speciments varied between 1 - 94, the average number was 12 per day. Among the artificially polluted letters various

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materials as talcum, milk powder, chalk, gypsum, and others were recognized. Later on the number of prepared letters was reduced to 1 - 2 per week in the first months of 2002. The HDF was also involved by a similar way but in less extent. In the same interval 6 times 8 special suspicious items were stopped, transported to the special diagnostic laboratory. Moreover the cost of movement of specially equipped emergency and field investigation teams ("HAZMAT groups"), the safety transportation and the microbial investigations caused more than thirty million HUF excess expenses. During this time more than 40 presentations were held for the high level military commanders in coordination of the Medical Directorate for the prompt information of the commanders in the HDF, and among different civil offices country side from the members of civil public health authorities up to the governmental level. What lessons have been learned - what have been done and what should have been done Although several steps were prepared during the last years - actually our situation can be considered as a position of the result of a 'so called' asymmetric development at NBC medical defense. The asymmetric development is not a Hungarian specialty only, but can result in certain systemic security risk for everybody. The Medical Directorate worked out a NATO based provisory new medical support doctrine, and started to integrate the NBC defense principles into the preventive medical support system in accordance with standards and recommendations. We have successfully conserved some elements of former NBC medical concept and built in the new preventive medical support - the two level system of mobile field deployable laboratories, well harmonized to the structure of the levels of medical support. About the practical value of mobile container laboratory we collected fresh experiences on the basic element of this system - a general purpose, mobile laboratory of hygiene - actually is functioning in Pristina and earlier was tested also in Albania - convincing us about the utility of our concept. It possessed a great importance because one part of the practical NBC medical defense activity has been planned and integrated into the infrastructure of the 2nd level of field deployable laboratory of preventive medicine of HDF. The system preparedness greatly depends on a health technical revitalization project. A new multipurpose element is under development for sample collection and preparation, too, in a close coordination with other NATO nations. During the last year in collaboration with civil epidemiological authorities legally reorganized - the formerly separated national and military infectious disease notification system and merged them in the same integrated one. The applied common Early Warning Surveillance and Response System was proven to be an adequate system for the requirements of the European Union as declared by an EU supervisory expert group. The modified national reporting system for communicable diseases and the epidemiological surveillance system in Hungary includes also the flow of military data. The integrated system shall be able to work as a "real time" epidemiological network in the next future. Accepting the importance of education and training a new university level of military postgraduate educational element, a military medical cathedra was established two years ago at the "Zrinyi Miklos" National Defense University in collaboration with the Semmelweis Medical University, and a new organization of the Military Medical Training Center has been also launched.

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The Medical Service on the basis of Institute of Health Protection established a new PCR diagnostic laboratory, as a provisory part of stationer Level 4th microbiological laboratory. The HDF made some effort to develop a HHTK a lateral flow tests for rapid identification of biological agents by the NATO. During these works we had to face with a lot of administrative and practical problems, so we can declare, that it is easier to produce a quiet efficient simple biological weapon than to try its identification. The problems are closely connected to the formerly mentioned asymmetries. To work with dangerous bioagents is problematic enough, because of their BSL classification. The lack of standards and different controls makes their identification even more difficult. Recently we became convinced that the best way for our mutual security could be to overcome the asymmetric development in the frames of a NATO project of a high level of preparedness in the field of microbiological diagnostic. We should to organize a NATO reference laboratory network system and we ought to develop preformed system of dangerous international sample transportation for emergency situations including the exchange of laboratory data. The special sample types could get high priority, it might be useful to work out certain contracts in advance and lay down a clear mechanism of financial processes and legal conditions. It is far over the national responsibility but if we want to think about national security against bioterrorism we need to think about reality of this suggestion. In the XX century- nearly at the end of the I.WW the pandemic influenza outbreak - the Spanish flu - had come upon the mankind causing minimally 20 - 40 million deaths. This epidemic event also shocked the whole world, and resulted a building up of a global influenza virus control network with national diagnostic laboratories in order to prevent similar events. If it was necessary, complete public health institutes were organized and established all around the Eastern European region. The project was financed everywhere by the Rockefeller Foundation, therefore in several countries the National Institutes of Public Health are located in the Rockefeller street. This analogy shows us, that if we wish to overcome quickly and safely the danger of interstate differences of identification of biological agents within the NATO, the reestablishment of a contemporary molecular diagnostic system according to the concept of the old multinational influenza laboratory network system would be the proper solution. The network could provide uniformity in laboratory capabilities of the member states. The network might include identical military BW diagnostic methodology in every NATO states, which would work continuously, equipped with the same, reagents, reagent kits, control strains from the same quality-controlled sources, and the staffs could pass the same training courses for the same procedures and laboratory methods. Their proficiency might be checked time to time by 'round robin tests'. Headquarters can be ordered as the top of the lab-network being a real or a virtual laboratory center, which might lead the network and might support, supply the national laboratories with information and collect their results and information. The Center would be responsible for the range of diagnostic capability, for the training of specialists, for planning, and it could collect and store unidentified and suspicious samples under proper packaging and transportation conditions. We am sure that only such a well disciplined multinational military laboratory network could result a rapid improve the global safety against the bioterror attacks and it could cope with the increased security requirements. If we would summarize our activity, we could express that we stepped ahead in certain aspects in the territory of national security against bioterrorism, the multi and intersectorial talks started among different organizations of the whole society. Something after a long period has

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happened, the acceptance of CDC, WHO recommendations have started to implement. We hope this way is congruent with the other nations and our efforts will result a significantly safer world. We also hope this way is congruent with the other nations efforts and will also cause a significant vulnerability reduction of Hungary. References [1] [2] [3] [4] [5] [6]

Public Health response to biological and chemical weapons. WHO Guidance. 2001. Tucker, J. Historical Trends Related to Bioterrorism, An Empirical Analysis. Journal of Emerging Infections 5 vol No.4. / 1999. p.498-505 Patterns of Global Terrorism 1998 US State Department www.state.gov/www/global/terrorism/1998Report/intro.html Patterns of Global Terrorism 1999 www.state.gov/www/global/terrorism/1999Report/appb.html Patterns of Global Terrorism 2000 www.state.gov/www/global/terorism/2000Report NATOAJP4.10

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Emerging Infectious Diseases and their Surveillance in Lithuania Loreta ASOKLIENE Centre for Communicable diseases Prevention and Control, Kalvariju str. 153, LT-2042 Vilnius, Lithuania Abstract. The legal framework of communicable diseases surveillance system is set out in Lithuanian Republic Law Amendment to the Law on Prevention and Control of Communicable Diseases in Humans, No IX-649, 13 December 2001. National Centre for Communicable Diseases Prevention and Control is responsible for routine surveillance of communicable diseases at national level, except HIV and AIDS, STD and tuberculosis. The organizational structure of surveillance is based in 11 regional Public Health Centers and 37 microbiological laboratories. Laboratories don't have capacity to test biosafety level 4 facilities. Preparedness to microbiological emergencies is also very important item in all communicable diseases surveillance system. There are some institutions responsible for legislation, organizing measures and technical support for cluster's allocation and liquidation. All responsibilities for every institution are set out in Minister's of Health and Minister's of National Defense order "Due functions of national institutions, which are participating in clusters of emerging and re-emerging communicable diseases allocation and liquidation".

General Background Lithuania is a small country on the East of the Baltic Sea. It's area is 65.2 x 103 km2. Lithuania is a country with 3.4 mln. inhabitants: urban population - 2.3 million, rural population - 1.1 million. For some years there have been registered a negative population increase: -1.3 for 1000 inhabitants. There are 10 administrative divisions (regions): Alytaus, Kauno, Klaipedos, Marijampoles, Panevezio, Siauliu, Taurages, Telsiu, Utenos, and Vilniaus. Every region is divided into smaller districts and there are 44 districts at all. The capital of Lithuania is Vilnius. The country has borders with the following countries: Belarus, Russia (Kaliningrad Oblast), Poland and Latvia. When the country accedes to the EU, there will be a new European Union border. In this context, it will be important to strengthen surveillance and early warning systems. 1. Communicable Diseases in Lithuania About 600 thousand people falls ill with communicable diseases are registered every year in Lithuania. It composes about 15 percents of all registered diseases.

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In 2001 the majority of infectious diseases notified in Lithuania were "airborne diseases" (83%). Parasitic diseases account for 7% of notified communicable diseases, acute intestinal diseases - for 2%. 2. Communicable Disease Surveillance and Control System 2.1. Legal Framework The legal framework of communicable disease surveillance system is set out in Republic of Lithuania Law on Amendment to the Law on Prevention and Control of Communicable Diseases in Humans, No IX-649, December 13, 2001. This Law shall set forth the basics in the management of prevention and control of communicable diseases in humans, dispute settlement and damage compensation and liability for the violations of legal acts with regards to the issues involving the control and prevention of communicable diseases in humans. The law shell set forth rights and obligations of natural and legal persons in the sphere of the control and prevention of communicable diseases and specific characteristics of the funding of prevention and control of communicable diseases and compensating of the costs thereof [1]. There are many other standard documents relevant to communicable diseases surveillance. Main of them is listed below: • State Strategy of the Prevention and Control of Emerging and Re-emerging Communicable Diseases and Measures Plan, 1999. • Law on Public Health, 2002. • Law on Public Health Monitoring, 2002. • National Public Health Strategy, 2001 and Action Plan, 2002. • Order of Health Minister "Due Mandatory Recording of Epidemiological Objects, Procedures of their Recording and Transferring of Information", 2002. • Order of Health Minister "Due urgent information on communicable diseases rendering procedures confirmation", 2002. • Order of Health Minister "Due procedures of communicable diseases epidemiological surveillance organization", 1998. 2.2. The State Strategy of the Prevention and Control of Emerging and Re-emerging Communicable Diseases The State Strategy of the Prevention and Control of Emerging and Re-emerging Communicable Diseases (hereinafter - State Strategy) and the Plan of the State Strategy Measures for implementation thereof was approved by the Government in 1999. The main aim of the State Strategy is to reduce morbidity with communicable diseases, disablement and mortality from these diseases in Lithuania [2]. The main objectives of the State Strategy are listed below [2]: • To evaluate communicable diseases surveillance system. • To ensure a good epidemiological situation with vaccine managing diseases. • To protect Lithuanian people form importation of infectious disease and their spread in the country.

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• • • •

To stop an epidemic spread of TB. To stop an epidemic spread of HIV infection. To reduce morbidity with STD. To stop outbreaks of gastrointestinal infectious diseases.

The list of the mandatory State programmes of surveillance of communicable diseases is specified in the Plan of the State Strategy Measures. Surveillance programmes of Immunoprophylaxis, Hospital Infections, Rabies, TBE and Lyme Disease, Trichinosis, Intestinal Infections, Viral Hepatitis, Tuberculosis, AIDS, STD is prepared according to the State Strategy. Influenza Surveillance, Emerging and Re-emerging Communicable Diseases Importation and Spreading Prevention, Parasitic Diseases Surveillance, Rodents Transmitted Communicable Diseases Surveillance Programs, Measles Eradication and Congenital Rubella Syndrome Eradication Plans will be prepared and/or approved in 2003. 2.3. Institutions, Responsible for Surveillance of Communicable Diseases National Surveillance of communicable diseases is carried out by Centre for Communicable Diseases Prevention and Control. There is a line of accountability from the Ministry of Health, via the State Public Health Supervision Service to the Centre for Communicable Diseases Prevention and Control and regional Public Health Centres and there branches [3]. Centre for Communicable Diseases Prevention and Control is the State institution established by the Ministry of Health, which organises and performs the functions of communicable disease (with the exception of AIDS, HIV, STD, and TB) prevention and control. The Centre methodically directs outbreak and epidemic prevention operations in Lithuania, coordinates the activities of all the Lithuanian institutions concerning prevention of communicable diseases. It is authorised to collaborate with the institutions of the World Health Organisation, European Union and foreign authorities, which regulate and coordinate the surveillance and control of communicable diseases. Centre for Communicable Diseases Prevention and Control, within the sphere of its competence, is authorised to represent the Republic of Lithuania in these institutions [1]. Lithuanian AIDS Centre is responsible for epidemiological surveillance of AIDS, HIV infection and STD, Republican Hospital of Tuberculosis and Lung Diseases - for surveillance of tuberculosis. Health Emergency Centre is responsible for epidemiological surveillance in prisons and armed forces [3]. The organizational structure of epidemiological surveillance is based on 11 regional Public Health Centres (intermediate level) and 44 their branches (local level) and 37 microbiological laboratories at public health and personal health care institutions. National Virology Laboratory is based at Lithuanian AIDS Centre and National Bacteriology Laboratory is based in Microbiology Laboratory of Vilnius Public Health Centre. Main functions of regional Public Health Centre are epidemiological investigation and management of communicable diseases cases, data registration and analysis, data reporting to national level, communicable disease outbreak investigation, preepidemic measures implementation at clusters, implementation of immunization programme, preparation and implementation of special programmes, public education [1, 3, 4].

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2.4. Epidemiological data reporting system Surveillance of infectious diseases in Lithuania is based on case reporting, supplemented by laboratory reporting for some organisms. The Ministry of Health has approved objects and procedure of mandatory recording, mandatory contents of information concerning epidemiological recording objects and mandatory transfer of information to the Public Health Centres. The objects of mandatory epidemiological recording are as follow [1,4]: • Identified agents of communicable diseases and cases of carrying the agents of these diseases. • Suspected cases of communicable diseases and cases of these diseases and deaths resulting thereof. • Cases of humans bitten (salivated upon), when they have been bitten (salivated upon) by animals suspected of having rabies. • Complications resulting from vaccinations. List of mandatory recording communicable diseases and agents of communicable diseases are approved by Ministry of Health. There are 82 communicable diseases and syndromes and 30 identified agents recorded in the State Registers of the communicable disease and communicable disease agents. Registers' regulations are approved by the Government. Centre for Communicable Diseases Prevention and Control is a Chief administrator of State Registers [1,4]. Reporting is based on individual cases and done by the physician who makes a report based on a clinical or laboratory confirmed diagnosis. The primary care institutions inform the local Public Health Centre about every confirmed or suspected case by telephone or fax the same day. If the diagnosis changes, primary health care institutions inform Public Health Centres about changed diagnosis and laboratory results by phone or fax during the same day. At the end of every month all reported cases are summarised on form No 4. District Public Health Centres via regional Public Health Centres send report to the Centre for Communicable Diseases Prevention and Control once per month [4]. Vaccination coverage is assessed at district level using local lists as the denominator. These data are forwarded to regional and national level monthly [4]. Nosocomial infections are reported by healthcare institutions via the local level to Centre for Communicable Diseases Prevention and Control [4]. TB is reported via the existing six TB centres to Republican Hospital of Tuberculosis and Lung Diseases. This hospital informs Centre for Communicable Diseases Prevention and Control about summarized data once per month [4]. HIV and AIDS cases are reported by the primary health care clinics to the Lithuanian AIDS Centre, where they are entered into an HIV/AIDS Register. Lithuanian AIDS Centre informs Centre for Communicable Diseases Prevention and Control about summarized data once per month [4]. 2.5. Data Analysis The summaries sent to national level are aggregated data giving numbers of cases broken down by age, sex, urban or rural and those who died. Summarized data by epidemiological signs are

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sent to national level once per year. Centre for Communicable Diseases Prevention and Control performs analysis for the whole country and produces a monthly, quarterly and annual summary of surveillance data. Dissemination of summarised notifications is sent from Centre for Communicable Diseases Prevention and Control via an e-mail bulletin every month. The bulletin is sent to Health Ministry, State Public Health Supervision service, regional Public Health Centres, other institutions, World Health Organization and neighboring countries (Estonia, Latvia, Belarus, Poland, and Kaliningrad). 3. Surveillance of Extraordinary Situation 3.1. Description of Extraordinary Situation Extraordinary epidemiological situations mean cases, when infected areas are announced and (or) spread of agents of communicable diseases of unknown origin, outbreaks of emerging communicable diseases or epidemics or cases, outbreaks or epidemics of acutely emerging or re-emerging diseases are recorded [1].

3.2. Investigation and Notification The definition of an outbreak is three or more cases. Outbreaks and epidemics are supposed to be identified and processed at regional level with assistance from the local level. Outbreaks should be investigated immediately and laboratory confirmed. During an outbreak case-based data should be collected. Outbreaks of food-borne diseases are investigated together with specialists of Food and Veterinary service. Centre for Communicable Diseases Prevention and Control consults regional Public Health Centres, coordinates outbreak investigation and informs Ministry of Health and State Public Health Service. Media should be informed too. Information about emerging communicable diseases and their outbreaks have to be sent to Centre for Communicable Diseases Prevention and Control by phone within 12 hours and by fax or e-mail within 72 hours. Information about re-emerging communicable diseases and their outbreaks have to be sent to Centre for Communicable Diseases Prevention and control by phone within 2 hours and by fax or e-mail within 12 hours. On weekends, holiday time and at nights all urgent information should be sent to the Health Emergency Center, which is the Centre on duty at Health Ministry [5]. Information about extraordinary situation should be sent to World Health Organization by requirements of acts of International Law.

3.3. Legislation and Surveillance of Health Emergencies Under the treat of biological agent use it was necessary to secure safety of country against above mentioned diseases importation and outspread among population and to organize operatively preepidemic measures from cluster's allocation and liquidation. On 4 July 2002 Minister of Health and National Defense Minister approved an order "Due the functions of governmental authority, participating in emerging and re-emerging communicable diseases

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clusters allocation and liquidation". All responsibilities for every institution responsible for legislation, organizing measures and technical support for cluster's allocation and liquidation are set out in this Order. After World Trade Center attack on 26 September 2001 Minister of Health formed the Health Ministry Centre of Extreme Situation Managing. This Centre would be activated and it would coordinate all activities, if emerging situation occurs. Authorities of this Centre are Health Ministry and subordinate institutions and supporting institutions are Ministry of National Defense, Ministry of Social Security and Labour, Ministry of Transport, Ministry of Economy, ministry of the Interior. This Centre has a Group of Experts for liquidation of consequences after biological agent use. Experts are specialists from Centre for Communicable Disease Prevention and Control, Health Emergency Centre and Laboratories. Functions of Group of Experts for liquidation of consequences after biological agent use: • Estimate an epidemiological situation. • Confirm a cluster of emerging and re-emerging communicable disease. • Participate in preparing and fulfillment measures for clusters allocation and liquidation. • Methodically consult and direct other institutions, participating in clusters allocation and liquidation. On 19 of October 2001 Director of State Public Health Supervision Service formed another group for managing of emerging epidemiological situation. This group considered the following issues: • Ability to recognize and confirm the case of deliberate biological agent use and to organize preepidemic measures. • System of information and decision coordination. • Country hospitals base for accommodation massive infectious disease causalities. • Microbiological diagnostic procedures and laboratories availability. • Notification system and public information. • Preepidemic measures organization.

4. International collaboration in surveillance field In 1998 Swedish infectious Diseases Control Institute did inventory of infectious diseases surveillance and control system in Baltic State. After inventory three Baltic countries were included into Extended Inventory Resources of Infectious diseases in Europe (IRIDE) network. Now Centre for Communicable diseases Prevention and Control participates in this network and updates data of Lithuania. Lithuania participates in CCEE-Baltic Communicable Diseases Network and in other WHO networks: vaccine managing diseases, AFP, diphtheria, meningococcal infection, influenza, rabies, and measles. Specialists from Centre for Communicable Diseases Prevention and Control participates in European Working Group for Legionella Infections (EWGLI) since 1998 and now in EWGLI-NET and in International Scientific Working Group on TBE (ISN-TBE). Lithuania takes part in some international programs:

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• • • • •

WHO Surveillance Programme for Control of Foodborne Infections and Intoxications in Europe. Task Force on Communicable Diseases Control in the Baltic Sea Region (AIDS, TB, antimicrobial resistance and nosocomial infections). International Health Regulation Revision. Project "Computerised Infectious Diseases Reporting System". Poliomyelitis Eradication Project.

5. Problems and Future Tasks of Communicable Diseases Surveillance in Lithuania In 2002 EU experts revised communicable diseases surveillance system in Lithuania. They gave some recommendations how to strengthen and improve an existing system. There is no transfer of complete individual records electronically; so that only summarized data are received at national level. This does not permit a detailed national analysis [6]. One of the most important future tasks for surveillance system evaluation is implementation of "Computerised infectious diseases reporting system". This system would help to strengthen surveillance system, better analyze the situation and implement preepidemic measures as soon as possible. There is a need to strengthen laboratory capacities, especially at nation level, because capacities of national laboratories are limited. There is no availability for testing level 4 facilities. The same was said in report of EU experts. In effective collaborating with Baltic States and other Europe countries there is a need of early warning and respond system for early outbreak and epidemic detection by European Parliament and Council Decision 2119/98/EC [6]. There are 85 epidemiologists in Lithuania at all and local Public Health institutions are lacking in specialists. As recommended EU experts, there is a need to develop specialists training [6]. There are a lot of plans how to improve an existing surveillance system. Hops, all these plans will be not on a paper, but in reality in nearest future. References [1] [2] [3] [4] [5] [6]

Lithuanian Republic Law Amendment to the Law on Prevention and Control of Communicable Diseases in Humans, No IX-649, December 13, 2001. Valstybes zinios 2001; 112-4069. State Strategy of the Prevention and Control of Emerging and Re-emerging Communicable Diseases and Plan of Measures, Decision of the Republic of Lithuania Government No 543, 7 May 1999. Valstybes zinios 1999; 41-1293. Lithuanian Republic Health Ministry Order No 614 "Due procedures of communicable diseases epidemiological surveillance organization", 23 October 1998. Valstybes zinios 1998; 95-2644. Lithuanian Republic Health Minister's Order No 673 "Due Mandatory Registration of Epidemiological Objects, Procedures of their Registration and Transferring of Information", 24 December 2002. Lithuanian Republic Health Minister's Order No 122 "Due urgent information on communicable diseases rendering procedures confirmation", 12 March 2002. Valstybes zinios 2002; 30-1095. Gair R. Communicable Diseases Surveillance in Lithuania. Final Report 2002.

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Biological Recognition Teams in the Polish Army Krzysztof CHOMICZEWSKI The General K. Kaczkowski Military Institute of Hygiene and Epidemiology, 4 Kozielska St., 01-163Warsaw, Poland Abstract. In past times the biological threats were treated very marginally among other problems connected with Armed Forces and civil population protection against mass destruction weapon. The attention was concentrated mostly on problems of protection against nuclear and chemical weapon. The turning-point in modification of this view were experiences from "Dessert Storm" operation, in which the risk of usage of biological weapon against the allied armies was most probably. After bioterroristic attacks in United States the relation to these problems changed considerably. Today the respectable people do not question the meanings of biological weapon and consider this weapon as one of the most important present threats for army and civilian population. The earliest conceptions about organization of modern protection system against biological weapon in Polish Armed Forces started at the Military Institute of Hygiene and Epidemiology in the early ninety years of last century. This process was activated in 1996, when cooperation with US military and civilian institutions were began after signature of an agreement between Polish Ministry of Defense and United States Department of Defense. The undisputed role in creating the protection against biological weapon were initiatives and support from National Security Bureau. At this time the idea of organization the modern BSL-3 microbiological diagnostic laboratory and mobile biological recognition teams was studied. This concept was confirmed. In the beginning of 2001 seven Biological Recognition Teams (BRT) of the Polish Armed Forces Medical Service were organized and by the end of this year fully equipped. At this time, additional BRT was organized and specially equipped to engage in field conditions together with allied forces out of Polish borders. The main tasks performed by BRT include: collection and preservation of samples of the presumably contaminated material, bio-securing and bio-safeguarding of the contaminated area, on the spot preliminary identification of the biological agent used, and transportation of the samples to selected laboratories or to the Microbiological Diagnostic and Research Center (MDRC). Currently, the system of protection of the Polish Armed Forces against biological warfare agents consists of eight mobile Biological Recognition Teams and the Microbiological Diagnostics and Research Center located at the Military Institute of Hygiene and Epidemiology in Pulawy.

In the past biological threats were treated very marginally among other problems connected with Armed Forces and civil population protection against weapons of mass destruction. The attention was concerned mostly on problems of protection against nuclear and chemical weapons. Signed in 1972 Convention of prohibition of production, storage and use biological weapons put to sleep the vigilance of international community. Soon, however it was proved

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that not all states follow the rules of Convention. Outside of all international inspection were terrorist formations interested in usage of biological factors on greater scale. After terrorists attacks on September 11, 2001 the viewing of these problems has changed considerably and bioterroristic attacks with Bacillus cmthracis spores in USA visualized to world opinion the scale of biological factor threats. Of 22 persons infected (11 of cutaneous anthrax and 11 of pulmonary anthrax), 5 died. Prophylactic antibiotic therapy was applied in 32 000 non-infected people The costs connected with decontamination of atttacked objects (buildings, personnel and environment) were huge. For example, cost of decontamination of Capitol Building only, were about 17 million USD. Today the reasonable people do not question the meaning of biological weapons and consider this weapon as one of the most important contemporary threats to military and civilian population. The undisputed role in creating the protection against biological weapons system in Polish Armed Forces was and is: the activity of the Military Institute of Hygiene & Epidemiology, cooperation with US Army, initiatives and support from National Security Bureau. The earliest idea about organization of modern protection system against biological weapon in Polish Armed Forces started in the Military Institute of Hygiene and Epidemiology in early nineties of the last century. In 1995 the first steps in co-operation with US military and civilian Institutions began. This process was activated in December 1996 after signing of the "Information Exchange Annex IEA-A-96-PO-1556, US-Poland Master Information Exchange Agreement Concerning Technologies for the Detection and Analysis of Biological Materials" between Polish Ministry of Defense and United States Department of Defense. At that time the idea of organization of modern BSL-3 microbiological diagnostic laboratory and mobile biological recognition teams was studied. This concept was confirmed by Ministry of Defense in 2000. At the beginning of 2001 seven Biological Recognition Teams (BRT) were organized. At the end of 2001, these seven BRT were fully equipped. Five of BRTs are based at the regional Military Units of Preventive Medicine in Modlin, Bydgoszcz, Gdynia, Wroclaw, Krakow and two at the Military Institute of Hygiene and Epidemiology in Warsaw and in Pulawy. Additional Biological Recognition Team was organized and specially equipped for deployment abroad together with allied forces. Biological Recognition Team is composed of: • Six members of the personnel: commander (microbiologist), four medical and veterinary officers (microbiologists, epidemiologist) and technician/driver. • Personal Protective Equipment: impermeable, level A vacuum suits complete with the self-contained breathing apparatus and the communication system. • Device for refilling air bottles and for checking the air-tightness of the PPE. • Air-filled tent for changing equipment. • Equipment and materials for collecting air, water, soil, food and tissue samples, for detection and provisional identification of pathogens. • Decontamination equipment. Detection and identification equipment consists of: • sample collection equipment and kits; • clinical microbiology equipment (portable microluminometer, etc). • PCR equipment; • biochemical tests and kits.

incubator, portable centrifuge,

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Decontamination equipment of Biological Recognition Teams consists of: • portable decontamination tent with a high-pressure shower; • high pressure portable decontamination device; • portable autoclave; • disinfectants active against vegetative and spore forms of bacteria. The tasks performed in the field conditions by the teams include: collection and preservation of samples of the presumably contaminated material, bio-securing and biosafeguarding of the contaminated area, on the spot preliminary identification of the biological agent used, and transportation of the samples to the Biological Threat Identification and Countering Center. Biological Recognition Teams are also in continuous readiness in a case of bioterroristic attack. Currently, the system of protection of the Polish Armed Forces against biological warfare agents consists of: • seven mobile Biological Recognition Teams; • network of BSL-2 and BSL-2+ microbiological laboratories at Military Institute of Hygiene and Epidemiology in Warsaw and Pulawy and at the Military Units of Preventive Medicine (Modlin, Bydgoszcz, Gdynia, Wroclaw, Krakow); • The Biological Threat Identification and Countering Center (BTICC) located at MIHiE in Pulawy. The BTICC constitutes the central part of the bio-defense system of the Polish Armed Forces. It is the unique in Poland microbiological BSL-3 laboratory, supported by seven BSL-2 laboratory stands. The permanent epidemiological monitoring of military units is conducted the group of experts in field of biological weapon were also appointed at MIHiE. The advisory and expertise of this group to Surgeon General of the Polish Armed Forces is undisputed.

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National Approach of Ukraine to Bioterrorism and Biowarfare Alexander KAPUSTIN Chemical and Biological Weapons Conventions Problems Sector, Ministry of Foreign Affairs of Ukraine Abstract The following aspects of the problem of biowarfare and readiness of state system of Ukraine to prevent and counteract the possible use of biological weapons by the terrorist are discussed in the report: 1. Current approach of Ukraine to the prospects of strenthening Biological and Toxin Weapons Convention. 2. National measures in the sphere of counteraction to the possible use of biological weapons on the territory of Ukraine in the context of events of Autumn 2001. 3. Procedures of interaction between the executive authorities of Ukraine in case of use on its territory of the biological weapons. 4. General information on sanitary-epidemiological surveillance system in Ukraine and its improvement in the context of complex measures to counteract terrorism. 5. Problems that should be resolved for the improvement of readiness of sanitary-epidemiological surveillance system of Ukraine to counteract the possible use of biological weapons by the terrorists. 6. Interest of Ukraine to the establishment and affiliation to the Internet network between the countries of Central and Eastern Europe for exchange of epidemiological data. 7. Procedures of export control of goods that could be used in the development of biological weapons. 8. Information on criminal responsibility for the bioterrorist activity and activity aimed at developing of weapons of mass destruction. 9. Organisation of biological protection system in Armed Forces of Ukraine.

After terrorist attacks of September 11, 2001 in United States and events of October November 2001 connected with the use of Antrax for terrorist purposes the character of perception by Ukraine of the threats for its national security has changed because the danger of terrorism as a whole and possible use of biological weapons by the terrorists in particular has passed from a theoretical plane in practical. In this connection the antiterrorist activity has become one of the priority directions of work for the state security authority as well as for other law enforcement authorities. Availability and cheapness of components of biological weapons as well as the scale of consequences of their use make its attractive for the use by the international terrorist organizations. What have been done in Ukraine to prevent possible proliferation and use of the biological weapons by the terrorists?

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In this connection I would like to concentrate on the following aspects of the mentioned problems: 1. National measures in the sphere of counteraction to the possible use of biological weapons on the territory of Ukraine in the context of events of Autumn 2001. 2. Procedures of interaction between the executive authorities of Ukraine in case of use on its territory of the biological weapons. 3. General information on sanitary-epidemiological surveillance system in Ukraine and its improvement in the context of complex measures to counteract terrorism. 4. Problems that should be resolved for the improvement of readiness of sanitaryepidemiological surveillance system of Ukraine to counteract the possible use of biological weapons by the terrorists. 5. Interest of Ukraine to the establishment and affiliation to the Internet network between the countries of Central and Eastern Europe for exchange of epidemiological data. 6. Procedures of export control of goods that could be used in the development of biological weapons. 7. Information on criminal responsibility for the bioterrorist activity and activity aimed at developing of weapons of mass destruction. 8. Organisation of biological protection system in Armed Forces of Ukraine. I would like to begin the consideration of the specific issues with the describing of the current approach of Ukraine to the prospects of Biological and Toxin Weapons Convention (BWC) strengthening. Ukraine was one of the co-authors of the Biological Weapons Convention and realizes the important role this international agreement has played during the recent decades in the system of international legal instruments network on disarmament and non-proliferation of weapons of mass destruction. Ukraine fully complies with its obligations under the Convention and has never had the intention to develop, produce, stockpile or acquire in any way the biological weapons, equipment or means of its delivery. Our country annually submits to the United Nations Secretariat necessary information on BWC implementation within the framework of confidence-building measures adopted by the Second and Third Review Conferences of the States Parties to BWC. Ukraine considers the Biological Weapons Convention as one of the important elements of the WMD non-proliferation regime and declares its readiness to join efforts aimed at resolving the problems and securing the successful implementation of the BWC on the hole. Our country welcomes completion of work of the Fifth Review Conference of the States Parties to the Biological and Toxin Weapons Convention and its decision to hold three annual meetings of the States Parties commencing in 2003 until the Sixth Review Conference, to be held not later than the end of 2006, aimed at elaborating new approaches for ensuring full compliance of the States-Parties with the BWC provisions. In accordance with the results of the second part of Fifth Review Conference the proposals for the meetings of the experts and the meetings of the States Parties will be developed at the national level. At the same time we regret that negotiations on elaboration of integrated BWC compliance control instrument were not fruitful. We also believe that the bilateral cooperation in the area of biological threat reduction should not substitute the multilateral efforts aimed at strengthening of BWC. Concerning the problem of bioterrorism, Ukraine is deeply concerned by the recent events in the United States and other countries of the world connected with the intensification

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of the terrorist groups' activities and use of biological agents with the aim of intimidation of population and its physical destruction. The science achievements and new technologies in the hands of irresponsible politicians or terrorists have added new threats to traditional ones. All this requires fundamental reconsideration of efficiency of protection against these new threats. In connection with the title of our workshop "Preparedness against Bioterrorism and Re-emerging Infectious Diseases - Regional Capabilities, Needs and Expectations in Central and Eastern Europe Countries" I would like to note that one of important aspects of the emerging-reemerging infections is possibility of arising of extreme epidemic situations as a result of bioterrorism. According to the published data of the American researchers, attack on the city with the population about 100 000, depending on the used agent, will result damage from 5 up to 25 billions US$ [1]. Use of the biological agents with the criminal purposes can have the various scenarios. Some of them from the terrorist point of view are rather simple, cheap and effective. In number of cases the antropogeneous sources of epidemic situations can be casual. For example, introduction on the American continent from Asia of effective carrier of many dangerous infections - mosquito Aedes albopictus with the Japanese automobiles, on the wheels of which the mosquitoes put their eggs. The character of other situations depends on qualification of the producers. In case of their high qualification the analysis of situation can be very difficult or even impossible. In other cases the scenario can have ecological and methodological mistakes, what will enable to put the correct diagnosis. Therefore from the very beginning of occurrence of the emerging-situation it is necessary first of all using epidemiological and clinical data in order to clarify a way of contamination: respiratory, alimentary or transmissive. Probably, earlier it is necessary to isolate etiological agent and to receive a PCR-product and IgM-antibodies. The electronic microscopia enables at the initial stage to classify a virus up to a level of family. After preliminary classification it is possible to define an opportunity of occurrence of that agent, way of contamination on the given territory, on the given season, among the given part of population with given clinical syndroms. Later the virus genom should be sequenced for comparison with the data base of known strains. This is the principle scheme of analysis, but let's return from the scientific issues of prevention of possible use of biological weapons by the terrorists to the common approaches to problem of bioterrorism. In this connection I'd like to note that other particular danger aspect of this problem is mass psychosis which can arise as a consequence of the biological weapons use. It is known, that erroneous alarms in connection with possible use of biological weapons represent a special kind of threats which are used by the terrorists. The fear of possible terrorist attacks with the use of biological agents can reach such a scale that it can turn into the same effective instrument of terrorists as a real use of these agents. The recent developments in the United States of America, connected with the cases of antrax contamination, confirm that. At the same time we cannot also ignore the threats resulting from the real use of biological weapons by the terrorists as well as in the military conflicts of different nature and intensity. Taking into account possible consequences of bioterrorism, the governments should stand ready to prevent such developments. The systemic approach to the problem of protection against biological weapons at the global level should envisage international interaction in all spheres as well as the effective utilization of professional personnel. The specific spheres of interaction should cover all aspects of protection against biological weapons beginning with the determination of future threats, problems of indication, timely notification, decontamination, development of efficient vaccines and some other areas of work. The main

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task is to develop and to implement the balanced and at the same time effective policy in this sphere. 1. Coming back to the first issue which was mentioned at the beginning of the report, namely to the question of national measures in the sphere of counteraction to the possible use of biological weapons on the territory of Ukraine in the context of events of Autumn 2001 I would like to note the following. With regard to the development in the world the wave of new form of terrorism distributions of agents of dangerous infectious diseases through the friable substances, in the context of events of Autumn 2001 President of Ukraine has charged the Cabinet of Ministers of Ukraine to determine the measures on prevention of penetration on the territory of Ukraine the agents of Antrax and other dangerous infectious diseases. According to this assignment Ministry of Ukraine of Emergencies and Affairs of Population Protection from the Consequences of Chornobyl Catastrophe, Ministry of Health of Ukraine, Ministry of Agrarian Policy, Ministry of Internal Affairs, Ministry of Environment and Natural Resources, State Committee of Communication and Information issued the joint Decree "On Measures of Prevention of Threat of Distribution of Dangerous Infectious Diseases Agents". This Decree approved the Integrated Scheme of Reaction of Authorities and Forces in Case of Detection of the Substances of Unknown Origin in the Mail Correspondence and Other Places, and also Temporary Instructions of Actions of the Population and Employees of the Authorities, Enterprises and Organizations in such cases. The above mentioned ministries created working groups for carrying out analysis and forecast development of situation on detection substances of unknown origin and preparation of proposals on realization of prevention measures. Information about cases of detection of unknown substances and measures of protection of the population from dangerous infectious diseases was sent to the Press Service of the Ministry of Emergencies which organized regular information of mass media and the population about the situation and measures were taken. Only during October 2001 there were 273 cases of registered detection of unknown substances which were isolated and sent for the laboratory analysis. For all period of work of groups there were 405 registered messages on detection of objects which are suspected to contain dangerous substances: 283 - in the units of "Ukrposhta", 70 - in the living buildings, 39 - in the official buildings of state authorities, 7 - in the points of the customs control, 6 - in the buildings of mass media. It is necessary to note that analysis of cases of detection of objects which are suspected to contain dangerous substances does not give the reasons to consider that it has systemic character. The measures were carried out have enabled to reduce the tension and to avoid destabilization of situation in society.

2. Procedures of interaction between the executive authorities of Ukraine in case of use on its territory the biological weapons The procedures of interaction between executive authorities in case of detection of agents of dangerous infectious diseases and biological substances were adopted by the Integrated Scheme of Reaction of Authorities and Forces in Case of Detection of the Substances of Unknown Origin in the Post Correspondence and Other Places.

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The Ministry of Emergencies has prepared and entered into force the comprehensive Plan of actions of the ministries, other central authorities, Council of Ministers of Autonomous Republic of Crimea, regional, Kiev and Sevastopol City State Administrations and local authorities on providing with medical and biological protection of the population and territories, where were determined: the basic directions of work of authorities on detection and identification of dangerous biological substances; the procedures of informing of the population in case of detection of agents of dangerous infectious diseases and biological substances; the task on providing of institutions of public health system with disinfection, rodent control, medical and preventive means, appropriate diagnostic systems and equipment, means of individual protection. Besides in Ukraine there was created the Unified State System of Prevention and Reaction on Technogenic and Natural Emergencies. It also includes the system of medical support for the period of emergency. By definition, the medical support system is the complex of constant actions aimed at giving immediate medical aid to the victims in the emergency zone and their delivery to the medical institutions. At the same time it is necessary to take into account absence in the List of emergency situations of Ukraine as well as other CIS countries conception of bioterrorism, and the fact that such phenomena as infectious deseases of human and animals, contamination of agricultural plants are referred to the natural phenomenas or negative medical consequences of technogenic disasters. In opinion of some ministries, in particular the Ministry of Emergency, it would be expedient to develop the state program of counteraction to the acts of terrorism, including bioterrorism.

3. General information on sanitary-epidemiological surveillance system in Ukraine and its improvement in the context of complex measures to counteract terrorism Coming back to the title of our workshop which contains the concept of re-emerging infectious diseases, I would like to note: one of the most important problems of emerging and reemerging infections is great danger because of its unpredictability and ability to cause extraordinary epidemic situations. The recent and strike example of occurrence of such epidemic situation is outbreak of Western Nile Fever in period of Summer-Autumn, 1999 in Astrakhan, Volgograd and Krasnodar regions of Russian Federation. It was also difficult to explain the fact of occurrence at the same period of outbreak of Western Nile Fever with the high level of death rate in New York and its vicinities. In 1996 big outbreak of this infection also with high level of death rate appeared in Romania. The similar extraordinary epidemic situations will appear apparently under mysterious circumstances in the future. As it was shown by the example of Western Nile Fever outbreak on American continent, such epidemic situations may emerge outside usual areals of infectious diseases. It requires creation and maintenance in appropriate conditions of effective system of epidemiological monitoring. By definition, sanitary protection of territory of Ukraine is the system of general state medical and sanitary measures (organizational, sanitary and hygienic, medical and prophylactic, antiepidemic) aimed at preventing penetration and spreading on the territory of Ukraine quarantine diseases (Cholera, Plague, Yellow Fever), Virus Hemorrhagic Fevers (Lassa, Ebola, Marburg), malaria and other dangerous infectious diseases, which are

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transferred by the mosquitoes (Denge Fever, Chicuncunia, Rift Valleys, Western Nile, Horse Encephalomielitises - Western American and Venezuelan, encephalitises - Japanese, Californian, Saint Louis, Murray Valleys), and also at localizing and liquidating of centers of these diseases. The procedure of such measures is determined by the number of legislative, normative and methodological documents. These documents first of all include the laws of Ukraine "On providing of sanitary and epidemiological well-being of population", "On protection of population from infectious diseases"; Rules of sanitary protection of the territory of Ukraine; Decree of MOH of Ukraine "On improvement of anticholeric measures in Ukraine", "Instruction on organization and realization of anticholeric measures, clinical and laboratory diagnostics of Cholera"; Decree of MOH of Ukraine "On extraordinary situation on plague and necessary antiepidemic measures" as well as other decrees and instructions. For realization of complex of antiepidemic measures and prevention of occurrence and spreading of especially dangerous infectious diseases, including possible acts of bioterrorism, Ukraine has the state sanitary-epidemiological surveillance system. Saninary-epidemiological monitoring is carried out by the sanitary-epidemiological service of the Ministry, of Health of Ukraine. The sanitary-epidemiological service of MOH consists of sanitary-epidemiological stations and scientific and research institutes of epidemiological profile. With the purpose of detection of biological pathogenic agents in maximum short terms in the system of MOH as part of networks of surveillance and laboratory control the system of organization of specific indication of bacterial (biological) means was created (exists since 1979 - Decree of MOH of USSR issued on 10.07.1979 -No152-c). Institutions of the state sanitary-epidemiological service of MOH (sanitary-epidemiological stations), scientific research institutes of epidemiological profile of MOH and AMS of Ukraine are involved in this activity. The system of organization of specific indication of bacterial (biological) means includes three categories of institutions: 5 centres of specific indication of biological pathogenic agents, namely - Central SanitaryEpidemiological Station of MOH, L.V.Gromashevskogo Institute of Epidemiology and Infectious Diseases of AMS of Ukraine (Kyiv), Institute of Microbiology and Immunology of AMS of Ukraine (Kharkiv), Lviv Scientifis and Research Institute of Epidemiology and Hygiene of MOH, Ukrainian Scientific and Research Antiplague Institute of MOH (Odesa); 33 leading institutions, namely: Republican Sanitary-Epidemiological Station of Autonomous Republic of Crimea, 24 regional and 8 city sanitary-epidemiological stations, and also 27 duplicating sanitary-epidemiological stations; 652 city and regional sanitary-epidemiological stations. According to the Decree each category of institutions carries out its functional tasks, from sampling, indication and identification of biological pathogenic agents till the laboratory control of food and water. In accordance with the data of MOH for today in Ukraine there are 2706 microbiological laboratories (777 - in sanitary-epidemiological service, 735 - in medical and prophylactic institutions and 991 - in other specialised institutions). In the context of events of Autumn 2001 special attention was given to the issue of improvement of system of indication and identification of biological pathogenic agents. Normative documents concerning the work of institutions of state sanitary-epidemiological service within the regime of functioning of Unified State System of Prevention and Reaction on Technogenic and Natural Emergencies were developed. MOH of Ukraine prepared the draft decree on improvement of functioning of the system of indication of biological pathogenic agents. According to this decree the country should be divided into 6 regions in accordance with the number of appropriate Centres of

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indication on the territory of Ukraine. MOH also prepared Regulations on the Centres of Indication and Regulations on Leading Institutions of Indications. Moreover in October 2001 Ministry of Health of Ukraine elaborated and adopted Comprehensive Plan of organizational measures on maintenance the medical and biological protection of the population and territories. This plan assumes maintenance of operative communication, informing and interaction with the ministries, other central agencies and local authorities. Comprehensive Plan includes: providing the personal of different organisations with the training on improvement of actions in case of biological pathogenic agents contamination or threat of acts of terrorism; control of reserve of medicines, including diagnostic, desinfection means and individual means of protection; strengthening of the state sanitary-epidemiological surveillance on facilities of increased epidemiological risk.

4. Problems that should be resolved for the improvement of readiness of sanitaryepidemiological surveillance system of Ukraine to counteract the possible use of biological weapons by the terrorists Although the structure and criability of laboratories of the state sanitary-epidemiological service of Ukraine basically has been preserved as was confirmed by the research of unknown substances in Autumn 2001, that was carried out in all regions of the country by the laboratories of especially dangerous infections of regional, Kiev and Sevastopol city sanitaryepidemiological stations, unfortunately it is necessary to note, that these institutions can not ensure thorough fulfillment of all their functions. First of all because of constant lack of immunobiological diagnostic means for indication and identification of biological pathogenic agents, that could be explained by the fact, that means of express diagnostics practically are not produced in Ukraine. It is spent annually about 2 million US$ in order to provide laboratories with necessary quantity of these means. Second, the equipment of microbiological and virological laboratories of sanitary-epidemiological stations and research institutions of epidemiological profile that were supplied in 1970-th are morally obsolete. As a consequence quite often the results of indication are subjective and its terms are long. According to the opinion of experts, urgent modernization of laboratories and equipment requires about 8,5 million US$. Equipment of 652 city and districts sanitary-epidemiological stations also does not correspond to requirements of today. Modernization of this equipment requires about 5 million US$. Besides training of the personnel of institutions of the system of indication and identification of biological pathogenic agent is practically suspended (last training of the experts of centres of specific indication was carried out in 1983 on the basis of Rostov Antiplague Institute). The analysis of situation in institutions of sanitary-epidemiological service and scientific research institutes of epidemiological profile related to the safety of functioning of sanitary-epidemiological control system shows that the imperfect organization of safe functioning of institutions of this system does not correspond to the modern threats. For the reduction of these threats it is necessary to carry out the complex of special measures that should include: resolving the issue of target financing of sanitary-epidemiological institutions; providing these institutions with appropriate typical modern premises in accordance with requests for regime of work with biological agents of I - II groups of pathogenicity (national classification); providing the laboratories which work with biological pathogenic agents with the modern laboratory equipment, diagnostic means and means of individual protection, laboratory animals, modern electronic systems of protection and supervision; training of

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experts of appropriate profile in the national and foreign educational and research institutions; providing the institutions of sanitary-epidemiological service with qualified personnel. In this context it is important for our country the development of cooperation between Ukraine and United States in the sphere of biological threat reduction. Within the framework of development of such cooperation the American side expressed its readiness to grant the aid for creation of modern system of phisical protection of some objects in Ukraine where work with bioagents of double use are conducted. Now we are at the final stage of preparation of the text of Agreements between the Department of Defence of the United States of America and the Ministry of Health of Ukraine on cooperation in the area of prevention of proliferation of technology, pathogens and expertise that could be used in the development of biological weapons. Conclusion of such agreement provides the legal basis for cooperation in this area. Ensuring of sustainable functioning of sanitary-epidemiological surveillance system is possible only under conditions of supply with modern equipment of its institutions, first of all the Centres of indication and leading sanitary-epidemiological stations. With this purpose the Ministry of Health of Ukraine developed the program of complex measures of material support and target financing of the Centres and Leading Institutions of indication and identification of biological pathogenic agents for 2002 - 2005. The program was sent to the Cabinet of Ministers of Ukraine and the Ministry of Emergency. However till now the problem of granting of target financing does not resolved. I would like to emphasize once again, that it is necessary for the State to have a permanent network of surveillance and the laboratory control which would has possibility in short time frames to conduct necessary researches and to provide with the recommendations population and authorities concerning their further actions. For those purpose modernization of laboratories and equipment, and also appropriate training of personnel are necessary. 5. Concerning the fifth issue mentioned at the begining of our workshop I will be brief. In opinion of the ministries and other governmental agencies which are involved in the activity aimed at preventing the possible acts of terrorism, Ukraine would be interested in affiliation to the Internet network between the countries of Central and Eastern Europe for exchange of epidemiological data.

6. Procedures of export control of goods that could be used in the development of biological weapons Regulations on the Procedures of Control on Export, Import and Transit of Goods that Could be Used in the Development of Chemical and Biological Weapons (Decree of the Cabinet of Ministers of Ukraine No384 issued on 22.04.1997) as well as the lists of such goods completely correspond to the basic principles and the lists recommended by the Convention on Prohibition of the Chemical Weapons and appropriate documents of the Australian Group and are constantly updated in accordance with the requirements of such regimes. According to this Regulations all agents of the human, animals and plants diseases, its genetically modified forms, fragments of genetic material, equipment and technologies that could be used for the development of the biological and toxin weapons are the subject of control. Today the list of agents of diseases that could be used for the development of the

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biological weapons include 20 types of viruses, 4 species of ricketsia, 16 species of bacteria and 44 types of toxins. Now State Service of Export Control carries out the work aimed at more full defining of biological agents of the control List.

7. Information on criminal responsibility for the bioterrorist activity and activity aimed at developing of weapons of mass destruction Any development, manufacture, stockpiling or use of biological weapons is the result of decisions and actions of individual persons, whether they be officials, representatives of the companies, weapons experts or terrorists. At the same time the international convention that prohibit this kind of weapons almost does not assume an individual responsibility. Thus the states are put before necessity to bring in its acts the appropriate provisions which would establish the criminal responsibility for the activity aimed at developing of the weapons of mass destruction. As I have already noted, there are no conception of "bioterrorisms" in Ukraine' legislation as well as appropriate provisions that would determine the responsibility for earring out such kind of crime. For today it is possible to use some related articles of the Criminal Code of Ukraine in case of possible bioaggression. However in opinion of experts the efficiency of these articles in proving for fighting bioterrorism is low. In particular the Criminal Code of Ukraine contains following articles that to certain extent refers to the criminal responsibility for the bioterrorist activity and proliferation of weapon of mass destruction: Article 258 ("Act of Terrorism"), Article 261 ("Attacks on the facilities on which there are items that constitute the enhanced danger to the surroundings"), Article 321 ("Illegal production, making, purchasing, transportation, sending, storage for selling purposes, or sale of poisonous and drastic substances"), Article 326 ("Violation of rules related to handling of microbiological or other biological agents or toxins"), Article 333 ("Illegal exportation outside Ukraine of raw material, materials, equipment, technology for creation of weapons as well as military and special enginery"), Article 439 ("Use of weapons of mass destruction"), Article 440 ("Development, production, purchasing, stockpiling, sale or transportation of weapons of mass destruction"), Article 441 ("Ecocide"). Article 258. Act of terrorism 1. An act of terrorism that is the use of weapons, explosions, fire or any other actions that exposed human life or health to danger or caused significant pecuniary damage or any other grave consequences, where such actions were performed with a view to violate public security, intimidate population, provoke an armed conflict or international tension, or to exert influence on decisions made op actions taken or not taken by government agencies or local government authorities, officials of such bodies, associations of citizens, legal entities, or to attract attention of the public to certain political, religious or any other convictions of the culprit (terrorist), and also a threat to commit any such acts for the same purposes, - shall be punished by deprivation of freedom for a term of from five to ten years. 2. The same acts, committed repeatedly or by a group of persons upon their prior conspiracy, or where these actions have caused significant property damage or other grave consequences, - shall be punished by deprivation of freedom for a term of from seven to twelve years. 3. Acts provided for by paragraph 1 or 2 of this Article, where they have caused death of people, - shall be punished by deprivation of freedom for a term of from ten to fifteen years or life deprivation of freedom. 4. Establishing, leading or participating in a terrorist group or terrorist organisations as well as providing with logistical, organisational or any other assistance in order to facilitate the establishment or operation of a terrorist group or terrorist organisation, - shall be punished by deprivation of freedom for a term of from eight to fifteen years.

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5.

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Any person other than an organiser or leader shall be discharged from criminal liability for any acts provided for in paragraph 4 of this Article, if he (she) has voluntarily reported it to a law enforcement authority and assisted in termination of existence or operations of such terrorist group or organisation, or in uncovering crimes related to creation or operation of such terrorist group or organisation, unless his (her) actions contain no element of any other offence. Article 261. Attacks on the facilities on which there are items that constitute the enhanced danger to the surroundings. Attacks on any facilities on which any radioactive, chemical, biological or explosive materials, substances, or items produced, stored, used or transported therein, for the purpose of seizure, injury or destruction of any such facilities, - shall be punished by deprivation of freedom for a term of from five to twelve years. Article 321. Illegal production, making, purchasing, transportation, sending, storage for selling purposes, or sale of poisonous and drastic substances. 1. Illegal production, making, purchasing, transportation, sending, storage for selling purposes, or sale of poisonous and drastic substances, other than narcotics, psychotropic substances or their analogues as well as any such acts in regard of any equipment devised for the production or making of poisonous or drastic substances, where these acts were not duly authorised, - shall be punished by a fine up to 50 tax-free minimum incomes of citizens, or deprivation of freedom for a term up to three years. 2. Violation of rules related to production, making, purchasing, storage, dispensation, inventorying, transportation or sending of poisonous or drastic substaces, other than narcotics, psychotropic substances or their analogues, - shall be punished by a fine up to 100 tax-free minimum of citizens, or deprivation of freedom for a term up to two years. Article 326. Violation of rules related to handling of microbiological or other biological agents or toxins. 1. Violation of rules related to storage, use, accounting, transportation of microbiological or other biological agents or toxins, and any other rules related to handling of same, where it caused the risk of death of people or any other grave consequences, or caused any harm to the victim's health, - shall be punished by a fine up to 50 tax-free minimum incomes of citizens or correctional labour for a term up to two years, or limitation of freedom for a term up to three years, with or without the deprivation of the right to occupy certain positions and engage in certain activities for a term up to three years. 2. The same action that caused death of people or any other grave consequences, - shall be punished by limitation of freedom for a term up to 5 years, or deprivation of freedom for the same term, with the deprivation of the right to occupy certain positions or engage in certain activities for a term up to three years. Article 333. Illegal exportation outside Ukraine of raw material, materials, equipment, technology for creation of weapons as well as military and special enginery. Violation of established regulations on exportation outside Ukraine of raw material, materials, equipment or technology that can be used for the development of missile, nuclear, chemical or other types of weapons, as well as for production of equipment for military and special purposes, - shall be punished by a fine of from 100 to 200 tax-free minimum incomes of citizens, or limitation of freedom for a term up to three years, or deprivation of freedom for the same term. Article 439. Use of weapons of mass destruction. 1. The use of weapons of mass destruction prohibited by international treaties consented to be binding by the Verkhovna Rada of Ukraine, - shall be punished by deprivation of freedom for a term from eight to twelve years. 2. The same action that caused death of people or any other grave consequences, - shall be punished by deprivation of freedom for a term of from eight to fifteen years, or life deprivation of freedom. Article 440. Development, production, purchasing, stockpiling, sale or transportation of weapons of mass destruction. Development, production, purchasing, stockpiling, sale or transportation of weapons of mass destruction prohibited by international treaties consented to be binding by the Verkhovna Rada of Ukraine, - shall be punished by deprivation of freedom for a term of from three to ten years. Article 441. Ecocide. Mass destruction of flora and fauna, poisoning of air or water resources as well as any other actions that may cause an environmental disaster,- shall be punished by deprivation of freedom for a term of from eight to fifteen years.

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As a whole Ukraine' legislation concerning the issue of use and proliferation prevention of weapons of mass destruction (including biological weapons) completely corresponds to the existing principles and norms of international law. 8. Organisation of biological protection system in Armed Forces of Ukraine Resolving the problems connected with the biological protection in Armed Forces of Ukraine is charged on the Nuclear, Chemical and Biological Protection Forces (NCB Protection Forces) and Military Medical Service. According to the Status of Internal Service of Armed Forces of Ukraine adopted by the Law of Ukraine issued on March, 24, 1999, any military unit has service of nuclear, chemical and biological protection. NCB Protection Forces carry out: • constant supervision over environment with the purpose of early detection of the fact of weapons of mass destruction use and determination of the border of contamination zone; • special processing of the contaminated territories, technics and buildings. Specific measures which are duty of the Military Medical Service of Armed Forces of Ukraine include: • carrying out the preventive vaccination of military staff under epidemic reason; • carrying out the laboratory diagnostics of biological pathogenic agents; • providing of military men with the means of preventive measures (antibiotics) and control for its use; • organization and carrying out antiepidemic measures aimed at detecting of the source of infections, localization and elimination of centers of infectious diseases which have emerged as the result of use of biological means. As a conclusion I would like to note that in the XXI Century biological threat in the form of bioterrorism became more dangerous and possible. In this connection I would like express the hope that international community will be able to elaborate the definitly new conception of counteraction to this type of agression as well as to ensure the comprehensive strengthening of the regime of prohibition of using the pathogenic agents as the means of warfare.

References [1] [2] [3]

Kaufman A. F., Meltzer M. I., Schmid G. P. The economic impact of bioterrorist attack: are prevention and postattack intervention programs justifiable? // Emerg. Infect. Dis. - 1997. - Vol. 3. - P. 83-94. K. HoBbie H BHOBL BOSHHKaromHC BHpycHbie HH^CKUHH. // Bonpocbi BHpycojiorHH. - 2000. - No 4. - C. 4-7. HeKpacoea JI. C., MapuntoK B. B., KocmwteHKO H. M. OpramaauJH saxoaie canixapHOi oxopoHH xepirropii VKpaiHH BUI. aaneceHHa xa poanoBciozpKeHHa KapaHTHHHHX XBOpo6 xa IHUIHX oco6jiHBO He6e3neHHHX iH^eKuiK. // CynacHi in^eiou'i. - 2000. JSf° 4. - C. 4 - 7.

PartS

Risk Assessment, Crisis Management and NBC Training

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Preparedness Against Bioterrorism and Re-Emerging Infectious Diseases J. Kocik et al. (Eds.) IOS Press, 2004

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Effective Risk Management in the Human Factors Assessment of Chemical/Biological Threats Andrew H. BELLENKES School of Aviation Safety, Naval Postgraduate School (Code 10) 1588 Cunningham Rd, Monterey, California USA 93943-5002

Abstract. The employment of chemical and biological agents as weapons of mass destruction poses a viable, continuing threat in asymmetric warfare. As such, strategic decision-makers must account for a comprehensive set of risks and responses associated with the plethora of hazards facing those responding to a chemical/biological event. Until now there has been no standardized set of processes established whereby threats to responders could be adequately assessed and controlled. There is, however, a process already used widely by defense and industrial organizations that has proven very powerful in identifying and minimizing hazards, and in doing so increasing the level of overall safety and 'operational readiness'. Operational Risk Management (hereafter ORM) is a proven schema for planning that could be adapted for use by chemical/biological response decisionmakers. This chapter provides a review of the processes and tools associated with ORM, and how the five steps in the ORM process can be effectively used to (a) determine the degree of risk in terms of the vulnerability of the target population, (b) determine the probability that specific agents are involved in a given event, and (c) predict the severity of risks associated with the hazards. With this information, it is then possible for decision-makers to prioritize these risks, develop and implement risk control options against each of the threats, and then ensure that these controls remain in place and have the best desired protective effect. This information, in turn, allows for more effective management of the many consequences resulting from a chemical/biological event. With ORM, comprehensive pre-event plans and risk models can be created, tested and the weak-points identified and corrected before the possible implementation of the plan (when it would already be too late). Specific operational scenarios will be used to demonstrate the effectiveness of ORM in apriori planning and practice.

Introduction "By its nature, the uncertainty of war invariably involves the acceptance of risk. Because risk is often related to gain, leaders weigh risks against the benefits to be gained from an operation." (NDP-1: Naval Warfare). "We rely on the judgment of individual Commanders to balance the requirements of mission success with the inherent risks of military action. Naval leaders have always practiced risk management in their operational decision-making. However, the approach to risk, and

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degree of success in dealing with it, has varied widely depending on the leader and his/her level of training and experience. The principles of Operational Risk Management can be taught and effectively applied throughout the Navy and Marine Corps to enhance the decision making capabilities of our personnel." (OPNAVINST 3500.39: Introduction to Operational Risk Management). The global scope of terrorist activity has necessitated the careful development and implementation of numerous homeland defense programs that are as diverse in nature and scope as are the threats. The tragic events of 11 September 2001 and the bio-terrorism emerging since then clearly reveal the desire of terrorists to inflict as much pain, damage, and fear as possible into a target population, so much so that even the threat of using terror tactics is sufficient to prompt great expenditures in time, fiscal and human resources. The global conflict against terrorism is made all the more challenging by the notion that terrorists are able to procure (and have the potential to use) chemical and biological weapons of mass destruction (WMD). Although their use is limited by the ability to clandestinely transport and effectively deliver such weapons, the capabilities of terrorists continues to increase [1,2], and they will use them against both civilian and military targets [3,4,5,6]. It is the primary goal of any organization's decision-makers to establish and maintain the highest levels of 'operational readiness' whereby any organized institution or system is prepared to deter and if necessary react to these as well as other threats. One way to achieve such operational readiness is through the use of effective risk management; that is, apriori planning designed to provide security by (a) preventing attack and (b) in the event of attack, minimizing injury/loss [7]. Central to the risk management 'process' is the requirement to identify (and control for) the plethora of 'human factors' associated with such planning. This task is made all the more difficult in that decision-makers must not only account for those human factors associated with the terrorists who would employ WMDs (i.e., psychosocial, organizational, and political dynamics, available technologies for weapons design, delivery, and employment, etc.), but also with those which help define target population readiness (i.e., hazard identification, detection, and handling, incident response readiness, special countermeasures, etc.) [8]. Operational Risk Management (hereafter, ORM) has become recognized as one of the best means minimize risks (thereby, losses) associated with many types of hazards, both natural and man-made. ORM is a decision-making tool that can be used by personnel (decision-makers) at all levels of an organization or system, (not just those performing supervisory or management duties) to increase operational effectiveness. The use of the ORM process increases the ability to make informed decisions by providing the best baseline of knowledge and experience available. ORM use also minimizes risks to acceptable levels by systematically applying controls to each risk that is not acceptable [9]. Indeed, ORM has become an integral part of military and industrial management doctrine because it has forced decision-makers to ask a critical question: are decisions being made and implemented in such a manner so as to maximize readiness whilst minimizing unnecessary risk? It also provides decision-makers with a standardized, effective means to answer that question [10]. This chapter first reviews how ORM has been used most successfully by both the military and industry. That is followed by a brief overview of the 5-step ORM process. In this regard, there will be a discussion how to identify various types of threats (both external and internal), how to select and implement appropriate controls to set in place to counter these threats, and then how to ensure that these controls are have the hoped-for effect. As shall be seen in the next sections, ORM has already been very successfully used by both military and civilian organizations; it is a proven quantity. Thus, it becomes even more paramount that all

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medical and safety personnel, especially those in decision-making positions associated with planning for WMD events, learn and employ ORM as a part of their daily operations. 1. ORM: A History of Success ORM is not a new idea; it is an established, proven process that has been widely employed by academia, industry, and the military. It has not always been called "Operational Risk Management" per se, however the practice of assessing and managing risk has long been a major tool in predicting the success or outcomes of a particular activity. A major problem in risk management, however, lay in the lack of process standardization, and with it the inability to effectively promulgate strategies associated with the practice of risk management. 1.1. Military The United States Army first recognized the need to create a process during their reviews of battlefield 'accidents' (hereafter referred to as 'mishaps') those mishaps with little or no direct relation to combat. Such mishaps could impact field operations and in doing so, hurt operational readiness. Thus, in the late 1980's, the Army established a formal set of procedures to identify risks, the hazards associated with these risks, and then create a means of controlling these risks...all of this in the name of operational safety. The result was a substantial decrease in non-combat-related mishaps. Once the success of the ORM was established, the Army then began to ensure that ORM became a 'process', a way of thinking inculcated in the culture or the organization, rather than merely another standardized way of doing business. Thus, ORM was expanded from the battlefield to include other critical operational activities including all levels and phases of decision-making, training, personnel assignments, ground, aviation, workplace, and maintenance activities [11]. The other U.S. armed services likewise found that the processes and practices associated with ORM could be applied to their operational culture. In 1997, the Chief of Naval Operations and Commandant of the United States Marine Corps created an 'Instruction', OPNAVINST/MCO 3500 [2], in order to introduce ORM and standardize ORM-related practices throughout the Fleet. In this instruction, Sailors and Marines [12] were introduced to the notion that 'uncertainty' and risk are inherent in almost any activity, and as such must be considered when planning for operations, rather than analyzed post-hoc; that is, in response to mishaps. At about the same time, the U.S. Air Force similarly implemented ORM in their 'way of doing business'. Air Force leadership promulgated ORM information to their troops via 'AFP 91-214, Operational Risk Management Implementation and Execution' [13]. The Air Force Chief of Staff has repeatedly stressed the need to create an environment whereby ORM is taught in a continuum from basic training up through supervisory and command levels. As with the Army, Coast Guard and Naval Services, the Air Force couched risk assessment in terms of a means to best identify hazards and set in place controls that could effectively prevent or minimize the impacts of these hazards (such as chemical and biological weapons [14])All of the armed services have been integrating ORM into their respective cultures, stressing the 'top-down, bottom-up' chain-of-command responsibilities to make it function effectively and in a timely manner. Sets of lessons-learned and 'best practices' databases have been established to aid in the overall training of military personnel of all ranks and levels of

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responsibility, both in classroom and via distance learning. For all of the services, the use of Internet technologies has proven invaluable in promulgating ORM training. For example, the Navy and Marines have created two highly successful Internet-based ORM training programs, one for all personnel (ORM University) and the other specialized to medical personnel (ORM for Docs) [15]. 1.2. Industry As with the military, leadership in industry has long been plagued with workplace hazards, some of which have resulted in tragic mishaps. The original need to employ effective risk management was therefore (and continues in-part to be) driven by the concept of workplace safety. There are a large number of organizational risks inherent to business and industry; many are specific to the nature and mission of the particular organization in question. These hazards could range from those that could physically endanger individuals and/or small groups of workers to those safety and non-safety variables that could imperil the status, even the existence of the company (i.e., poor financial management, hostile take-overs, etc.). Reason [16] likewise makes this 'local-global' distinction, but couches the nature of the hazard and risks as a function of human error effects on the 'victim(s)' of accidents. He notes that mishaps could impact either single (or small groups of) victims (individual accidents) or an industry as a whole entity (organizational accident). Whilst the former will affect the individual and near-associates/family, the latter form of accidents can negatively impact an entire organization. Reason makes it clear that whilst both forms of 'accidents' can affect an organization's ability to function, it is the less-frequent but catastrophic organizational accident that involves several levels of a company and its staff/employees. It is on this basis that Reason developed his widely used human error models and taxonomies of human error that have proven so critical to hazard identification in effective risk management [17]. It is not only the identification of hazards which have proven so critical to industrial safety management, but also the notion that not everyone, whether worker or management, perceives risks in the same way. Indeed, there appears to be a significant relationship between how one perceives risk, how much risk one is will to take (that is, accept for oneself and/or others), and the likelihood that a mishap will occur [18]. From this, one could conclude that an organization whose culture fails to allow for accurate risk perception may eschew effective and/or timely risk management, and in doing so, increase the chances for a mishap. However, the requirement for risk assessment and control has become inculcated in industrial standard operating procedures, and the use of ORM has found its way into a number of diverse industries. Of these one of the most active uses of ORM can be found in the area of food safety, as evidenced by a recent special report by the U.S. Food and Drug Administration [19]. In summary, ORM has been shown to be an effective tool for use in managing risk and in doing so, minimizing the opportunities for hazards to result in organizational mishaps. As such, ORM promotes operational readiness. It is the many strengths of this process that lead the United States General Accounting Office to first promote using ORM in planning for homeland defense. In 2001, the Director of Defense Capabilities and Management [20, 21] noted that ORM could be used to (a) assess the likelihood of terrorist activities against a target (threat assessment), noting especially the capabilities, motivations, and results of such an attack actually occurring, (b) identify the weaknesses in organizations, targets, and systems that could be exploited by terrorists (vulnerability assessments), and (c) prioritize the list of possible targets based on their criticality to national security.

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To understand how ORM could be best used in homeland defense issues and, more specifically, as a weapon against biological/chemical threats, one must first understand how ORM works. The following section therefore briefly describes the five primary steps involved in the ORM process. 2. ORM Process-the Five-Step Risk Management Paradigm 2.1. Identifying

Threats/Targets/Hazard

The first step in the ORM process is to identify the often numerous hazards facing an individual, organization, or system. One normally does this only when immediately confronted with a situation or environment where risk is associated with an action or inaction (such as driving in the fog or flying a combat mission). However, on a more global level, operational readiness must be ensured against potential as well as real threats. It therefore behooves decision-makers to account for as many threats as possible. These threats normally can be generically defined as any weapon, system, procedure, or policy that, either by use, inappropriate use, or lack of use could lead to (a) injury or harm, (b) property damage and/or (c) mission/performance degradation. 2.7.7. There are several tools that one can employ as part of the ORM threat identification process; some of which involve unrestrictive association and hypothesis generation ('Brainstorming' and 'What If analyses) and the generation of visual guides (Flow Charts, 'Cause-Effect' and 'Affinity' diagrams). An effective ORM process employs many of the tools, thereby maximizing the chances of identifying as many real and potential threats as possible. For descriptions of these and other threat identification tools, the reader is directed to the work of the Naval Safety Center Risk Management Division [22]. When identifying threats, decision-makers must note that threats can originate both from sources outside (external) or from within (internal) a system or organization. This discussion will be limited to external and internal threats posed by chemical and biological terrorism.

2.1.2. External Threats Although the strategic nuclear threat diminished with the end of the bipolar Cold War, weapons of mass destruction (WMDs) designed for use during that period continue to pose serious threats both to military troops and civilian populations. In this post-Cold War milieu, terrorists (state-sponsored or independent special armed forces, cells, or individuals) could potentially come into possession of such weapons. It is for this reason that Chemical and Biological Defense program decision-makers must thoroughly assess the unique nature, scope, and impacts of terrorist-based (external) WMD threats. In his discussion on how to prepare for expected bio-terrorism attacks, Moser, White, Lewis-Younger, and Garrett [1] note that the use of biological agents in particular, whilst somewhat difficult to deliver, may be attractive to terrorists because of the 'invisibility' of the attack, the delayed onset which could hinder or even preclude treatment of those exposed, and the inability to treat certain agents. With these factors in mind, the planner (when creating the

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'Event Probability' axis of the threat identification risk matrix), must account for such diverse factors including the ability of the terrorists to create, transport, and deliver a certain agent, the likelihood that a particular agent will affect a given population within a given period of time. For example, the Director of a major city health facility and his/her staff may decide that the threat posed by Human pathogens such as Anthrax or botulinus toxin is greater in the short run (say, within one-year) than that posed by the more exotic Ebola virus or Venezuelan equine encephalitis within the same period of time. This does not suggest that Ebola or equine encephalitis do not pose a threat. Rather, the ORM matrix accompanied by timely medical intelligence can aid the Director in deciding the likelihood that these or any other pathogens may constitute a 'clear and present' danger. With that information, the Director can then proceed to determine the appropriate measures to minimize or prevent the effects of a bioterrorist attack employing these agents. One can also see that it is imperative that this threat identification process be as exhaustive as possible.

2.1.3. Internal Threats Even if an external threat is known and has been correctly identified, that information is without worth unless an organization is prepared to effectively counter (if not prevent) the attack. However, unlike the external threats posed by the terrorist, internal threats are inherent in the operational readiness of an organization and as such may be more difficult to identify. The attempt to examine ones own readiness could be impaired by bias for ones organization or fear that such an internal readiness 'audit' might reveal critical problems. It is therefore highly recommended that when performing an ORM assessment of internal threats, some members of the team conducting the assessment should not come from within the organization itself. This type of unbiased 'outsider's look' is common in the military where members of one unit are invited to conduct as 'survey' of another unit's procedures and practices. Examples of internal threats include (but are not limited to): • •

•

•

Personnel Factors: Response teams are too few in number, lack the requisite skills, training, or motivation. Training Factors: Response training is outdated, inaccurate, cannot be transferred to 'real-world' events, fails to account for all patient, environmental, and logistic conditions and contingencies. Resource Factors: Supplies, tools, support gear, instrumentation, hardware, software, and logistical support are inappropriate, insufficient, of poor quality, or unavailable. Cultural Factors: Organization is unprepared and/or unable to quickly and effectively respond to events, lacks the expertise to manage activities, leadership fails to define, promulgate and support a proactive as well as reactive set of standard operating procedures. Organization fails to employ risk management.

Some specific internal threats posed by a bioterror event may include [23]:

•

"Shortfalls of ICU beds, ventilators, and other critical care needs

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Shortages of chemotherapeutic agents Needs for ancillary or nontraditional treatment centers High demand for mortuary and/or funeral services High demand for social and counseling services Shortages of health care workers due to absenteeism" 2.2. Assess Threats Risk(s) Once the threats have been identified, the planner must then determine the level of risk associated with each hazard, particularly with regard to both the severity of consequences resulting from exposure to the threat (here, a biological or chemical terrorist event) and the probability that a loss will occur should an event take place. The probability can vary as a function of a host of environmental and human factors variables. Decision-makers must begin the assessment by establishing a set of assumptions regarding, for example, bioterrorism. Some of these have been outlined by Jagminas, Marcossi, and Mothershead [23] and may include the following: • • • • •

"With or without advanced warning, the actual time and location of the release of a biological agent most likely will be covert. Terrorists desiring maximum effect will opt for aerosolized release of the pathogen. Exposed individuals will have minimal physical and immunologic protection. Signs and symptoms of illness will be delayed from hours to weeks and initially may mimic minor nonspecific illnesses or naturally produced disease syndromes. Arrival of assistance from state or federal agencies will be delayed from 24-72 hours after request and may take longer for full operation." EVENT PROBABILITY A

B

C

D

1

2

3

4

II

2

3

4

5

III

3

4

5

6

IV

4

5

6

7

THREAT SEVERITY I

Table 6. ORM Threat Matrix

An invaluable tool in threat assessment, the 'threat matrix' [21], employs such assumptions in order to prioritize threats an example of which is shown in Table 6. Each threat is classified as a function of its perceived level of 'severity' as well as the probability that the threat may evolve into an actual event. As depicted in Table 6, a threat matrix contains four subjective classes of threat severity: (I) Catastrophic-may result in loss of personnel (death) and/or loss or grave damage to facilities and resources, (II) Critical-may result in severe injury or severe illness or major damage and/or severe degradation in the effective use of facilities, resources, and related

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assets, (HI) Marginal-may cause minor injury, minor illness, or minor damage and/or minor degradation in the effective use of facilities, resources, and assets, and (IV) Negligible-a disruptive threat, but one that would not cause illness or physical damage, and minimal degradation to the effective use of assets. A lack of standardization in the characteristics defining each of these classes can result in some confusion. Thus, it becomes imperative that, an organization performing ORM should establish these definitions at the outset of the analysis and maintain them throughout the entire process. The probability that an 'event' will occur is couched in terms of likelihood over time: (A) Imminent-defmitely will occur immediately or within a short period of time; expected to occur frequently, (B) Likely-may occur within a short period of time; expected to occur several times, (C) Probable-may occur in time, and (D) Unlikely-not likely to occur. Much of this information comes from security and intelligence updates. As such information is volatile, it is imperative that an organization conducting ORM ensure that the data used to identify threats be timely and accurate. Once threat severity and probability have been established, and the threat placed in the matrix, each threat is then assigned a 'Risk Assessment Code' (RAC). The RAC is a standardized way of prioritizing threats un such a manner as to best decide which threats pose higher risk than others. Table 6 lists the RAC codes for each level of combined threat severity and probability. A threat assigned RAC 1 or 2 is considered a critical threat and should be acted upon immediately. RAC 3 threats are serious threats. RAC 4 threats pose a moderatelevel of risk. RAC 5 threats are considered moderate. RAC 6 and 7 threats hold the lowest priority on this matrix and are considered of negligible risk. Defining threats in this manner can help decision-makers to better assess the entire range of real and perceived threats and, as we shall see in the Section 2.3, plan counter-threat measures. The ORM matrix thus provides planner with a threat-risk metric that, in turn, facilitates the prioritization of various threats per unit time. It is within this framework that assessments of target vulnerability and criticality can also be made. A target 'vulnerability assessment' identifies any weaknesses that can be exploited by terrorists, and in doing so, also suggests options to eliminate or minimize the negative impacts of those vulnerabilities. At the same time, the target 'criticality assessment' systematically identifies and evaluates a target's assets, importance or function, and rates the target in terms of attack probability. The product of performing a threat assessment is a prioritized list of threats that, in turn, provides decision-makers with information regarding the amount of risk posed by each threat. For decision-makers, this step in the ORM process is especially critical, as it not only describes the threat, but also provides a foundation upon which subsequent 'controls' could be applied to mitigate the effects of a biological or chemical event. 2.3. Make Threat (Risk) Control Decisions Having identified one or more threats, the ORM process next requires decision-makers to make decisions about how to set in place risk-mitigating 'controls'. A control is anything (i.e., a policy, procedure, piece of hardware, weapon, etc,) that eliminates threats or reduces the degree of risk of exposure to said threats. That is, a control (a) lowers the probability of a bioterror event occurring, (b) decreases the severity of effects of an event should it occur, and/or (c) limits the exposure of victims to the event. There are three primary types of controls that can be used against a chemical or biological threat: (a) Engineering Controls - Controls that reduce risks by the engineering of

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system design, the selection (or substitution) of materials that minimize or preclude exposure to an agent (when technically or economically feasible), (b) Administrative Controls - Controls that reduce risks through specific administrative actions, and (when appropriate) (c) Protective Controls - the use of protective equipment and systems that would create barriers between personnel and the threat(s) at hand. Engineering and Administrative controls (a and b) are preventive controls, designed to preclude exposure to attack. Protective controls (c) would be implemented should an event actually occur, and would be designed to limit exposure to the threat. Section 2.3.1 discusses these controls in detail. There exist a number of areas of concern in which controls against a bioterror event wouldhave to be implemented [23]. These include: • Alerting and notification • Containment • Mass prophylaxis • Facility and/or medical personnel protection • Decontamination • Triage • Treatment • Mass Fatality Management • Psychological Issues • Legal and Forensic Issues In order to make effective control decisions, decision-makers must first start with the most serious risk first and select controls that will reduce the probability of event-related losses occurring or the severity of loss should the event never-the-less occur. Once these controls have been selected and are implemented, the planner is then able to balance the costs and benefits of using a particular control. Should the risks associated with a certain control outweigh its benefits, then the control is eliminated and another set in place. The most critical aspect of this step is that decision-makers must ensure as best as possible that the controls selected can indeed address the threat(s) noted from previous steps in the process. Thus, decision-makers should be flexible enough to accept and reject controls as necessary, especially once an attack has occurred. It is also imperative that as decisions about the appropriate controls are made, the decision-makers must also consider the level of authority associated with implementation of said controls; they must ensure that high-risk control decisions are made at the right level.

2.3.1. Tools to Minimize Risk from Threats In making these control decisions, decision-makers must be certain that the decisions they make will, in the end, mitigate the effects of those threats that have been defined and assessed as real or perceived. To do this, decision-makers have a wealth of tools and skills that they can employ to facilitate the control decision-making step. Let us assume that Hospital X lies in a rural medium-sized town, but is the primary health care facility for an entire region. The hospital is located on a large open campus, and consists of a large main steel, glass, and adobe building in which are housed all surgeries, patient rooms, clinics, laboratories, and management offices. Staff and support facilities are housed in modern, adobe and glass dormitory-like structures adjacent to the main building. At

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any given time, there are at least 200 patients and staff within the facility. Hospital security has been slightly increased since the terrorist attack of 11 September 2001, and consists primarily of civilian security staff on 24-hour patrol. There is no defined fixed perimeter around the facility and vehicles are allowed to come with a few feet of the main structure. Medical intelligence information and recent events suggest that there is a moderate to high probability that some type of bio-terrorism event is being planned against a civilian target, and that the target may be a major rural health care facility. In response to this information, Hospital X decision-makers have already conducted ORM steps one and two, and have determined that their there is a high probability that their facility could be a target. Based on intelligence data, they have also determined that there are several possible pathogens that could be introduced into the hospital environment. At the top of their list is 'N', a highly deadly agent that can be easily transported, anonymously introduced into the hospital's environment, and for which there remains no treatment. Decision-makers now must decide on controls that, if put into place, can either prevent an attack of 'N' from occurring or, should an event occur, minimize the effects of 'N' on the Hospital and it's environs. Decision-makers used the following tools to help them decide on the best controls to employ: •

• •

•

•

•

Training/Education. All hospital staff could be briefed on the situation, noting the nature and effects of 'N'. They could be trained to recognize the symptoms of 'N', both in early and advanced stages. Staff could also be instructed regarding what procedures and precautions to take should they suspect the presence of 'N'. Limit Exposure. A 'mishap plan' should be in place whereby patients and staff will receive minimum exposure to 'N' should its presence indeed be detected. Technology. There may be different technologies that could be used to prevent or minimize the effects of 'N'. These could include the design and use of isolation systems whereby airtight doors would separate all hospital spaces and room environmental systems could be modified to isolate and/or cleanse contaminated air. The training in and use of personal protective gear could be employed to minimize exposure and spread of 'N'. Hardware designed to detect 'N' and related agents could be installed. A suitable caution/warning system could be slaved to the sensors thereby providing real-time alerts to the presence of such threats. Space isolation security systems could also be operated in tandem with the security detection network. Hospital staff could wear small personal agent detectors. Security. Hospital security could adopt procedures and practices to minimize the opportunity for a terrorist to introduce 'N' into the hospital environment. This may involve the erection of physical security barriers and monitoring systems around the campus and within all facilities. Security staff would have to be trained to recognize and respond to a terrorist attack. Intelligence. Medical and non-medical intelligence information both play invaluable roles in deciding what controls to implement. This information is volatile and changes frequently. Therefore, the hospital should have an 'Intelligence' staff that would monitor, gather, and interpret intelligence information as frequently as deemed appropriate for the situation at hand. Logistics and Resources. Hospital supply officers should ensure that there are sufficient financial and operating resources available to support efforts to prevent an

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attack using 'N'. They should also facilitate the development and/or acquisition of resources that, should 'N' be detected, could promote rapid and effective response to the event. Written Policies and Standard Operating Procedures (SOPs). It is most critical that all of the procedures and duties and responsibilities of hospital staff be described and promulgated both in writing and during frequent training. These policies and procedures (including the use of ORM) should be maintained as part of a 'PreMishap Plan'; a document outlining all of the information required to respond to an event. Hospital decision-makers and management should make these policies and SOPs available at any time to all staff. They should be maintained and updated as required. If training and awareness are effective, a Pre-Mishap Plan should provide excellent direction to all event responders.

2.4. Implement Threat (Risk) Controls Once the control of set of controls have been chosen, they must then be implemented and monitored for their effect(s) against the threat(s). Effective and enduring application of controls to a given threat requires that a control 'infrastructure' be created and maintained by the highest levels of organizational authority. This control infrastructure is built upon three primary foundations: (a) make the case for implementation clear, (b) establishing a chain of accountability in the use of said control, and (c) ensuring that as long as the control is 'in place', it will receive full support from all levels of authority.

2.4.1. Make the case for implementation clear It is most critical in this step to ensure that those who are implementing and/or are affected by the control fully understand what the control is what the costs and benefits of using it may be. Indeed, it is easy to incorrectly assume that just because decision-makers understand the use of a particular control, that all who are affected by that same control will be equally clear about why and how it is to be employed. This does not mean that all who influence or are affected by a given control must understand the rational behind the decision for it's use; rather, it is imperative that those who use or are affected by controls understand in a clear and concise manner how they are to be used, what positive and negative outcomes may result. For example, those receiving injections against smallpox should be made aware in the clearest terms that while there is a very small chance that they will contract rather than avoid smallpox, the chances are far greater that the injection will help them survive should they face the disease. Clearly, if the likelihood of being exposed to smallpox is sufficient enough to cause alarm, and if the rationale for it's use is couched in this manner, the benefits of having the injection will, in most cases, outweigh the costs.

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2.4.2. Establishing a chain of accountability in the use of said control Those who use or are affected by the use of a control should be aware of to whom in their chain of command or authority they should report regarding the positive and negative results of control implementation. Depending on where in the chain of authority that person lies, he or she could then make a decision on whether or not to continue to use the control, withdraw it, or modify the way it has been implemented. If that individual is, by virtue of his/her authority in the chain, unable to make and implement those decisions, accountability then moves laterally or vertically in the chain. It is most critical, however, that the chain of accountability be clearly promulgated throughout the affected organization.

2.4.3. Provide support to control use With the decision to accept the use of a control, one also assumes the responsibility to provide those resources required to implement and maintain a control. For example, if decision-makers decide to build a protective stone wall barrier about the perimeter of Hospital X, then those responsible for carrying out that task should be provided with the funds, equipment, and materials necessary to build and maintain that wall. Without that support, the control becomes ineffective. Support for a control should be withdrawn only if the control in question is found to be ineffective, or when the costs of using the control outweigh the benefits. Resources could then be redirected to another possibly more viable control.

2.5. Supervise-Monitor the Effect(s) of Control Implementation Decision-makers must be wary of allowing complacency to affect their performance. Once a control or set of controls is in place, a planner may feel that 'this problem is solved..now to move on to the next'. Such an attitude can prove dangerous, as the use of a given control may fail to prevent or reduce exposure to a threat. Indeed, it is possible that the use of a control might increase the probability of an event. For example, the perimeter wall around Hospital X may have provided 'cover' for approaching terrorists. Had the wall not been there, the terrorists would have had to cross a highly exposed open area, thereby making them more vulnerable to detection. Thus, once controls have been implemented and are in use, it remains necessary for decision-makers to monitor the results of control use. In this way, decision-makers are able, if necessary, to remove, replace, or modify a control. Decision-makers must therefore ensure that controls are effective and in place and be aware of any changes to threat priorities and probabilities (as described earlier) that may necessitate performing the entire 5-step process again. This could result in the re-evaluation of the original control decisions that, in turn, could lead to the addition, withdrawal, or alteration of controls already in place. Indeed, if the result of this re-evaluation is that controls are not effective, or if changes to the nature and scope of threats become apparent, it will be necessary to repeat the entire 5-step ORM process, looking for new hazards. Although it is the final step in the ORM process, supervision is not relegated just to the monitoring of controls. Rather, each of the steps in ORM must be supervised by a credible, knowledgeable authority of the ORM process. As can be seen in Figure 8, supervisory oversight is recommended at all steps, from threat (hazard) identification through the implementation of controls. The authority who oversees the process may or may not be the same authority who monitors the controls.

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Figure 8. The Risk Management Paradigm. Decision-makers must supervise each of the steps in the ORM process.

In some cases, a planner lower in the authority chain may be the trained ORM 'expert'; the person who actually manages ORM training and use for a system or in an organization. That person may not, however, be accountable for making and implementing control decisions. In the end, having an ORM 'expert' oversee the process (and not the controls) as it evolves can only benefit the organization facing one or more threats, as this form of oversight could minimize any bias-effects that could result in an otherwise effective control being rejected (or an ineffective or harmful control being implemented).As we have seen, ORM is a 5-step process by which threats are first identified and then are assessed for the risk(s) they pose. Decision-makers next must make and implement control decisions which will result in the mitigation of these threats. Finally, decision-makers must continually supervise the process to ensure that the controls are 'functioning' as planned.

3. The Three 'Levels' of ORM A final factor to consider is the amount of time an organization has to identify the decisionmakers and have them perform the 5-steps described here. If a terrorist event is imminent, decision-makers may have to establish and monitor controls in a very short time. However, in other cases, when an activity is being planned for the future, planner may have the luxury of time to perform a more detailed ORM assessment of threats. This 'temporal' element in the process defines three 'levels' or ORM, and these are considered next.

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3.1. Time-Critical ORM You have been informed that a number of individuals working at a nearby factory have all suddenly started displaying the same symptoms within a few days of each other: skin infections, most of which were painless ulcers approximately 1-3 cm in diameter, with a dark area or necrotic skin at the center. Medical examinations have revealed swollen lymph glands in most of the cases. From recent medical intelligence information about bioterror events involving anthrax, you consider the possibility that the symptoms described may be a result of cutaneous anthrax. You have not heard any reports of anyone suffering from similar symptoms at your facility. Still, the proximity of the factory where these symptoms were reported suggests that your facility may also be vulnerable. You and the decision-makers at your facility do not have a great deal of time to go through the ORM process. If your facility management had not done so already, they would now have to perform a quick, rudimentary risk assessment known as 'time-critical' ORM. This assessment would involve having a short as-you-go review of the five steps. That is, decisionmakers would have to make immediate decisions (short-fuse planning) based on a cursory version of the process during the execution phase of operations/training and for crisis response planning. This type of ORM can also be referred to as 'intuitive' ORM, for it often involves weighing risks and executing immediate control decisions. Time-critical ORM is often employed when one is faced with an unexpected threat or set of threats. This, time-critical ORM can be high in risk, as there is minimal time to garner details about the threats, and controls may be implemented without fully knowing if they can be supported or even effective. Still, the same 5 steps are carried out, and monitored. If a threat is not mitigated by one control, another may have to be immediately substituted. Again, without the benefit of time, 'snap' decisions may reflect inadequate or faulty information about threat severity or probability. In some cases, there may not be sufficient time to modify or substitute controls. This may, in turn, prevent the event from occurring, and it may even preclude effective response.

3.2. Deliberate ORM You have been informed that in seven days, your Battalion Aid Station will be moved forward in support of a combat element. Although not at the center of the area of operations, you realize that you will be facing an enemy that has been known to use chemical and biological weapons. You and your staff now have some time to perform a 'deliberate' ORM assessment of this move; that is, you are able to perform a more detailed threat analysis. Deliberate ORM employs the same 5 steps discussed above, but having more time to assess threats allows decision-makers to better understand the nature of the threats, and as such can better choose those controls that are more likely to either prevent or minimize the effects of an event. This process level is used when there is a good understanding of the issues based on experience.

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3.3. In-Depth ORM There has been a change of plans; your Battalion Aid Station is not due to move forward for three months. Now you and your decision-makers have even more time to perform an ORM analysis. In this case, you will probably elect to carry out an 'in-depth' ORM analysis. As with time-critical and deliberate ORM, in-depth ORM uses the five steps described earlier. However, as more time is available for planning, in-depth ORM stresses the first two steps of the process; that is, decision-makers spend a greater amount of time researching possible threats for their severity and probability of occurring. Further, a more in-depth analysis of target vulnerabilities can be made using the results of tools such as scenario modeling, testing, research and intelligence data. The application of these tools is especially critical when threats are not easily identifiable or well understood.

4.

Conclusions

At a time when there is an increase in the likelihood that terrorists wielding chemical and biological weapons and will use them against both civilian and military targets, it becomes imperative to make comprehensive preparations against such an eventuality. This operational readiness against threats can help ensure the safety and continued operation of any system or organization. The ORM schema presented here provides decision-makers with a standardized approach to confronting possible or real threats and the risks associated, in particular, with weapons of mass destruction. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]

Moser, R., Whilte, G.L. and Lewis-Younger, C., abd Garrett, L.C. (2001). Preparing for expected bioterrorism attacks. Military Medicine, 166:5, 369-374. OPNAV Instruction 3400.1 0F: Chemical, biological and radiolgical (CBR) defense requirements supporting operational fleet readiness. Washington, D.C.:Department of the Navy (1996). Lunch, W. (2002). Bioterrorism: Then and now. NRA News, (Jan.). Loy, J.M. and Ross, R.G. (2001). Defending the homeland. Government Executive: Daily Federal. (10/29). Rose, S. (1989). The coming explosion of silent weapons. Naval War College Review. (Summer) 339354. Gander, T.J. (1992). Chemical warfare today. Jane's Intelligence Review , (6), 284-287. Bellenkes, A.H. (2002). The Human Factor in Homeland Defense. NPS Research, 6, 16. Bellenkes, A.H. (2002), Operational Risk Management as a Weapon for Homeland Defense. Proceedings of the 73rd Annual Congress of the Aerospace Medical Association, Montreal, Canada. OPNAV Instruction 3500.39/Marine Corps Order 3500.27: Introduction to Operational Risk Management. Washington, D.C. Department of the Navy (1997). Bellenkes, A.H. (2000), Operational risk management as an international standard in aviation safety. Proceedings of the 48th International Congress of Aviation and Space Medicine. Rio de Janeiro, Brazil. U.S. Army Safety Center (2002). Risk Management: Philosophy. Ft. Rucker, Alabama. http://safety.army.mil/pages/rm/phil.html. Medical Operational Risk Management (ORM) in: Marine Forces Reserve HM/DT Sustainment/Enhamcement Training (SET) Program. Washington, D.C.: Headquarters, United States Marine Corps (2002). USAF Instruction AFP 91-214: Operational Risk Management and Execution. Washington, D.C.: Department of the Air Force. USAF Operations in a Chemical and Biological (CB) Warfare Environment, Planning and Analysis. Air Force Handbook 32-4014 (1), Washington, D.C.: Department of the Air Force (1998). Bellenkes. A.H. (2000). Operational risk management training for naval aeromedical personnel. Presented at the Meeting of the Navy Aerospace Medicine Strategic Planning Group, Houston, Texas.

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[16] [17] [18] [19] [20] [21 ] [22] [23]

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Reason, J. (1999). Managing the Risks of Organizational Accidents. Aldershot, U.K.: Ashgate Publishing Ltd. Reason, J. (1990). Human Error. New York: Cambridge University Press. Embry, D., Kontogiannis, T., and Green, M. (1994). Guidelines for Preventing Human Error in Process Safety. New . New York: American Institute of Chemical Engineers. Food Safety and Security: Operational Risk Management Systems Approach. Washington, D.C.: U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition (2001). Decker, R.J. (2001). Homeland Security: Key Elements of a Risk Management Approach. Washington, D.C.: United States General Accounting Office, GAO-02-150T. Decker, R. J. (2001). Homeland Security: A Risk Management Approach can Guide Preparedness Efforts. Washington, D.C.: United States General Accounting Office, GAO-02-208T. An excellent overview of risk management tools is available on the Naval Safety Center ORM website at http://www.navalsafetycenter. navy.mil. Jagminas, L., Marcozzi, D.E., and Mothershead, J.L. (2001). CBRNE-Biological Warfare Mass Casualty Management, http://www.emedicine.com/emerg/topic896.htm.

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Crisis Management: Expediting Information and Resource Flow via the Global Incident Analysis and Alerting System (GIAAS) for CBRN Richard PRICE (1), John WOODALL(2), Sergey NETESOV(3) (1) Applied Science & Analysis, ASA, PO Box 1144, Aberdeen, Maryland USA 21001; e-mail: [email protected] (2) Universidade Federal do Rio de Janeiro, Instituto de Ciencias Biomedicas, 21941-590 Rio de Janeiro, RJ, Brazil; e-mail: [email protected] (3) State Research Center of Virology and Biotechnology, VECTOR, Koltsovo, Novosibirsk Region, 630559 Russia; e-mail: [email protected]

1. Introduction Today our available systems for chemical, biological, radiological (CBR) crisis management are convoluted and fragmented. There is no single system to provide all information needed to rapidly assess each situation and help bring a CBR crisis under control. Where would one go to determine needed actions to minimize effects of an outbreak and to avoid miscalculations and an escalation to armed conflict; to find and understand the region's geopolitical situation; the suspected movement and capabilities of known terrorist organizations and individuals within the region; the intelligence and interpol type organizations within the area; for identification of best laboratories for specific reported outbreak; whether or not the reported disease is endemic to the area and for having a thorough knowledge of the disease within a region; for available information on designated quick reaction medical and operational teams and agricultural specialists specific to each type of crisis; for available pharmaceutical stockpiles across a region and constraints to use of those stockpiles; for airlift and air traffic control specialists; etc.? Today - there is no single organization or geographical location to answer all stated questions. The outbreak of Rift Valley Fever (RVF) in Yemen and Saudi Arabia is an example of a crisis management situation caused by a natural medical disaster that could have had serious international consequences. The WHO provided a press release on 29 September 2000 saying they and others were working the outbreak. This was 3.5 weeks after ProMED Mail had reported the first information on the outbreak. What the press release did not address were the multitude of questions that, in a more heightened tension arena, must be answered, immediately, to help preclude miscalculations and avoid possible conflict. These questions which were addressed by ASA to the CBMTS family of professionals on 5 October 2000 included, in part: 1. Was RVF considered endemic to the area or have there been outbreaks in the past? The answer: No. This was first outbreak of RVF outside of Africa.

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2. Are morbidity and mortality characteristics consistent with previous outbreaks? Typical mortality rate for RVF is 1%, according to CDC. Reports from Saudi Arabia and Yemen indicate mortality rate of from 10% to 20% for this outbreak. This would be a red flag for the Threat Analysis / Assessment and Intelligence Analysis groups - if they were available. 3. Has RVF been considered a BW agent? Yes, it is on the proposed BTWC lists. 4. Have there been problems between the countries? Yes, border and economic issues. 5. Would either country have the internal capability to develop, manufacture or acquire a sufficient amount of RFV agent for dissemination across border? Yes. 6. Did either country acquire RFV cultures from known BW agent culture dissemination sources, such as American Type Culture Collection? Unknown. 7. Within the region, which countries may have acquired RFV for experimental work and who may have supplied RFV cultures to them? Suppliers - many. 8. Terrorism: what individuals, non-government organizations, and state sponsored organizations, known to have expertise in chemical and biological terrorism (CBT), are known to have an interest in or actually have operatives in the region? Unfortunately, intelligence information is not shared. To help overcome these and other deficiencies, we propose developing an independent system, the Global Incident Analysis and Alerting System (GIAAS), to provide immediate information and analysis as well as alerting functions to those responsible for readiness and response to major CBR incidents, whether these incidents are natural or man-made.

2. Proposed GIAAS System We would use 12+ Incident Analysis and Alerting Centers (IAACs), each responsible for a specific geographical area and receiving/analyzing their area data from Regional Alert and Information Centers (RAICs), located within a specific region within a geographical area. RAICs would receive data from the local Area Information Centers (AICs), the focal points for individual reports within a portion of a region. IAAC shift or duty teams would be international and would include specialists across the medical spectrum, pharmaceutical stockpile programs, airlift/air traffic control, intelligence, information, Interpol, plus others as identified. Parallel to, but reporting through the lAACs are the International Centers for Disease Research, Analysis and Reporting (ICDRAR), an interconnected series of internationally recognized Laboratories established to provide a prime laboratory focal point for each specific region. Each ICDRAR will also have a dual responsibility for a specific portion of emerging and re-emerging disease spectrum. Each ICDRAR accepts information flow of significance to their laboratories' designated expertise and/or area of responsibility. The ICDRARs rapidly digest data and provide initial analysis to the GIAAS via their IAAC. The initial analyses by the AICs, RAICs and IAAC are enhanced with additional data requested by the concerned ICDRAR. The area IAAC is expected to know the available, required pharmaceutical stockpile items plus other rapid response supplies and equipments and designated response teams. An example of a decision mechanism and communications at the IAAC/ICDRAR levels would be that raw reports coming in through the RAICs/AICs would be automatically filtered against an A List of agents of concern, along with all outbreaks of unidentified etiology. Duplicate reports would be identified based on the identity of numbers of cases/deaths and location, and

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removed. The resulting product would then be sent to non-government infectious disease or toxin specialists for evaluation. Thus, for example, a report coming in containing the keyword "anthrax" would immediately be routed to the designated anthrax specialist on duty (of course, some specialists could cover more than one disease agent or toxin). In the case of a zoonosis, it would be necessary to have the report seen by a public health veterinarian. The on-duty team (shift) members would add their preliminary evaluation to each report, at the same time consulting by the fastest possible means with any other specialist they consider might have useful advice. They would also give a preliminary hazard rating, using a system such as the RED-YELLOW-GREEN or the 1-3 or 1-5 star system, of the danger of the outbreak spreading. This would be updated in the light of further incoming reports. The actual evaluation of whether the outbreak is likely due to bio- or agro-terrorism would be done by another group who scans the evaluated reports, composed of Interpol and intelligence community specialists. An Example of an IAAC candidate: Singapore's DSO with its excellent facilities, professional staff, and communications hub, could function as the IAAC for South East Pacific area with RAICs in Australia, Indonesia, Malaysia and Thailand. ICDRARs could be located in China, Australia and Japan. VECTOR in Novosibirsk might have an ICDRAR and IAAC colocated. CDC in Atlanta might also serve double functions. There are many possibilities. An Example of an ICDRAR Laboratory candidate: VECTOR, the State Research Center of Virology and Biotechnology, Koltsovo, Novosibirsk Region, Russia which was established in 1974 to conduct basic and applied research on extremely pathogenic viral agents such as Marburg, Ebola, Lassa and other viruses related to potential BW agents and to evaluate the potential threat posed by these agents. VECTOR is near the geographical center of Russia and 900 km from Mongolia and China. VECTOR is well suited for effective collection of viral/bacterial strains and establishing, using specific diagnostic procedures for study/analysis of specimens from Asian Russia, Central Asia FSU republics, Mongolia, neighboring countries. VECTOR consists of six scientific research institutes with 1200 of its professional staff either directly involved in or in support of scientific research. This staff includes specialists in genetic engineering, molecular biology, epidemiology, immunology, virology, theoretical virology, ecology with experience in highly dangerous viruses research and production of diagnostic/prophylactic preparations for public health/veterinary requirements. VECTOR'S Collection of Cultures of Microorganisms comprises over 10,000 deposit entries including various viral strains (such as the national collection of variola virus strains and strains of viral BSL-4 pathogens). This Collection is affiliated with the European Culture Collection Organization (ECCO). VECTOR is a WHO Collaboration Center for orthopoxviruses diagnosis and is a repository for variola virus strains and DNA. VECTOR also has maximum biological containment laboratory facilities available. VECTOR'S Disease Spectrum Responsibilities could include but not be limited to: Arboviruses such as West Nile virus and tick-borne encephalitis virus which is endemic in Russia; the Crimean-Congo hemorrhagic fever virus; and Omsk hemorrhagic fever virus which are endemic in Siberia and/or in European Russia. Also Marburg and Ebola filoviruses and Orthopoxviruses:smallpox virus, monkeypox, cowpox and other poxviruses and bacteria and parasites, as specified.

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3. GIAAS Communications: Reporting and analysis systems Basic to our premise of a fast, reliable, responsive system is that no communication from any GIAAS level is ignored and all communications flow uninterrupted. The uninterrupted flow is considered 'raw data' until picked up by an IAAC. Each reporting level within the GIAAS is responsible for the rapid and best analysis possible with the assumption that the local area has the best knowledge of who the players are in the area; whether or not the outbreak appears to be endemic to area; whether the incident appears to be natural or man made, deliberate or an accident. We expect the GIAAS will need access to the following restricted networks: • the WHO Outbreak Verification List (OVL), which collects reports from GPHIN (see below) and its offices covering 192 countries worldwide. Currently restricted to ministries of health, WHO offices & Collaborating Centers; • the Global Public Health Intelligence Network (GPHIN), operated by Health Canada, which scans current media outbreak reports on the Internet several times a day. Restricted to Health Canada and WHO/HQ, Geneva; • the Global Emerging Infections System (GEIS), operated by the US military; • the Epi-X, operated by CDC. Restricted to state and local public health epidemiologists and laboratories in the USA. The GIAAS will also subscribe to these public networks: • ProMED-mail, the independent, free, network with more than 27,000 subscribers in over 155 countries worldwide. • MITRE Text and Audio Processing (MiTAP, a prototype system available for monitoring infectious disease outbreaks and other global events. MiTAP focuses on providing timely, multi-lingual, global information access to medical experts and individuals involved in humanitarian assistance and relief work. MiTAP currently [October 2002] stores over one million articles and processes an additional 2,000 to 10,000 daily. This is a free internet-based service to the public health community. The GIAAS would also share databases with any future BTWC, as well as the in-place OPCW group, and the WHO, CDC and other organizations with similar missions. An Example: ProMED as a Communications model has been selected to be the model for the GIAAS' requirement for a fast, reliable, responsive system. ProMED often reports outbreak data well ahead of reports of outbreak by official sources, WHO or CDC. Examples include plague in India on 15 February 2002 followed by WHO on 1 March; yellow fever in Bolivia on 23 March 2002 which had not yet been officially notified to WHO by 31 June, and the RVF outbreak in Saudi Arabia and Yemen which was reported on by ProMED as early as 4 September 2000, well ahead of others. ASA detailed this outbreak because of bioterrorism implications and this outbreak specifically provided the impetus to pursue this paper. Other examples: October 2001, WHO published in its weekly Epidemiological Record (WER) and its Outbreak News page on the Internet, a report of the case on anthrax in a Florida USA magazine photographer. This was first report by WHO of anthrax in 2001. Yet at least 13 outbreaks of anthrax in humans in 11 countries, including the USA, were reported in the news media in the preceding 12 months. In the 12 month period ending 31 March 2002, human cases of botulism (South Africa, UK, USA), cowpox (Ukraine), monkeypox (Congo), plague (Brazil,

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Congo, India, Kazakhstan, Mongolia, Uganda, USA) and tularemia (Kosovo and USA) were reported in the media. Only the outbreak of plague in India was reported by the WHO. The World Animal Health Organization, OIE, reports outbreaks in livestock. Reports on its List A diseases are posted on its website when received. But anthrax is an OIE B List disease, it is only reportable annually. OIE does not cover cowpox, monkeypox, or plague. There is no official world combined list of on-going outbreaks of diseases in humans and animals caused by agents and toxins which are also on the bioterrorism/agroterrorism list. However, there is an unofficial one, which also reports infectious diseases of crop plants, thus covering all of the bases. ProMED posted ALL of the disease outbreaks mentioned above in its daily e-mailings to its over 27,000 subscribers. Their cross-agency, interstate, international membership is unique and not matched by any official system. Very importantly, ProMED maintains over 20,000 archived reports and comments which can be searched by keyword. For example, anyone hearing of a case of anthrax in Kazakhstan can immediately access the ProMED data base to verify that anthrax has frequently been reported there, is endemic, and occurs at predictable times of year. ProMED also has a roster of moderators that include infectious disease specialists, virologists, bacteriologists, parasitologists, epidemiologists, veterinarians and plant pathologists. All reports are screened by the moderators before posting. When in doubt the moderators try to obtain further information from the source or country concerned. If this is not possible, ProMED will post the current report while requesting additional information. 4. Three phase approach for GIAAS development/ implementation All phases of development of GIAAS will be based on a thorough knowledge of the existing experience of different countries with their informational/analytical networks for disease reporting.

4.1. Phase I GIAAS Proof of Concept. For each geographical area an IAAC will be identified. Initially, if a specific identified geographical location can not be established in a timely manner, an internet nodal point will be constructed to serve as an interim IAAC for the area. The IAAC for each area will assist the GIAAS in identifying and securing agreements with a representative number of RAICs and AICs within each region. We recognize that the Centers will initially be part of worldwide network on a voluntary basis to establish Proof of Concept while providing a much needed capability; however, these Centers must eventually be established via intergovernmental agreement and funding (not later than Phase III). For Proof of Concept the GIAAS in coordination with all GIAAS entities, will develop and test specific scenarios in command post type exercises (CPXs). From these numerous tests of the system the GIAAS will develop uniform reporting, requests, analysis, and decision making procedures.

4.2. Phase II GIAAS Interim Implementation and partial funding. The GIAAS will Incorporate agreed upon changes as a result of observations and recommendations from the CPX's. During Phase II, the

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IAAC/RAIC/IAC layout will be finalized and the system will be thoroughly tested from the individual reporter to the AIC and through the RAIC to the IAAC. Additional and current communication and reporting systems, i.e., disease reporting, crisis management, communications into and out of the GIAAS will be incorporated. During this phase the GIAAS will be continually updated based on knowledge gained via CPXs and recommendations from the field. The GIAAS system will be implement at least to the communication nodal points in each area, and organized per the ProMed model until funding is achieved. In preparing for Phase III GIAAS full implementation the GIAAS will document and package GIAAS requirements and proof of concept along with system testing results. During this phase, a cadre of professionals will be identified to work in the system to gather, analyze and disseminate information on a rapid basis. Initially this may be done via internet only and from Labs and other Centers who volunteer their resources until funding sources are identified. 4.3. Phase III GIAAS System Implementation and full funding. The GIAAS will be prepared to present documentation and proof of concept to the United Nations with simultaneous transmissions to concerned government entities such as public health authorities, organizations such as CDC, and independent agencies such as Interpol, ICAO, WHO, OIE, FAO, etc. A worldwide symposium will be called to finalize details of the GIAAS system and to request support both for funding and for professionals who would assist with the GIAAS operations both administratively and in the 7/24 operations of the system. The GIAAS will continue to develop and train a cadre of professionals (volunteers) who would work and test the system continuously. At an agreed time, the system becomes operational. Plans for the system will include operations both with and without official support and funding. 5. Conclusion We have presented a three phase approach to building and operating a global network to quickly and reliably assess outbreaks and epidemics. These assessments will include political realities and evaluations of natural and man-made outbreaks as well as other natural and manmade disasters across the CBR spectrum. The GIAAS will remain dynamic throughout the building of this much needed system and it will continually evolve as requirements and capabilities dictate.

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Civilian Relief after Release of Weapons of Mass Destruction: Need for a New Task Force 'Scorpio' Jack WOOD ALL* Director, Nucleus for Investigating Emerging Infectious Diseases, Dept. Medical Biochemistry, Federal University of Rio de Janeiro, Brazil. As the theater hostage crisis in Moscow showed, you can't effectively treat gas victims without knowing what the gas is. What if the Bali nightclub bomb had been a chemical weapon — who could have identified the chemical agents? The same problem on an even larger scale will face humanitarian agencies responding to the civilian crisis if weapons of mass destruction are used in the Gulf area, or if stocks of them are destroyed by sabotage or aerial or ground bombardment. In either case there is likely to be a messy outcome in which chemicals (gases) and biologicals may be mixed together, possibly with the addition of radiation from 'dirty' bombs, implicating maybe thousands of civilians in addition to military. The military can be expected to take care of their own, but may well find themselves unable to cope with a mass of civilian casualties. NGOs such as the International Red Cross and Red Crescent will be anxious to help, but will be extremely concerned about sending their personnel on what might be a suicide mission into a contaminated area. Consider the scenario of a UAV (unmanned aerial vehicle) armed with a spray tank of toxin, hit by a missile over a border area in the Middle East, contaminating hundreds of local residents. Who could or would identify the toxin? In 1990, shortly before the Gulf War began, the problem was addressed by Swiss Disaster Relief in collaboration with the World Health Organization (WHO). SDR has extensive experience in taking relief to disaster areas worldwide. The standard configuration of SDR comprises physicians, public health specialists, veterinarians, specialists in nuclear and chemical weapons and explosives, together with backup staff for decontamination, communications, logistics and even flight control. The unit, about 20 strong, had access to a MD-80 aircraft with Red Cross markings. When on maximum alert, the members could be fetched by military helicopter from wherever in Switzerland they might be, and could take off for anywhere in the world at 24 hours notice. In order to upgrade the unit for dealing with biological weapons, specialists in anthrax, botulinum toxin and hemorrhagic fever viruses were recruited, together with their portable detection equipment. Team members were vaccinated against, among many other diseases, smallpox (planned but not actually implemented), anthrax (3 doses at 2 week intervals), tularemia (1 dose) and Venezuelan equine encephalitis virus (1 dose), and assured of a supply of botulin antiserum. It later transpired that the team was more fully vaccinated than the coalition forces. They trained together in personal protective suits. The resulting group was christened Task Force 'Scorpio' [1]. Its mission — as a neutral, internationally acceptable team -- was to fly immediately to wherever weapons of mass

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destruction might have been released and have affected the civilian population, measure the level of contamination, and tell humanitarian organizations what the contamination was caused by and when it would be safe to send in relief. The Secretary-General of the United Nations was the authority that would have dispatched 'Scorpio'. Fortunately, its services were never needed. After the Gulf war, 'Scorpio' was disbanded. Over the intervening years, the 'Scorpio' model has been discussed in the context of instituting regional teams based around the world, able to reach any corner of the earth rapidly, and with the appropriate language capabilities. For example, a Latin America-based team with Spanish speaking personnel would reach Central or South America faster than a team from Switzerland, and without jet lag. An Arabic-speaking team would likely be more acceptable and useful in the Middle East. WHO has studied the question, but has decided to rely on their roster of more than 100 experts worldwide in nuclear, biological and chemical (NBC) threats. However, these experts are not vaccinated against all the appropriate agents, have not trained together in protective suits, do not have decontamination and logistic backup, and could not be deployed in 24 hours. They will be appropriate for accompanying humanitarian relief teams to refugee areas, but not for rapid, on-the-spot identification of the type and level of contamination. Since 1991, the military in many countries have upgraded their NBC detection and response capabilities, but little has been done for civilians. However, in 1998, the EuroAtlantic Disaster Response Coordination Centre (EADRCC) was established at NATO HQ in Brussels, Belgium [2]. Its work is carried out in close cooperation with the United Nations Office for the Coordination of Humanitarian Affairs (UN-OCHA). It has created a nonstanding Euro-Atlantic Disaster Response Unit (EADRU), composed of appropriate specialists who are on call for disasters, specifically affecting civilians. Subsequent to the events of September 2001 in the USA, the EADRCC has been tasked by the Euro-Atlantic Partnership Council (EAPC) to coordinate, upon request of the stricken nation, international assistance from the 46 EAPC countries to help deal with the consequences of terrorist attacks in the same way as it does in the case of natural and technological disasters. But assistance to countries outside the EAPC is dependent on political clearance, which could take time. In 2002 Australia inaugurated its Incident Response Regiment, with 300 soldiers and 10 civilian specialists capable of responding to a NBC event affecting Australians at home and anywhere in the world [3]. Canada has a NBC response team headquartered at CFB (Canadian Forces Base) Borden (central Ontario) able to reach any point in Canada within 12 hours [4]. A question that remains to be resolved is whether appropriate specialists from such task forces would be able to remain on site after the initial determination of the type and extent of contamination, to monitor, for example, changes in wind direction bringing further contamination, or whether there was a mixed CBW release in which the symptoms of the biological agent might not become apparent for some days. If 'Scorpio' could be reconstituted today (March 2003), there might yet be enough time to boost the team's vaccinations and get it ready to meet the potentially dire need of civilians in the Gulf region or elsewhere. As it. appears that Switzerland is not prepared to provide the service this time, perhaps a NATO member state could step forward to fill the gap.

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References [1] [2] [3] [4]

Steffen R, Melling J, Woodall JP, Rollin PE, Lang RH, Liithy R, Waldvogel A. Preparation for emergency relief after biological warfare. J Infect. 1997;34:127-132. NATO. Euro-Atlantic Disaster Response Coordination Centre www.nato.int/eadrcc Accessed 13 Mar 2003. Dept. of Defence, Australia. Incident Response regiment (IRR) www.defense.gov.au/terrorism/response.html Accessed 13 Mar 2003. Richard Kokoski 1999 (pers. comm.)

*Leader of the World Health Organization's delegation to the Biological Weapons Convention 3rd Review Conference.

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Consequence Management of a Bioterrorist Incident (CMBI) J. Theodore AHRENS Headquarters, U.S. European Command Northrop Grumman Information Technology Abstract. The CMBI project is analyzing the required crisis response and consequence management processes to respond to a bioterrorism incident and recommend steps to improve U.S. European Command (USEUCOM) response plans and procedures. The initial step has analyzed the potential magnitude of the medical and health service response requirements. This paper describes the threat-based methodology developed and presents emerging casualty and fatality results.

1. Background "We saw terrorism escalate towards mass casualties. That reflects the fact that the motives of most of these terrorist groups now are ideological, religious, messianic, apocalyptic. The goal is no longer self-restraint, but to kill as many people as possible. "[1] Participants at an August 2001 Consequence Management Symposium at the U.S. Army War College "agreed on the need for a comprehensive national strategy for territorial security ... the strategy would identify the roles and missions of the diverse agencies addressing different components ... identify the fault lines ... and fill identified gaps with procedures and resources necessary to meet the given threat."[2] Attendees identified the potential requirement for a surge capacity to respond to a terrorist incident. They emphasized the criticality of synchronizing the roles and missions of the diverse players. The U.S. European Command (USEUCOM), located in Stuttgart Germany, is the headquarters for all U.S. military forces in Europe. The area of responsibility (AOR) for USEUCOM encompasses 13 million square miles and includes 93 countries in Europe, parts of the Middle East, and most of North/Sub-Saharan Africa. The USEUCOM Special Assistant for Security Matters recognized that a terrorist incident within the Command could result in a surge of requirements for medical and health care resources, particularly considering the size of the Command. He asked the Judge Advocate to review existing bilateral agreements and Memorandums of Agreement which address response to a bioterrorist incident, focusing on "fault lines" where additional coordination with nations in the region is required. The Command Surgeon summarized the gravity of the potential bioterrorist threat, stating, "it is quite likely the threat of the future will be the simultaneous employment of multiple chemical and biological agents that are engineered to evade detection and negate vaccines and medicines."[3] U.S. forces must be prepared to respond to a bioterrorist incident directed against U.S. forces or national interests, on a U.S. installation/base, or vessel in this AOR and to assist, if requested, the response efforts of countries within the AOR. The Consequence Management of

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a Bioterrorist Incident (CMBI) project has three co-sponsors, the Special Assistant for Security Matters (response to terrorist incidents), the Judge Advocate (legal issues), and the Command Surgeon (medical preparedness). The purpose of the project is to analyze the consequence management processes to be invoked in response to a bioterrorism incident and recommend steps to improve the Command's response posture. This analysis will ultimately document planned Courses of Action (COAs), identify overlaps/shortfalls in response mechanisms, delineate the roles and responsibilities of the various U.S. and host-nation agencies involved, identify additional resource requirements, and provide alternatives for streamlining bioterrorist incident consequence management response plans and procedures. The current project focuses on U.S. installations in four countries - Germany, Italy, Turkey and the United Kingdom. These countries were chosen because the preponderance of U.S. forces is stationed there. A follow-on study will analyze deployed forces, U.S. personnel on host nation installations (e.g. embassies, liaison personnel), and the other countries in the AOR. This paper presents emerging results of the first step - analysis of the potential magnitude of the medical and health service requirements to respond to a bioterrorist incident affecting U.S. personnel.

2. Resource Sizing Scenario The first step in reviewing the robustness of consequence management processes is conducting a threat-risk assessment. The scenario chosen focuses on a worship and study conference held every October at a German hotel. This event was selected because of the predictability of the venue and because a terrorist group might very well target Americans, particularly Christians. Table 7 displays the number of people who recently attended that particular conference from countries within the USEUCOM AOR. It is anticipated that all attendees would be infected due to the length of the conference (five days) and the particular virulence/ infectivity of the pathogen selected. Table 7. Infected Personnel by Country Country Belgium Germany Israel Italy Netherlands Spain Turkey United Kingdom

# Attendees 36 261 1 25 17 2 4 70

Figure 9 displays the distribution of American attendees from cities in Germany, ranging from three attendees from Dexheim to 42 from Heidelberg. The release of a communicable pathogen, such as smallpox, could spread very rapidly. This analysis only focuses on Americans attending the conference; the analysis does not consider hotel staff or the sizeable number of non-conference attendees who may be staying at this hotel at the time of the terrorist attack (October is a peak month for walking through the hills and hiking through the woods which surround the hotel.

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Figure 9. Conference Attendees by City in Germany

The distribution of American attendees at the conference (attendees from U.S. installations in Germany are reflected in Figure 9) will be important for evaluation of the geographical adequacy of the installation medical/health care facilities and proximate host nation resources listed in the applicable bilateral agreements and Memorandums of Understanding. The distribution of attendees by German state is important legally because memorandums of agreement are specific to each individual German state. Table 8 displays the number of affected conferees from each state in Germany. Table 8. Affected U.S. Conferees by German State State BadenWiirttemberg Bavaria Hessen Rheinland-Pflaz

Conferees 72 57 81 51

3. Smallpox Epidemic smallpox reports date back to ancient Egypt and China. The age distribution of cases parallels the age distribution in the affected population. The disease is easily confused with influenza, meningitis or other infections, thereby potentially delaying recognition of a terrorist attack and identification of the pathogen involved. Historically, mortality rates have ranged from 30 to 50 percent of those affected. The only known reservoir is human. The World Health Organization conducted a massive eradication program from 1967 to 1977, finally declaring

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the disease eradicated in 1980. Two known repositories of the virus still exist - one in Russia and one in the U.S. The Soviet Union is reported to have conducted research into the weaponization of smallpox in spite of being signatory to the 1972 Biological Toxin Weapons Convention. Ken Alibek, a Soviet defector to the U.S., documented his efforts to weaponize the India-1 strain because it was "highly virulent and ...stable enough to retain its infectious qualities over time"[4]. That strain has a mortality factor of 50 percent. Alibek supervised the testing of the aerosolized smallpox weapon at Vector's explosive chambers in Kolsovo Siberia. Recently released Soviet documents link a 1971 smallpox epidemic in Aralsk Kazakhstan with the openair testing of smallpox at Vozrozhdeniye Island.[5] The Aralsk epidemic was characterized by a form of smallpox differentiated by an increased incidence of hemorrhagic symptoms and a higher fatality rate. In a November 2001 interview with the Moscow News, General Pyotr Burgasov, one of the leading Soviet bioweaponeers, stated, "anthrax isn't worth much ... it doesn't spread. But smallpox - that's a real biological weapon." [6] The Soviet Union instituted the okhotnik program specifically to "develop a smallpox-based biological weapon containing virulence genes from Ebola hemorrhagic fever virus ... transmissibility of smallpox with the lethality of Ebola ... between 90 percent and 100 percent fatal."[7] The U.S. is concerned about the existence of smallpox seed cultures in other nations beside the two internationally sanctioned repositories. The UNSCOM inspection team found a freeze drier in Iraq labeled "smallpox" in 1995. Dr. Hazem AH, the director of the former Iraq virus weapons program, is a pox virologist trained in England. Smallpox was chosen for the resource-sizing scenario because of concern about the results of reintroduction of this pathogen to a largely unvaccinated population and because there will be delayed recognition of the terrorist attack until symptoms develop.

4. Casualty Situation Template Predictions about the potential spread of smallpox are problematic because of the globally unvaccinated population, questions about the long-term efficacy of the vaccine that is currently in use, and because of changes in the overall health of the population increasing possible side effects of the vaccine. Predictions are also complicated by a lack of knowledge of the strain (potentially hybrid) which may be selected for release, and the questionable effectiveness of any ring vaccination strategy which might be instituted. The ring strategy requires vaccination of all potential contacts in a circle going out concentrically from each infected patient. 4.1. Illness Distribution over Time The U.S. Soldier and Biological Chemical Command (SBCCOM) developed a template for the anticipated presentation of smallpox patients over time and the anticipated progression of these patients through the medical system. The SBCCOM anticipated presentation and progression of the virus (per 1000 infected on day-1) is displayed in Figure 10.

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Figure 10. Smallpox Presentation and Progression over Time

4.2. Stages of Smallpox Infection The data shown in Table 9 displays the anticipated clinical progression of the variola infection (smallpox). This data was developed by the U.S. Office of the Surgeon General.[8] Table 9. Stages of Smallpox Infection (U.S. Army Office of the Surgeon General) Day of Symptom 1-11 12-14

Symptom None

Disease Progress Virus Replication

First Symptoms

15

Rash Rash Rash Rash Rash Rash

Fever, backache, headache, nausea, malaise, enanthem Macules Papules (bumps, pimples) Vesicles (blisters) Pustules (pus-filled blisters) Scabs Scars

16-18 19-20 21-24 25-30 31- on

5. Infected Patient Workload 5.1. Introduction This section will analyze the estimated patient workloads. It is anticipated that fatalities will be minimized and that disease progression can be attenuated if vaccine is administered within four days of exposure. In this section, total vaccine efficacy on the infectee is assumed (fatality rate is set to 0 in order to focus on the potential infected patient workload). Smallpox is a highly transmissible pathogen. Each subsequent group of infectees is called a "generation", i.e. those originally infected are members of the "1st generation", those infected by members of the 1st generation are categorized as members of the "2nd generation", etc. Estimates for smallpox communicability range from 1:3 (very stringent quarantine strategy) to 1:25 (rapid onset of symptoms and/or high virulence of the strain released). The communicability rate used in this scenario is 1:5, meaning that each patient, on average, infects five additional people. This was the communicability rate experienced during the 1963 Wroclaw Poland epidemic, one of the most recently documented outbreaks. The cause of that epidemic was not recognized until 43 days after the first reported case. It is important to realize

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that mobility in Poland in 1963 was highly restricted - expected communicability today would be considerably higher because of the mobility of the population. The German state of Baden-Wurttemberg was selected for detailed analysis. Subsequently, analysis was extended to Germany and to the entire area of responsibility. Ring vaccination is not considered in this section.

5.2. Baden-Wurttemberg Seventy-two people from Baden-Wurttemberg attended the conference. It is assumed, for purposes of this scenario, that all 72 were infected. Figures 1 la to lld reflect the progression of smallpox through the population starting with the original infectees. For the first generation (Figure 1 la), it is anticipated that patients will present from day 7 to 21 after exposure with the peak on day 12(16 patients). All 72 patients in Baden-Wurttemberg will be in treatment from days 21 to 26.

Figure 1la. - 1st Generation Caseload

Note the chart in Figure 1 lb. Those infected in the second generation begin presenting to the clinic on day 13. The peak for presentation is on Day 18 when 79 patients present. All 360 patients infected in the second generation are in treatment from days 27 to 32. The last patient completes treatment and is released on day 46.

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Figure 11b. 2nd Generation Caseload

Progression of the disease during the third generation is shown on the chart in Figure 11c. Those infected during the 3rd generation begin presenting to the clinic on day 19. The peak for presentation is on Day 24 when 396 patients present. All 1800 patients infected in the third generation are in treatment from days 33 to 38. The last patient completes treatment and is released on day 52.

Figure 11c. 3rd Generation Caseload

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As noted in the chart in Figure 11d, those infected during the 4th generation begin presenting to the clinic on day 25. The peak for presentation is on Day 30 when 1980 patients present. All 9000 patients infected in the fourth generation are in treatment from days 39 to 44. The last patient completes treatment and is released on day 58.

Figure 11d. 4th Generation Caseload

Figure 12 displays the total caseload during the first 60 days after infection for BadenWurttemberg.

Figure 12. Infected Caseload - First 60 days for Baden-Wiirttemberg

There are four peaks for presentation at the medical facilities - on days 12, 18, 24, and 30. The peak for patients in treatment occurs on day 36 when 11,049 patients are in treatment. Recall that all these patients can be traced back to the original 72 infectees.

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5.3. Germany Figure 13 displays the total caseload during the first 60 days after infection for Germany. There were 261 attendees from Germany. The maximum number of patients presenting is on day 30 when 7256 patients present at local clinics. The peak for patients in treatment occurs on day 36 when 40,054 patients are in treatment.

Figure 13 Infected Caseload - First 60 days for Germany

5.4. Area of Responsibility Figure 14 displays the total caseload during the first 60 days after infection for Europe. There were 416 attendees from throughout Europe. The maximum number of patients presenting is on day 30 when 11,565 patients present at local clinics. The peak for patients in treatment occurs on day 36 when 63,841 patients are in treatment.

Figure 14. Infected Caseload - First 60 days for Area of Responsibility

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6. Infected Patient Workload with Ring Vaccination 6.1. Introduction This section will present the results of an excursion using the ring vaccination strategy. As described earlier, this strategy requires vaccination of all potential contacts in a circle going out concentrically from each infected patient. This assumes that patients will be identified early enough (up to four days after exposure), that the vaccination used will be effective against the strain of smallpox released, and that vaccinations can be administered quickly. Using the ring strategy, all potential contacts are vaccinated regardless of possible contraindications. For purposes of this excursion, it is assumed that vaccinations administered to contacts of firstgeneration patients will be 10 percent effective, second generation patient vaccinations will be 25 percent effective, and third-generation infectee vaccinations will be 50 percent effective. Results will be presented for the German state of Baden-Wurttemberg, for Germany, and the area of responsibility. 6.2. Baden-Wurttemberg The effectiveness of instituting the ring vaccination strategy in Baden-Wurttemberg is shown in Figure 15.

Figure 15. Effectiveness of Ring Vaccine (Baden-Wurttemberg)

The number of patients presenting on day 12 is the same (all 1st generation). With ring vaccination, the number of patients presenting on Day 18 is 90 percent of those who would have presented without ring vaccination, on day 24 it is 68 percent, and by day 30 the number is 34 percent. The number of patients in-treatment on day 36 (the peak for patients) is less than half.

6.3. Germany The effectiveness of using the ring vaccination strategy in Germany is shown in Figure 16. The total number of patients in treatment on day 36 drops from 40,094 to 16,396.

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Figure 16. Effectiveness of Ring Vaccine (Germany)

6.4. Area of Responsibility Figure 17 displays the impact of using the ring vaccination strategy in the area of responsibility. The total number of patients in treatment on day 36 drops from 63,841 to 26,132, a drop of approximately 59 percent.

Figure 17. Effectiveness of Ring Vaccine (Area of Responsibility)

7. Total Patient Caseload 7.1. Introduction Although initial analysis has focused only on the patients with smallpox, there will be, nonetheless, a concomitant surge of "worried well", those patients who have some symptoms, but whose diagnosis will not be smallpox-related. This demand on medical resources needs to be taken into account. Accordingly, this section will present the "worried well" results for 3 areas: Baden-Wurttemberg, Germany, and the entire area of responsibility. For purposes of

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this analysis, an effective ring vaccination strategy (as described in paragraph 6 above) is assumed. 7.2. Baden-Wurttemberg The magnitude of the worried well and the resultant impact on total patient workload in BadenWurttemberg is shown in Figure 18. Recall that this is the result of the original 72 infectees who attended the conference. The peak patient caseload (including 3426 worried well) occurs on day 31 when 6978 patients are in treatment.

Figure 18. Total Patient Caseload (Baden-Wurttemberg)

7.3. Germany The chart in Figure 19 depicts the impact of worried well on the total patient load for Germany. Note that the original 261 attendees result in 12,420 worried well presenting on day 31.

Figure 19. Total Patient Caseload (Germany)

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7.4 Area of Responsibility The magnitude of worried well and the resultant impact on total patient workload in the area of responsibility is shown in Figure 20. On Day-1, 416 people attended the conference. The peak patient caseload (including 19,796 worried well presenting) is on day 31 when 40,320 patients are in treatment.

Figure 20. Total Patient Caseload (Area of Responsibility)

8. Fatalities The U.S. Army Office of the Surgeon General projects that 70 percent of unvaccinated people developing smallpox will survive. This scenario, therefore, used a 30 percent fatality rate for the four generations analyzed. Figure 21 reflects the number of anticipated fatalities for BadenWUrttemberg, Germany, and the area of responsibility. The fatality rate experienced may be considerably higher if an enhanced strain (the India-1 and Hong-Kong strain have a 50 percent fatality rate) is released.

Figure 21. Projected Fatalities

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9. Treatment Network and Protocols 9.1. Introduction A notional health care network has been developed by SBCCOM as part of the modular emergency medical system being developed to respond to domestic emergencies in the U.S. This system is designed to augment local response efforts, and provide rapid organization of assets. The Acute Care Center (ACC) "provides rapid in-patient treatment for a large population of severely ill suffering from an agent of bioterrorism."[9] The Neighborhood Emergency Help Center (NEHC) "provides high volume casualty reception centers performing victim triage and dispensing prophylactic medications and self-help information."[10] The Medical Command and Control Centers (MCCs) are developed to provide command and control for up to 5 ACC/NEHCs. The ring vaccination strategy presented in paragraph 6 above is implemented in developing the requirements for the health care network for this section. Fatality rates of 30 percent have been applied. For purposes of this section, each of the ACC and NEHC modules have been developed which can treat up to 1000 patients each. The SBCCOM protocols designate the personnel requirements by type of personnel required by MCC, ACC, and NEHC module. Each MCC module requires 16 personnel; each ACC requires 706 personnel, and each NEHC requires 110 personnel. These modules are developed for purposes of assessing resource requirements (expandable in sub-modules to treat 50 patients each). For purposes of this scenario, patients who are in the chronic recovery stages are treated in the ACC whereas patients requiring only supportive care are treated in the NEHC.

9.2. Baden-Wurttemberg Recall that the 72 people from Baden-Wiirttemberg who attended the conference eventually led to almost 7000 U.S. patients in treatment. Figure 22 reflects the number of treatment facilities which would be required to treat this anticipated patient load. Treatment of these patients will require up to 2 ACCs and 5 NEHCs.

Figure 22. Treatment Network (Baden-Wurttemberg)

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9.3. Germany Figure 23 reflects the number of treatment facilities that would be required to treat the anticipated patient load within Germany. Treatment of these patients will require 7 ACCs and 19NEHCs.

Figure 23. Treatment Network (Germany)

9.4. Area of Responsibility The 416 people from Europe who attended the conference eventually lead to over 40,000 patients in treatment, of which almost half are worried well. Figure 24 reflects the number of treatment facilities that would be required - 11 ACCs and 30 NEHCs.

Figure 24. Treatment Network (Area of Responsibility)

10. Medical and Health Service Requirements 10.1. Introduction Treatment of smallpox patients focuses on supportive treatment and antibiotics. Included in the treatment protocol are the following actions: "(a) isolation of the patient to prevent

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10.2. Baden-Wurttemberg transmission of the smallpox virus to non-immune people, (b) monitoring and maintaining fluid and electrolyte balance, (c) skin care, and (d) monitoring for and treatment of complications."[8]. The medical treatment protocols developed by SBCCOM for smallpox have been used for this study. Four types of medical personnel -- nurses, physicians, physician extenders, and emergency medical technicians/paramedics -- have been selected, for purposes of this study, to represent the requirements for personnel to treat the surge of patients.

Figure 25. Medical Personnel Requirements (Baden-Wurttemberg)

Figure 25 reflects representative personnel required to man the treatment network for BadenWurttemberg. There is a requirement for 396 key health care workers to treat the 7000 patients resulting from the original 72 conference attendees from Baden-Wurttemberg. Note, in particular, the need for nurses.

10.3. Germany Recall that 261 persons stationed in Germany attended the conference. Manning of the treatment network to treat the resultant epidemic in Germany will require over 400 physicians and physician extenders in addition to close to 700 nurses. Figure 26 reflects the requirements for selected medical personnel to treat the projected patient load.

Figure 26. Medical Personnel Requirements (Germany)

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10.4. Area of Responsibility The 416 attendees to the conference came from 8 countries. Manning the treatment network for the entire affected area of responsibility will require almost 700 physicians and physician extenders (day 31) and almost 1200 nurses (day 40). Refer to Figure 27 to view the number of key medical personnel required.

Figure 27. Medical Personnel Requirements (Area of Responsibility)

11. Way Ahead This paper has documented an analysis of the potential medical requirements needed to respond to a bioterrorist incident. The paper has documented the methodology used to develop requirements for medical and health care facilities. The first indicators of a bioterrorist incident will probably occur at clinics. It is likely that the indicators will be associated with a slowly evolving event that develops into a medical/public health crisis. Clinics used to treat U.S. forces and their dependents are not designed to handle surge requirements such as those which could be associated with a bioterrorist incident. U.S. forces and their dependents are a relatively young and healthy population. The concept of health care provides for evacuation of the seriously sick through numerous echelons. In case of a communicable disease, evacuation may not be possible, particularly if a quarantine is imposed. Local crisis response and consequence management plans in the U.S. rely on layered reinforcement from state and national assets. The Centers of Disease Control maintain a National Pharmaceutical Stockpile to supplement requirements at health facilities throughout the U.S. These assets may not be available to respond to an incident in this AOR. The next step in the overall study will be to catalog the medical assets available in terms of infrastructure, personnel, equipment, and selected supplies. Both U.S. assets and host nation assets covered under host nation agreements will be included. Existing contingency plans will be analyzed and synchronized as possible. U.S. plans will include Department of Defense, Department of State, and other U.S. government documented potential courses of action. Host Nation Agreements and Memorandums of Understanding will be analyzed, where available, as will individual country consequence

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management plans. Other U.S. domestic plans will be analyzed, e.g. the Federal Response Plan and the recently drafted U.S. Model Hospital Act. The last step will be to develop recommendations for improvements to existing consequence management plans. These recommendations will address jurisdictional issues, conflicts in existing plans, as well as overlaps and shortfalls in medical and health service resources needed to respond to a bioterrorist incident.

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]

Bremer, L. Paul, "The Future of Terrorism: What Are the Next Threats?", http.weforum.org/site/knowledgenavigator.nsf. Tussing, Bert B. and Colonel Jeffrey C. Reynolds, "Consequence Management Symposium", Issues Paper 09-01, September 2001, p. 2. Hepburn, Colonel Byron C, "Chemical-Biological Attack: Achilles Heel of the Air Expeditionary Force?", USAF Counterproliferation Center, Maxwell Air Force Base, September 1999, p. 4. Alibek, Ken, Biohazard. New York, p. 112. Tucker, Jonathan B. and Raymond A. Zilinskas: "The 1972 Smallpox Epidemic in Aralsk, Kazakhstan, and the Soviet Biological Warfare Program", Monterey Institute of International Studies, 2002. Orent, Wendy, "Smallpox: A Deadly Recipe", Los Angeles Times, July 28, 2002, p. Ml. Tucker, Jonathan B., Scourge, New York, 2001, p. 159. Grabenstein, LTC John D., "Treatment Protocol for Smallpox," email on 20 September 2002. LTC Grabenstein is currently the DoD POC for the administration of the Smallpox vaccine SBCCOM: "A Mass Casualty Care Strategy for Biological Terrorism Incidents: Acute Care Center", Aberdeen Proving Ground, December 1, 2001. SBCCOM: "A Mass Casualty Care Strategy for Biological Terrorism Incidents: Neighborhood Emergency Help Center", Aberdeen Proving Ground, May 1, 2001.

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GM Modified Food/Feed as Biowarfare Tomasz TWARDOWSKI Institute ofBioorganic Chemistry PAS Noskowskiego 12/14, 61-704 Poznan, Poland

Abstract. In today's world, the consumer is the top interest. Furthermore, today's consumer needs safe and high quality products and service. In the case of food [and feed as well], it is often formulated as "From farm to fork. From fork to farm." These high expectations correlate with very different aspects of modern biotechnology: biomedical, agricultural, environmental, legislative, regulatory, consumer acceptance and educational issues, as well as many other. However, we have to take into account that food/feed has different value in different parts of the world. For instance, it varies in EU, North America, OECD in comparison with 3rd World Countries. Novel technologies give possibilities to treat food/feed not only as political weapon, but also as biowarfare.

1. Introduction The aspects of biomedical, agricultural and environmental biotechnology are very different. For instance, we cannot compare the needs of the society for biotech products of rich countries like EU, North America or OECD to those of the 3rd World Countries. Similarly, there are different interpretations and weights of regulatory, consumer acceptance, and educational issues between wealthy consumers and people from the 3rd World Countries [1]. The international tension concerning the potential use of genetic engineering as a tool for production of biological weapons is tremendous. It is common opinion of experts as well as of lay people that genetically modified organism can be used for positive and negative purposes. In numerous instances, all different GMO: bacteria, animals and plants are in the centre of interest. However, the process of genetic engineering used for human gene therapy plays important role as a resource for science fiction literature and movies. Similarly, the flow of genes more often occurs in literature than in nature. One's imagination is much more effective than reality in creating monsters and danger. Nowadays, the consumer is the top interest. He needs safe and high quality products and service. It is a common idiomatic expression used in modern agrobiotech businness: "From farm to fork. From fork to farm". In correlation, we have different points of view concerning modern BIOTECHNOLOGY. We can distinguish several different biotechnologies: • RED - commonly related to medicine and pharmacy; • GREEN - related to agrobiotechnology; • NANO - meaning molecular manipulation on the 10-12 level; • TERRO - related to terrorism. The need for GMO biosafety research is evident and unquestionable. However, there is a diversity of effects: from pure science through public perception to economy. Consider the

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fact that the products of genetic engineering related to sophisticated methods and tools are available in ordinary supermarkets and every consumer has to vote every day on their quality and acceptability. The biosafety of everyday products is out of discussion. However, the consumer's opinion does not have to be based on scientific facts. This could be evaluated and predicted from in silico, in vitro and in vivo experiments. There is no possibility to recognise the common opinion and the public acceptance in advance. The potential impact of public opinion is unpredictable. However, financial effect of the lack of public acceptance is easy to determine and prediction is very accurate. The direct financial losses and, in consequence, the indirect social effects could be tremendous. 2. GM food/feed In 2002, over 58 mln ha of GM plant [at least] were produced around the world. Public acceptance of genetic engineering is already minor and society's awareness is relatively low [2]. The preparation of GM plant [3,4] is relatively simple and makes several options for the introduction of biowarfare: - identification of function (e.g. toxicity or allergenicity); - determination of protein; - protein sequence determination -findingthe gene; - gene sequencing and functional characterisation; - introduction of the specific gene into desired genome; - detection of new gene activity in vitro; - reconstruction of the plant; - checking the presence and activity of the protein in the plant; - multiplication of the plant and its stability. The preparation of the food containing poisonous products is as easy or as sophisticated as the preparation of any kind of genetically modified plant or animal. The production of this kind of biowarfare is relatively simple. However, the transportation and distribution are limited by several factors. The potential negative effect is gradual. Nevertheless, one should take into account the sociological effect of the information saying that "food is poison". Biowarfare will result in tremendous sociological effect when combined with novel and mostly unknown terminology, e.g. biotechnology, genetic engineering, genomics, toxicogenomics, bioinformatics, GM food/feed, etc. Sociobioterrorism - could be a product of the combination of terrorism, biowarfare and sociological effect on a large scale. Let us make a comparison of potentially poisonous GM food and anthrax terroristic attack in 2002 in the United States. In such case, we have to take into account the mass media and public reaction. The similarity to anthrax effect is striking. It is worth to mention that during the mass panic of anthrax attack, 5 people died and 22 have been hospitalized as anthrax victims. The human life is the highest value, but... much more people are killed on the road every weekend. However, the cost of the panic was incredible, estimated in billions of US dollars. Such amount of money could save many human lives.

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The target for terrorists is general - the society. The large scale destruction of the society is the best goal of the terror. As in the case of anthrax, very few people died, but the panic was a global effect. The national biosafety system cannot be limited to physical status and health, but the sociological sense of security of the society is also of crucial significance. For lay people regulatory regime and governmental policy on biosafety are of key value. General knowledge of legislation system is relatively low in society. Furthermore, the cooperation of every single person with the state authorities is very important, particularly for monitoring and inspections, as well as for providing information. The overall approach of the government towards biotechnology and biosafety shall reach all citizens and shall be given in plain language. As for the potential impact on human health is concerned, we are not limited to physical health; we shall take into account the mentality and well being of humans. The potential influence on the environment of human health must be assessed. A flexible, creative, and rapidly responsive biomanufacturing infrastructure is an essential part of an effective overall strategy for bioterrorism preparedness and biological defense. A variety of approaches and technologies are evolving to provide the capacity to bring innovations in biological threat detection, prophylaxis, and therapeutics from the laboratory bench to the advanced development and ultimately to the enduser and/or the marketplace. Genetically modified food and feed as biowarfare - exercise scenario will include the following aspects: • scientific reality is a fact because of advanced research; • sociological effect will be tremendous; • preparedness against this kind of biological weapon is almost none; • further development is unknown. To minimize the effects of biological attack of terrorists, health care professionals and public health authorities must be aware of the threat of biological warfare and terrorism and have an increased index of suspicion that such an attack can occur. As a final effect, total panic of the society could be expected. In conclusion, biodefence matters, but is not limited to: intelligence, health system, and technical know-how. We need public AWARENESS program dedicated to extremely delicate nature of human behavior. Within this program, the specialized personnel have to be trained to recognize and treat real casualties of biowarfare properly. These people must be able to act as instructors/trainers to apply preventive measures to react rationally and to avoid panic. There is a shared belief that biotechnology communication is both necessary and beneficial, and that any progress depends on the availability of information, exchange of opinions, and restoration of trust. Bilateral communication is needed to bridge the gulf between the experts in the field and the general public. The creation of public awareness is organically associated with several definitions and clear answers for, at least, the following questions: - why communicate with society about biotechnology and biowarfare, particularly? - what to communicate?, or: do we need a censorship system? - who should be responsible for the information system and who should communicate - how to do it? It is the last but not least important question. Especially in the food area, the biotechnology controversy triggers a broader debate in which romantic ideas of naturalness and traditional forms of production oppose technological advances in the food production process. Finding a successful approach to the biotechnology

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controversy could serve as a 'lightning rod' which points the way for dealing with different but related issues. 3. Conclusions The large scale, global commercial use of different GMO produce global and diversified effects that are very significant for business world. The cooperation of producers, government agencies, non-governmental organisations and many others is a privilege and a must. However, the public and the media mind rather bad than good news. The implementation of management scheme in the case of panic of the society requires significant improvements. The general education and the knowledge of basic facts are probably initial steps in preventing the tragic effects of biosocioterror. The effects of biosocioterror could be tremendous and they have to be recognised as direct and undirect effects. Particularly, the undirect effects, due to the activities or to the goods or services, that are not delivered to the society could be incredibly high. For example, we can loose the positive effects of new drugs or we will spend huge amount of money on nonsense aims instead of spending them on valuable goals. This process requires the sophisticated system of evaluation of GMO towards the ordinary members of the society, all the steps should be understandable by lay people. The decision making process must be simple and transparent, but shall meet the needs of industrial groups as well. The danger of biosocioterror is a reality. We have to take this into account. The objective of this Advanced Research Workshop has been clearly described: • modes of transmission • drug resistance • genetic engineering • transmission to other organisms (animals) • ecoterrorism • logistics, e.g. vaccination However, in my opinion, sociological effect has been missed. It is worth to mention that today we often talk about "the weapon of mass effect" and not about "the weapon of mass destruction". Obviously, the economical or social effects are also significant. According to the opinions of international and national crisis management experts expressed during many workshops and conferences, biological terror attack can quickly overwhelm human and logistic resources in unprepared local communities. Poorly prepared, chaotic emergency aid from the voivode or national level will delay crisis containment and, in case of highly contagious disease (e.g. smallpox), it can be a cause of its evasion of the control. What relevant central and local level institutions can do to prevent chaos in their activities? The perception of risk and a sense of lack of control are crucial factors withholding the public from readily accepting developments in biotechnology [5]. It is therefore required both to create the conditions which make informed risk-benefit comparison possible and to express a coherent model for policy making. The actual risk might not be the crucial indicator, but rather the question of who decides that the risk should be taken. That is, these are the deep value issues that lie behind these questions which are the determinants. Surveys and public consultation exercises have shown that a process of informing about the risk and possibilities of biotechnology fosters the perceptions of risk on the one hand, but increase the preparedness for acceptance on the other hand [5-7]. The responsible menagement system should be able to

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concentrate on large scale prophylaxis and preventive procedures, including crisis plans and emergency from local to central and total scenario. Panic, chaos and disorganization will be very dangerous, probably at the same level as physical terror attack. The debate needs to be based on sound foundations. There is no single rationality, but a wide range of possible competing rationalities which decide what proof can be provided on the basis of the available information. These competing interpretations have to be balanced in order to create a dialogue that is based on shared knowledge about what is going on. This is necessary in order to provide the long-term goals and values required to create continuity in the discussion. The preparation of biowarfare as food/feed is scientific reality. The sociological effect [that we can refer to as ,,biosocioterror"] must be tremendous. In my estimation, the preparedness against this kind of biological weapon is none today. The further development is unknown.

References [1] [2] [3] [4] [5] [6] [7]

Biotechnology - the Making of a Global Controversy", Eds: M. W. Bauer, G. Gaskell, 21 -94, 2002, Cambridge University Press with the Science Museum, London. T. Twardowski - ,,Public Perception and Legislation of Biotechnology in Poland", a chapter in: ,,Use of Agriculturally Important Genes in Biotechnology ", Ed. G. Hrazdina, IOS Press, NATO Science Series vol.319, 180-202,2000. T. Twardowski, A. Michalska - ,,KOD - korzysci, oczekiwania, dylematy biotechnologii", Wyd. Agencja Edytor, Poznan, 2001. (,,CODE - profits, expectations and dillemas of biotechnology", in Polish). T. Twardowski, E. Kwapich - ,,100 + 30 najczesciej zadawanych pytan na temat wspolczesnej biotechnologii", Wyd. Agencja Edytor, Poznan, 2001. (,,100+30 most frequently asked questions about biotechnology" in Polish). A chapter in: Biotechnology 1996-2000 the years of controversy", Eds. G. Gaskell, M. Bauer, 251-257, Science Museum, London, 2001. A chapter in: ,,The European Biotechnology Directory", Bio Commerce Business Profiles 2000, vol. 2, 41-47. T. Twardowski - ,,The development of biotechnology in Poland", M. Dando et al. (eds.), in: Maximizing the Security and Development Benefits from the Biological and Toxin Weapons Convention, 273-276, Kluwer Academic Publishers, The Netherlands, 2002.

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Antibiotic Resistant Bacteria A PotentialThreat Marian NEGUT Cantacuzino Institute, SpL Independentei 103, Bucharest, Romania Abstract. The main implications in medical, veterinary and environmental fields and some objectives targeting the limitation of resistant bacteria are discussed in this paper. The magnitude and the implication of antibioresistance is an increasing and alarming threat as high pathogenic or genetically modified resistant can be used in aggressive purposes. Medical consequences of bio terrorist use of antibio-resistant organisms are pointed out.

1. Introduction In Congressional Briefing of AMS, in 1999, Levy S.B. was concluding "As the new century begins we are challanged by newly emerging infections and the decreasing effectiveness of our antibiotic arsenal" (19). Increasing of multidrug resistant organisms in the environment combined with medical invasive technology and large scale use of antimicrobials induced new pathologies with severe clinical pictures and unexpected epidemiological evolutions (8, 10, 17). Global emergence of antibiotic resistance has become a public health concern as medical expenses (hospitalization, therapy, complications) have increased dramatically. Hospital acquired infections with large extended spectrum of antibioresistance, mainly in intensive care units, cumulating predisposing factors, induced a high rate of mortality among debilitated patients (1, 5, 9, 11, 21). What was incredible before, happened in 2001. Bioterrorism has become a reality in 2001, following the tragic events of September 11. Since October 4 and as of December 7, 2001 a total of 22 cases of anthrax were recorded as a result of a bioterrorist attack (2). To the concern of emerging and reemerging infectious diseases the antibioresistance of highly pathogenic bacteria or genetically modified organisms amplifies the potential threat of bioterrorism using such organisms. 2. Antibiotic resistance - alarming increase of natural pathogens Regarding the natural pathogenic bacteria, at least two pieces of evidence are of important concern: Increasing rate of antibioresistance among natural pathogenic bacteria to old and recently introduced antibiotics (6, 8, 9, 19, 20). The colonising of the environment, mainly in hospital, with selected resistant strains that amplifies the risk of implication of such organisms in human and animal pathology (1, 5, 11, 14). Some examples of increasing antibiotic resistance are presented in Fig. 1. Well known as sensitive, bacteria species have been mentioned as resistant to the antibiotics of

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choice; more than 35 % of Str. pneumoniae and Neisseria meningitidis were reported resistant to penicillin, 10-40 % S. aureus strains to methicillin, and more than 10 % of isolates of Ps. aeruginosa were resistant to imipenem (24, 25, 26, 28). Many enteric pathogens as Salmonella, E. coli, Campylobacter, Klebsiella developed high resistance to floxacins and beta lactam antibiotics of IIIrd generation (5, 11, 14, 25).

Fig. 1

Fig. 2 Many authors reported high resistance of selected resistant flora colonising patients in intensive care units (1, 2, 5, 8, 9, 12, 16, 22) (Fig. 2). Acinetobacter, Pseudomonas and Enterobacteriaceae resistant to floxacins and cephalosporines of IIIrd generation are currently encountered and even resistance to cephalosporines of IVth generation was detected. Resistant Str. pneumoniae strains, mainly to penicillin but not only, were

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detected also in community acquired pneumonia (28). Of 456 Gram negative strains isolated from spontaneous flora of hospital environment and tested against 12 antibiotics (currently active on these bacteria) more than 50 % were resistant to 3 antibiotics, over 20 % to 5 antibiotics and around 5 % to 8 antibiotics. 3. Antibiotic resistance - implications The large impact of bacterial antibioresistance is presented schematically in Fig. 3. The current interference of the areas, mentioned in Fig. 3, public health, clinical, epidemiological, environmental, veterinary, amplifies the dimensions and the consequences of the spreading of antibiotic resistance.

Fig. 3 3.1. Clinical implications Fig. 4 shows the direct and the most striking impact of antibioresistance. Expensive therapy, complex and repeated laboratory investigations associated to a long duration of medical care involve very high costs of hospitalization. At the same time, the increasing failure of targeted therapy is reflected in unestimated (but high) mortality (16, 20). Expensive research and introduction of new antibiotics became a continuous escalade in the effort to minimize the failure of antibiotherapy. This policy amplifies the costs of hospitalization already high by side effects, complications and expensive medical care (14, 16, 20).

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Fig. 4

Fig. 5 3.2. Unpredictable public health implications Public health implications are strongly related to high risk and expensive hospitalization, new and severe epidemiological aspects and social involvements (Fig. 5). As mentioned in Fig. 4, hospitalization became one of the main implications of antibioresistance. As I mentioned before, the etiologic and associated therapy becomes very expensive as new generation antibiotics and long term administration are often required. Therapeutic failure increases the risk of complications and subsequent unpredictable losses. All clinical associated expenses as complicated paraclinic investigation - clinical laboratory, imagistic - long duration of housing, nursing as well intensive care surveillance - contribute to a very expensive hospitalization deeply reflected in public health budget (1,

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9,13,16,21). But no real estimation in public health is possible at present if a deliberate terrorist delivery of high resistant pathogenic bacteria occurs.

3.3. A modified epidemiology Particular aspects of hospital acquired infections caused by antibioresistant microorganisms are underlined in Fig. 6. Concentration of very active sources of infection in a limited space activate the transmission to subjects with debilitating condition. Even low pathogenic organisms from hospital environment can become aggressive generating long lasting epidemics. Resistant bacteria selected in hospital environment from inadequately treated patients or from unknown carriers conduct to unexpected but always complicated epidemiological evolutions. Specific hospital transmission, mainly air borne contamination, contributes to the extension and the duration of these epidemics (1, 5, 12, 13, 23). Social aspects arise from long term incapacities caused by prolonged hospitalization, lasting recovery and sometimes by short or long term disabilities. Some estimations of such aspects were made in present hospital practice and spreading of antibioresistance (3.4.12) but no predictable evaluation is possible in criminal spreading of multidrug resistant bacteria. Social involvements as a secondary step of bioterrorist attack using resistant bacteria even low pathogenic bacteria can generate important perturbancies.

Fig. 6 3.4. Uncontrolled spreading of resistant organisms in animals In Fig. 7, some aspects of amplified emergence of diseases transmitted from animals to man are schematically presented. As a direct result of increasing of antibiotic resistant microorganisms an important emerging epidemic of zoonotic diseases in animal and subsequently in man is predictable (18).

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Uncontrolled use of antibiotics in animals (therapeutic, prophylactic, promotion purposes) is already reflected in new pathologies in animals, uncommon epidemic forms, important losses and increased costs (18). But animal pathologies deeply reflected in human pathology and epidemiology alimentary, and environment pathways, amplified enormously in bioaggressive situations can increase the spreading of anthropozoonotic organisms at a level difficult to control (18,19).

Fig. 7

3.5. Antibiotic resistant organisms and the environment Some authors (10, 20) pointed out the importance of the environment in activating natural cycle of spreading resistant bacteria and modifying ecosystems (Fig. 8). Inadequately treated waste water discharges in surface sources is one of the most frequent way of spreading antibioresistant organisms from and to the community (10). This important link between human and animal that the environment realises is a key of modified ecosystems. Mainly abnormal colonisation is responsible for new pathologies in man and animals. As for newborns, colonisation with antibioresistant organisms causes long term perturbancies in children development.

Fig. 8

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4. Antibiotic resistant organisms: limitation of spreading Three important categories of measures were suggested by different authors and promoted at national, regional and even global level (6,7,9,13,19,20,21,28). - Surveillance Programme of the spreading of antibioresistance; - Control of antibiotic use; - Introducing alternate preventive / therapeutic methods.

4.1. Extended surveillance programme An active Programme of Surveillance of antibioresistant bacteria is presented in Fig. 9. Several authors pointed out that continuous surveillance is an ideal measure to know the trends of the epidemiology of bacterial resistance and to adopt the suitable measures as consequence (6,7,20). The data base collected from local laboratories at national level are currently reported and integrated in regional and continental reporting system. This active system of centralising regular information conduct recommendations, settlements, even decision at different central authority (9). European Antimicrobial Resistance Surveillance System developed an extended network covering Eastern European countries and Russia too (9,28). Any shocking changing in antibioresistant patterns has to be considered an alarming signal of an unusual picture of natural or induced spreading.

Fig. 9 4.2. Control of antibiotic use The most recommended measure of limitation of the extension at a large scale of antibioresistance refers to the limitation of the use of antibiotics in man and animals (1,3,4,7,21,23). In Fig. 10, some important measures are recommended for the use of antibiotics in

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ambulatory and hospital therapy. Control of antibiotic marketing is one of the most important measures that has to be promoted in developing countries mainly. The different policies in antibiotic marketing explain partly the increased incidence of antibioresistance of some organisms in South East European countries and Scandinavian countries for instance (9). Limitation of antibioprophylaxy in man and animals was also mentioned as a crucial measure in limiting spreading of antibioresistance. Recommendations, settlements, rules in antibiotic therapeutic use promoted a new medical competence "antibiomerapeutics". Well known measures / recommendations in veterinary use of antibiotics are also mentioned in Fig. 10. "Feeding antibiotics forbidden" was a measure promoted some decades ago, but even now it is not entirely applied.

Fig. 10

Fig. 11

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4.3. Alternative preventing / therapeutic methods Two large categories of measures are recommended for limiting antibiotic use (Fig.

11). - Vaccines and immunomodulators in therapy of chronic or reccurent infections; - Controlled colonising flora mainly in chronic enteric disorders. Also preventive vaccines were recommended in animals instead of antibioprophylaxy in epizootic outbreaks (27). References [I] ALB RICH. W.C., ANGSTWURM M., BADER L., GARTNER R. "Drug resistance in intensive care units" Infection, 1999, 27 Suppl 2:S19-S23. [2] BALLOW H.C., JONES N.R., JOHNSON M.D., DEINHART A.V., SCHENTAG J.J. AND SPAR STUDY GROUP "Comparative in vitro assessment of Sparfloxacin activity and Spectrum using Results from over 14.000 Pathogens Isolated at 190 Medical Centers in the USA" Diagnostic Microbiology Infectious Diseases 1997,29. 173-186. [3] BUKE J.P. "Rational approaches to combating resistance" Jtal. Journal Clin Practice Suppl. 2002, 125, 29-36. [4] COURVALIN D.G. "Better control of antibiotic resistance" Clin. Infections Diseases 2001, 33, 542-547. [5] DIRUSANO G.L. "Infection in the intensive care unit: beta lactamase mediated resistance among Enterobacteriaceae and optimal antimicrobial dosing" Clin. Infect. Diseases 1998,27, Suppl 1:S111-6. [6] FINCH R. "Antibiotic resistance - from pathogen to diseases surveillance" Clinical Microbiol. Infect. 2002,8,317-320. [7] GOOSENS H. "Antibiotic Resistance and Policy in Belgium" Verhandelingen - Koninkliijke Academie voor Geneeskunde van Belgie 2000, 62,439-469. [8] GUIRGUITZOVA B., CHANKOVA D., ZOZIKOV B., MINKOV N. "Les enterocoqnes comme uropathogenes Frequence disolement et sensibilite envers les substances antibacteriennes" Annales d'urologie 1998, 32, 15-9. [9] HANBERGER H., DIEKEN D:, FLUIT A., JONES R., STRUELENS M., SPENCER R., WOLFF M. "Surveillance of antibiotic resistance in European ICUs" Journ. Hosp. Infect. 2001,48, 161-176. [10] I WANE T., URASE T., YAMAMOTO K. "Possible impact of treated wastewater discharge on incidence of antibiotic resistant bacteria in river water" Water Science and Technology, 2001,43, 91-99. [11] JACOBY G.A. Genetics of extended spectrum Beta - lactamoses". Eur. J. Clin Microbiol. Infect. Diseases 1994, vol. 13, Suppl. 1, 2-11. [12] JONES H.R. "Impact of changing pathogens and antimicrobiol sceptibility paterns in the treatment of serious infections in hospitalized patients" Amer. Journ, Medicine 1996,100 (Suppl. 6A) 3S-12S. [13] KARLOWSKY J., SAHM D. "Antibiotic resistance - is resistance detected by surveillance relevant to predicting resistance in the clinical setting" Current Opinion in Pharmacology 2002, 2,487. [14] KESSLER E.R., FUNG-TOMC J. "Susceptibily of bacterial isolates to p-lactam antibiotics from US clinical trials over a 5 year period" Amer. Journ. Medicine 1996,100, Suppl. 6A, 13S-19S. [15] KLIETMANN F.W., RUOFF L.K. "Bioterrorism: Implications for the clinical microbiologist" Clin. Microbiol. Reviews, 2001, 14, 2, 364-381. [16] K.OLLEF M.H., FRASER V.J. "Antibiotic resistance in the intensive care unit" Am. Intern. Med. 2001, 134,298-314. [17] LANE H.C., FAUCI A.F. "Bioterrorism on the home front - a new challenge for American medicine in bioterrorism. Guidelines for medical and public health management" in Hunderson A.D., Inglesby V.T. O'Toole Eds JAMA Press. [18] LATHERS S. "Clinical pharmacology of antimicrobiol use in humans and animals" Journ. Clin. Pharmacology, 2002,42, 587-6000. [19] LEVY S.B. Antibiotic resistance: Microbs on the defense Infections Congressional Briefing - AMS, 1999, June 21. [20] LEVY S.B. Antibiotic resistance: consequences of inaction Clin. Infect. Dis. 2001, 33 Suppl. 3, S124S129. [21] MAN M.W., MEMISH Z.A. Antibiotic resistance. An impending crisis. Sandi Medical Journ. 2000, 21, 1125-1129. [22] MARSHALL A.S., ALDRIDGE E.K., ALLEN D.S., FUCHS C.P., GERLACH E.H., JONES N.R.

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Comparative antimicrobiol activity of Piperacillin - Tazobactam Tested against more than 5000 Recent Clinical Isolates from five medical centers. A reevaluation after five years. Diagn. Microbiol. Infect. Dis. 1995,21,153-168. [23] ORTQVIST A. Treatment of community - acquired lower respiratory tract infections in adults Europ. Resp. Journ. Suppl. 2002,36,405-535. [24] PEURA D. "Helicobacter pylori: rational management options" Amer. Journ. Med. 1998, 105,424-430 [25] TAMBIC A.A., TAMBIC T., KALENIK S., JANKOVIC V. Surveillance for antimicrobiol resistance in Croatia. Emerg. Infect. Dis. 2002, 8, 14-18. [26] UNGUREANU V., PANA M., GHEORGHE M., MIHALCU F., DOROBAT O., MITACHE E., LUCINESCU ST., VRANCEANU L. Study of the sensitivity to antimicobiol drugs of some S. pneumoniae strains isolated from different pathological states Romanian Arch. Microbiol. Immunol 1996, 55 (3), 241-251. [27] ZHANG - BARBER L., TURNER A.K., BARROW P.A. Vaccination for control of Salmonella in poultry. Vaccine 1999,17,2538-2545. [28] *** EUROPEAN ANTIMICROBIOL RESISTANCE SURVEILLANCE SYSTEM Annual Report 2001 On going surveillance of S. pneumoniae, S. aureus, E. coli, E. faeciun Bilthoven, The Netherland- 2002.

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Effective Training for First Responders Peter J. STOPA, Karen QUINN-DOGGETT, Richard A. VIGUS Edgewood Chemical Biological Center 5183 Blackhawk Road, Aberdeen Proving Ground, MD 21010-5424, USA

1. Introduction The March 1995 Sarin gas attack by the Aum Shinrikyo cult on the Tokyo subway system was a wake-up call to first responders and emergency planners worldwide. Prior to this incident, efforts toward preventing or responding to terrorist acts were focused on bombings, firearms, hostages, or sabotage. Most of the responders to this incident were ill-trained regarding the perils of chemical agents. They could not recognize the signs or symptoms of a chemical agent; did not wear effective personal protective equipment (PPE); and could not render effective first aid to the victims. Many became casualties themselves - becoming part of the problem rather than part of the solution. The United States Congress took notice. In response to the tragedy of this event, they passed legislation to train first responders to incidents involving Weapons of Mass Destruction (WMD). The Nunn-Lugar-Domenici Domestic Preparedness Program was designed to enhance the capability of federal, state, and local emergency response units to incidents involving WMD. This legislation tasked the U.S. Department of Defense to initiate training and sponsor exercises for these units. During the duration of the program (1996-2000), 105 cities were trained, and over 230 exercises were held. This chapter will describe the components of an effective WMD training program for first responders. Although the focus of this book is biological terrorism, the same principles apply to all forms of attacks involving WMD.

2. The WMD Delta The organizers of the training came to one conclusion at the outset of this task. Responders (police, firemen, emergency services, and medical personnel) know how to respond to mass casualty disasters and chemical spills. They are aware of decontamination procedures, medical treatment, and surveillance systems. Rather than teach them their jobs again, the organizers decided to concentrate on what was different about a WMD event vice a "normal" mass casualty event or chemical spill. Reaching out to the responder community, they organized a series of workshops that resulted in the development of the WMD Delta, i.e., those factors that were different in a WMD event. These differences are summarized on Table 10.

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Table 10. Comparison between a conventional HAZMAT (HMI) incident and one involving a Weapon of Mass Destruction (WMD) PARAMETER Deliberate Incident

HMI

WMD

*

V

Super Toxic Material Early Hazard Identification Mass Casualties/Many possible Fatalities

V * *

V

Mass Decontamination (>100) V

Unusual Risk to Emergency Responders and Civilians V

Crime Scene Evidence and Preservation

*

V

Major Interaction with Other Federal, State, and Local Agencies

*

V

Scene Communication Overload

*

V

Chaos

V

Resources Overwhelmed V

Secondary Device Designed to Kill Responders

*

V

Pre-Incident Indicators

V

Increased Time Required for Response

V

Dissemination Indoors

V

Major Psychological Impact V Long Term Health and * Restoration Concerns V * An asterisk denotes a characteristic that may have some degree of overlap between a classical HAZMAT incident and a WMD incident. However, there are differences. V The delta denotes a significant difference between a classical HAZMAT incident and an incident involving WMD materials.

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Let us further explore how a HAZMAT incident differs from a WMD terrorism incident, and explore the challenges and consequences presented by those differences. Deliberate attack: Many of you are already familiar with a HAZMAT response. HAZMAT incidents are generally accidents. A WMD terrorism incident is intentional, even if non-warfare gases, chemical, or biological materials are employed. Supertoxic material: Most of the typical military chemicals are significantly more toxic than industrial ones. For example, Sarin (GB) is close to 200 times more toxic than chlorine and is approximately 60 times more toxic than methylisocyanate. This is the chemical leaked in the HAZMAT incident in Bhopal, India, that caused 200,000 casualties; 10,000 were severely affected and 3,300 died. Biological agents have the potential to kill millions. Some of the biological agents are contagious. They not only infect the people initially exposed, but can travel through the infected people and infect people away from the initial release site. Small amounts of radiological material can contaminate large areas for years. Small quantities of WMD agents have the capability to produce large numbers of casualties (symptomatic and psychological) *Early hazard identification: Early identification of hazardous material is relatively easy. Many countries require placards on vehicles or shipping papers (manifests) that identify the material. Workers or the location of the incident can also provide information on the identification of the material involved in the incident. The WMD agents, on the other hand, will be difficult to identify (except by signs and symptoms, which will give you a clue) because none of the identifiers associated with an HMI will normally be present. Compounding the situation is the fact that while the signs and symptoms of most chemical releases will be evident immediately, those from a radiological release may be slightly delayed depending on the dose received, and those from most biological releases may be significantly delayed. *Mass casualties/many fatalities: In the vast majority of cases, a hazardous material incident does not result in mass casualties or fatalities. In the Tokyo incident, there were 5,500 casualties; all but 1,200 were psychological. However, a WMD terrorism incident has the potential to create mass casualties and many fatalities, particularly a biological attack. Some industrial incidents, such as the release in Bhopal, India of methyl isocyante, caused thousands of casualties; but this is atypical. Mass decontamination: A HAZMAT incident typically requires the decontamination of only a few people. Experience from the responders in our courses suggests that decontamination of 10 persons is typical while the decontamination of 45 people is perhaps the most that have had to be decontaminated after an incident. A WMD terrorism incident will require the decontamination of numerous individuals, perhaps hundreds or even thousands. Moreover, we may have to decontaminate large structures, buildings, and real estate in order to render area safe for human use again. Currently, there are no effective means to do this economically. Mass decontamination will require assigning some of the emergency responders to perform this very important task. Other units may be able to assist if they are properly trained and equipped. Please remember that another part of the HAZMAT team should be committed to establishing a separate decontamination corridor to provide technical decontamination for responders. Unusual risk to emergency responders and civilians: The WMD materials are extremely toxic; most are heavier than air and may be persistent. As a result, they pose a greater threat to responders than most hazardous materials for a longer period of time in the area where the responders must execute their mission.Because of their higher toxicity, the downwind hazard could be a greater risk than in a conventional hazardous material incident.

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*Crime scene and evidence preservation: Local laws dictate whether or not a HAZMAT incident may or may not be labeled as a crime scene. Factors such as environmental laws, criminal liability of the operators of vehicles, observance of regulations, etc., will dictate this. An act of WMD terrorism, even if it is a hoax, is a federal crime in many countries; therefore, the site of the incident mandates a crime scene designation and evidence preservation. These are key elements for apprehension and prosecution of the perpetrator(s). *Major interaction and coordination with local, state, and federal agencies: This may be required in a HAZMAT incident, but most definitely will be required during a WMD terrorism incident due to the complexity of the situation and the public interest. A WMD terrorism incident will require the Incident Commander to effectively use an Incident Command System and to assign areas of responsibility to other responding personnel. In a WMD terrorism incident, the Incident Commander will be faced with managing and coordinating multiple agencies - some requested, some not. In a WMD terrorism incident, one liaison officer and several assistants may be needed to serve on the Incident Commander's staff to assist in the management and coordination of all the responding agencies, and will be critical to the timely and efficient application of resources. The staging area(s) manager(s) will have to control a much larger number of resources than during a HAZMAT incident. Scene communications overload: Due to the large interaction among various agencies, communications overload will be the most significant challenge during and after a WMD terrorism incident. The Incident Commander must deal with communications overload immediately or he/she will soon become overwhelmed. While cellular phones may be useful during the early moments, cell sites could quickly become jammed by the media and others. Chaos: Because of the public's concern about the consequences of exposure to WMD materials, there will be more mass panic and hysteria in a WMD terrorism incident than in a HAZMAT incident. Fire companies and HAZMAT units alone will be unable to maintain scene safety and security at a WMD terrorism incident. The importance of proper and immediate employment of law enforcement to manage scene control in a WMD terrorism incident cannot be overemphasized. Public officials should also make a presence. This was very effective during the incidents in the U.S. during the Fall of 2001, and helped to minimize panic. Resources overwhelmed: In a HAZMAT incident, intervention can be handled methodically and deliberately by the HAZMAT team. Due to the number of victims, the catastrophic nature of injuries, and the speed with which WMD agents can kill, it is expected that the normally deployed assets, such as fire companies, HAZMAT teams, Emergency Medical Services (EMS) responders, Explosive Ordnance Disposal (EOD) units, and law enforcement units, will quickly be overwhelmed. Resources, which include hospitals, protective equipment, decontamination equipment, Pharmaceuticals, cover for victims after decontamination, and transportation for victims will likewise be overwhelmed. A WMD terrorism incident will demand immediate intervention on the part of the first responders if lives are to be saved. Secondary device designed to kill responders:Recent events suggest that emergency responders need to be aware of the threat of secondary devices or multiple WMD devices at or near the incident site. One should always assume in a terrorism incident that a secondary device might be present when responding to an incident scene. Secondary devices may be a conventional high explosive fill, or may be constructed to deliver additional WMD material. The threat of secondary devices presents unique tactical considerations. Pre-incident indicators: Before a terrorism incident, the law enforcement or intelligence community may have some indications and intelligence information about the

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possibility of a terrorism act. These indicators may take the form of phone calls to authorities describing the event or stating the reason for the attack, or might be the discovery of the dissemination device before it releases the agent into the atmosphere. There is also a possibility that a pre-incident call could be a hoax, or even a diversion, to tie up resources. Increased Time Required for Response: Due to the large numbers of casualties that may be involved, time in the hot zone may need to be increased for effective rescue and reconnaissance of the area. Also, the super toxic nature of the materials may require that additional time/steps may be needed for decontamination of responder personnel. All of these factors together result in an increased time that is needed in protective gear by the responder forces. Dissemination Indoors: To increase the potential for injury, terrorists may disseminate WMD materials indoors, causing a higher concentration and avoiding the effects of weather that may degrade and dissipate the agent. Most HAZMAT incidents take place outdoors. Major Psychological Impact: A WMD terrorism incident will create large numbers of psychological effects on casualties, thus magnifying the mass casualties situation. Long Term Health and Restoration Concerns: We are just becoming familiar with the long-term effects of exposure to conventional chemical warfare agents. There are still lingering effects in the population of people exposed to mustard agent some twenty-five years after the use of this agent during the Iran-Iraq War. Likewise, we still need to develop guidelines for how "clean is clean" since this will impact use of buildings or other assets after an attack. How low can we really detect chemical warfare agents? Are they still harmful? How clean is a room from anthrax? One spore? One thousand? And of course the use of radiological materials is of concern, although easy to detect.

3. Key Elements for Effective Training The increasing threat of the use of WMD materials in an act of terrorism places both civilian and military personnel at risk. Key components of the infrastructure, such as postal systems, water supplies, arenas, buildings, etc., can also be affected by such an attack. Regardless of what or who is attacked, the training approach described here provides a proven process for preparing both civilian and military responders to respond to asymmetric attacks involving WMD materials and for the mitigation of the impact of such an attack. The Anthrax attacks of 2001 in the U.S. confirmed that planning, training, exercising, and the use of appropriate resources could substantially reduce the impact of such an attack on both personnel and infrastructure. The target audience is the command staff, emergency responders (fire and rescue, HAZMAT, law enforcement, and security), medical responders (EMS and healthcare providers), and mutual aid responders. The program includes the following key elements: a. Command and Staff Workshop: This workshop is intended to develop an awareness within the command and staff of the implications of a WMD incident and to provide an overview of the WMD Training Program. The workshop is conducted in an interactive forum with lecture, discussion, case studies, and video clips. b. Baseline Exercise: The baseline assessment is accomplished with a facilitated tabletop exercise using a chemical agent scenario. The baseline will provide a focus for future preparedness efforts and allow the development of a strategy for enhancing preparedness. The baseline assessment will also include a review of any existing disaster preparedness plans and

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standing operating procedures (SOPs), and any previous vulnerability assessments that may have been conducted. c. Training: The training objective is to develop an awareness and understanding of the implications of a WMD incident to your installation and its mission. The training step will provide installation participants with a solid foundation and a much greater grasp of the unique considerations involved in responding to a WMD incident, prior to the planning step. The training includes six responder courses, covering the following topics: WMD signs and symptoms, the threat of WMD terrorism, detection, identification, sample collection, downwind hazard analysis, personal protection, decontamination, medical management of casualties, and consequence management tactics, techniques, and procedures. The training is conducted utilizing classroom lecture, videos, practical exercises, and tabletop exercises. The courses are described in detail in the next session. d. Planning Workshop: The planning phase involves either review and refinement of existing response plans/annexes by WMD experts, or joint development of response plans/annexes, if current plans do not exist. WMD planning assistance is conducted during a two-day facilitated workshop. The planning stage is critical in defining roles and responsibilities of each entity involved in your installation's response, and for conducting a comprehensive inventory of existing response assets and capabilities. The workshop provides an excellent mechanism for building an effective, executable Antiterrorism/Force Protection (AT/FP) plan that will be effective in responding to a WMD incident. Once the plan or draft plan is in place, the next step involves the conduct of a tabletop exercise to validate the plan and reinforce the training. e. Technical Assistance: The Technical Assistance effort complements the planning, training, and exercise phases in the WMD Installation Preparedness process by filling in any technical voids that exist. Technical Assistance ranges from vulnerability assessments of facilities to a WMD incident to equipment consultations. Technical Assistance is dependent upon the level of effort required. This effort can be conducted at any time during the process. f. Chemical/Biological (CB) Tabletop Exercises (TTXs): TTXs tend to focus on the command, control, and communication aspects of the response, and how various response functions combine into a total integrated system. The TTXs will assist installation participants and mutual aid partners in gaining a comprehensive understanding of an emergency response to a WMD incident, and specifically their role in that response. The TTXs also assist in focusing objectives for the field training exercise.

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Figure 28. Key elements of WMD preparedness

g. Chemical Field Training Exercise (FTX): A chemical incident functional or field training exercise should be conducted to test, to the maximum extent possible, all or some aspects of the AT/FP plan and its response to a WMD incident. The exercise will be tailored to meet the specific objectives of the exercise planning committee. The functional exercise provides a practical means to assess the plan's ability to be executed in an effective and timely manner. It provides insight into required changes to the plan, as well as areas of the response requiring additional work. h. Sustainment: The provider provides sustainment of training by leaving behind compact discs (CD) with all course, exercise, and planning materials. The materials can be used as part of an annual in-service training program. The training and exercises are continually updated to incorporate technical breakthroughs and improved response studies.

4. WMD Courses The WMD Training Courses are based on 26 performance objectives. These performance objectives were developed using various guidelines, established by the U.S. Government and professional organizations [1,2,3]. These courses are intended for personnel who have already been trained at one of the "standard" OSHA defined training levels: Awareness, Operations, Technician/Specialist, and Incident Commander. The WMD IP courses do not replace the OSHA training requirements, nor can they be substituted for credit. The WMD IP courses supplement the OSHA courses with additional, specialized WMD training.

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Figure 29. Elements of a successful WMD Training Program. The arrows on the slide show the WMD prerequisite training classes

a. For responders who are trained at the Awareness level, the Responder Awareness course provides 4 hours of WMD awareness instruction. The goal of this course is to enable students to recognize the signs and symptoms of chemical, biological, and nuclear/radiological materials exposure, recognize potential dissemination devices, to make proper notifications, and to take defensive actions to safeguard themselves. b. For responders who are trained at the Operations level, both the Responder Awareness and the Responder Operations are recommended. The Responder Operations course will provide the technical aspects of WMD incidents and the defensive actions required for responders to protect themselves. These courses provide 4 hours each, for a total of 8 hours of training. c. For responders who are trained at the Technician (or specialist) level, there are three courses offered:Technician-Emergency Medical Services (EMS): This course provides 8 hours of instruction on the technical aspects of WMD incidents and the defensive actions required by EMS responders to protect themselves as well as triage, treatment, and transport of victims. Technician-HAZMAT Level: This course provides 16 hours of instruction on the technical aspects of WMD incidents and the offensive actions required for FIAZMAT responders to protect themselves and their installation. This course covers the same subjects as the Responder Awareness and Responder Operations, but at a more advanced level. (3) Medical Facility Provider: This course provides 8 hours of instruction to emergency room physicians, nurses, and hospital safety administration personnel on the medical technical aspects of WMD incidents and the defensive actions required to protect themselves and their medical facilities as well as the proper medical management of victims. d. The Incident Command course provides 8 hours of instruction for incident commanders and key responders on the challenges, consequences, and special considerations of managing a WMD incident. This course requires one of four prerequisites: Responder Awareness, Responder Operations, Technician-EMS, Technician-HAZMAT, or Medical Facility Provider.

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e. In addition to the responder level training, we have prepared a 30-minute Employee Awareness video that provides basic WMD awareness to a diversified audience of employees at high-risk locations such as large, enclosed facilities with high concentrations of people. There is a separate module in the video that specifically targets 911 operators/dispatchers. 5. The Ultimate Goal of Training - Preparing the Response Plan Once the training is complete, the next step in the process is to develop an integrated response plan that covers all phases of an incident. The primary purpose of this plan is to safeguard lives, preserve health and safety, secure and eliminate the hazard, protect property, and mitigate the damage of the incident to the facility and the environment. Preparedness also maintains the confidence of the community in the responder element and also facilitates compliance with directives. All incidents have several phases to them. The first phase is Crisis Management. This phase consists of taking steps to prevent an attack, recognizing an attack, assessing the situation, and calling in the appropriate assets to confront the problem. At the local level, one can conduct a vulnerability assessment and determine response needs. The next phase is Consequence Management. It is this phase whereby one needs to develop response plans involving evacuation or sheltering in place, sampling and survey of the site, rescue and control of victims, decontamination of people and facilities, evidence collection, and medical intervention. The last phase, Mitigation and Restoration, is the phase whereby one returns the situation to normal. In a crisis the two most important phases are the first two: Crisis Management and Consequence Management. The relationship between these two phases can be summarized as per Figure 30 [4,5].

Figure 30. Relationship between Crisis Management and Consequence Management

As the figure shows, this effort requires considerable coordination among assets. Law enforcement plays the key role during the crisis management phase while the responder community plays the key role during the consequence management phase.

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Planning starts with the formation of a planning team. This team should consist of all of the stakeholders in the plan. The size of team will depend on operations, requirements, and resources. Examples of groups to include in the planning team include management, public affairs, legal, law enforcement, responders, and others.

Figure 31. Steps in an effective planning process

The planning team works together to perform a "front-end" analysis of the problem whereby potential vulnerabilities to various threats are determined. Response needs are then determined. It is this information that leads to the determination of appropriate, practical, and feasible response actions. These actions should then be documented to form the basis of the response plan. These actions are summarized in Figure 31. Once the plan has been developed and coordinated, it should be distributed for review and revised as needed. For a second review, conduct a tabletop exercise with management and personnel who have a key emergency management responsibility and have been trained on the plan. For example, in a conference room setting, describe an emergency scenario and have participants discuss their responsibilities and how they would react to the situation. Based on this discussion, identify areas of confusion and overlap, and modify the plan accordingly. Distribute the final plan to senior managers and key members of the emergency response element. Once the plan has been adopted, it should then be trained to all the stakeholders. Training builds competence and competence builds confidence. All players that might respond to a WMD incident should participate in established training and exercise programs to the extent possible, to understand each others' roles and responsibilities during and after the incident. If any shortfalls are found during the training exercises, the plan should be modified accordingly, and the new response should be implemented into the next cycle of training. Thus training and exercises becomes a continuous cycle of plan, train, exercise, and revise (Figure 32). An effective response to a WMD incident requires this [6].

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Figure 32. Effective elements of a WMD response - Plan, Train, Exercise, and Revise

6. Conclusions Prior to the initiation of this program, there was little, if any, consideration given to response against WMD agents. Through this process, which is a systematic analysis of concepts, plans, procedures and equipment, our first responders are able to enhance their effectiveness when dealing with chemical and biological terrorist incidents. This program showed that the training using this approach could provide emergency managers and first responders logical response approaches. These approaches can be used as a starting point to improve their overall preparedness for chemical and biological terrorism. This training proved invaluable during the anthrax attacks of the Fall of 2001. Many of the personnel involved in the response effort were trained under this program or actually served as trainers. Response plans were in hand and the professionals knew what their jobs would entail. The low death rate, minimal panic, and rapid response all proved that this program was effective.

References [1] [2] [3] [4] [5] [6]

Occupational Safety and Health Administration (OSHA) 29 Code of Federal Regulation (CFR) 1910.120 (Hazardous Waste Operations and Emergency Response). National Fire Protection Agency (NFPA) 472 (Professional Competence of Responders to Hazardous Materials Incidents) NFPA 473 (Competencies for Emergency Medical Services (EMS) Personnel Responding to Hazardous Materials Incidents, and the Joint Commission on Accreditation of Healthcare Organizations). Federal Emergency Management Agency, Federal Response Plan, Bulletin 9230.1-PL ,1992. Federal Emergency Management Agency, FEMA 229, change 11, 1997. Office of Personnel Management, WMD preparedness Executive Workshop Manual, p. 149, 2003.

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Part 4

Applied Research

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Brucellosis-A Biowarfare Threat and Public Health Concern Philip H. ELZER Louisiana State University AgCenter and School of Veterinary Medicine Department of Veterinary Science 111 Dalrymple Building, Baton Rouge, LA; 70803; USA Abstract. Brucellosis is a zoonotic disease with potential for use as a biowarfare agent. The genus Brucella contains six recognized species including B. abortus, B. melitensis, B. suis, B. canis, B. ovis, and B. neotomae. All of the brucellae are Gram negative, facultative intracellular pathogens, which survive and replicate in host macrophages. The hallmarks of animal brucellosis, in both domestic and wild animals, are abortion, infertility and reproductive failure. If used in an agroterrorist attack, these organisms could decimate a generation of livestock and companion animals. Fever, chills, malaise, arthritis, dementia, and possibly even death characterize the disease in man. Human brucellosis, also known as undulant fever or Malta fever, is caused by only four species of brucellae. B. melitensis is the most infectious to man in that 1-10 colony forming units are thought to cause disease followed by B. suis (1000-10,000), B. abortus (100,000), and finally B. canis (> 1,000,000 in an immuno-compromised individual). There are several animal vaccines that are safe and effective; however, they are all pathogenic to man. Currently there are no vaccines approved for use in humans. Brucellae are characterized as BSL-3 organisms due to their ability to infect humans through aerosol exposure, which makes them an ideal bacterial agent for use by terrorists. If the general public were exposed to this biowarfare agent, medical resources would be stretched 10 fold to take care of the large number of people that would be debilitated by this organism. With the recent tragic events in the United States and throughout the world, three species of Brucella are considered "agents of mass destruction." The need for a human vaccine is paramount.

There are six recognized species in the genus Brucella characterized by their host specificity and ability to cause chronic infections in many animals and man. Brucellae are know to cause abortion and infertility in wild and domestic ungulates [1,2,3]- In addition to the agricultural economic loss associated with brucellosis, several of the brucellae are zoonotic agents. After the tragic events associated with September 11, 2001, in the United States, three species of Brucella are also considered "agents of mass destruction." Therefore the development of a safe and efficacious vaccine for all ungulates and humans is a paramount goal for brucellosis researchers. The properties of the ideal biological weapon are that the agent should be highly contagious and consistently produce a known disease or syndrome. It is best if it can be disseminated throughout the environment, i.e. aerosolized; and it needs to be stable under production, storage, and delivery to target. However the organism should have a short survival time in the target area so it is not a threat at later time points to delivery personnel. It is preferable that the target populations have little or no natural resistance. Brucellae are the

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consummate biological warfare agents in that they meet all of these criteria. They are highly infectious (see Table 11), can be easily aerosolized, are stable during production; and due to its sensitivity to direct sunlight, it is destroyed in the environment over time. Since there are no human vaccines against brucellosis, most, if not all populations, have little or no natural immunity to this organism. Brucella species were weaponized in the United States following WWII. These species were field tested in cluster bombs in 1955. However all of the munitions using this agent were destroyed in 1969. If used in an agroterrorist attack, these organisms could decimate a generation of livestock and companion animals. With the concentration of livestock, lack of genetic diversity, increased farm sizes, importation of animals, and increased international travel, agriculture around the world is very vulnerable to a terrorism attack. The effects of agroterrorism would be immediate in that there would be mass hysteria manifested by panic buying of the stocked products. There would be demoralization due to the loss of food, plants, companion animals, and economic devastation. This economic destruction could become worldwide if a country's main agricultural export product or products were boycotted by numerous nations [4]. All of the brucellae are Gram negative, facultative intracellular pathogens, which survive and replicate in host macrophages. In man, brucellosis, also known as undulant fever or Malta fever, is caused by only four species of brucellae (B. abortus, B. melitensis, B. suis, and B. canis). Human infection is caused by ingestion of infected raw milk products, exposure to infected animals, and aerosolization of the organism. Brucellosis in man is characterized by a cyclical fever that starts two to three weeks post-exposure. Night sweats, headaches, backaches, and general malaise are symptoms associated with acute infection. Chronic brucellosis can lead to a debilitating condition, including arthritis, dementia and even death. Patients with chronic brucellosis have frequent relapses, and 2/3 of these individuals develop psychoneurosis. Human brucellosis can be treated with the administration of tetracycline or doxycycline in combination therapy with rifampin or gentamicin [5]. Due to the potential use of biowarfare agents, public health officials need to be constantly aware of possible intentional exposures to infectious agents. There are needs for rapid and specific detection devices for environmental releases and medical samples. Improved surveillance and diagnostics with effective communication amongst numerous regulatory and emergency agencies is necessary to contain and control an exposure or outbreak. An example of this is illustrated by a case report of a 38 yr old woman from New Hampshire, USA, who was thought to have been exposed to Brucella spp. This report highlights several aspects of the needed public health response to a possible biowarfare agent [6]. The CDC classifies B. abortus, B. melitensis and B. suis as "agents of mass destruction" and as category B organisms. Brucella canis, a less virulent strain, can cause human disease but only when contracted by an immuno-suppressed individual. B. melitensis is the most infectious to man in that 1-10 colony forming units (cfu) are thought to cause disease followed by B. suis (1000-10,000 cfu), B. abortus (100,000 cfu), and finally B. canis (> 1,000,000 cfu) in immuno-compromised individuals (Table 11). Brucellae are characterized as BSL-3 organisms due to their ability to infect humans through aerosol exposure, which makes them an ideal bacterial agent of mass destruction [7]. If the general public were exposed to this biowarfare agent, medical resources would be stretched 10 fold to take care of the large number of people that would be debilitated by this organism. Currently there is no approved vaccine for human use, and a vast majority of the animal vaccines are virulent to man. Thus, there is a need to find a safe and efficacious vaccine that can be used in humans [7].

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Table 11. Human Susceptibility to Brucella spp

goats, sheep

Human Virulence HIGH

Number of organisms 1-10

B. suis

swine

High-Moderate

1,000-10,000

B. abortus

cattle

Moderate

100,000

B. canis

dogs

Low/immuno-

> 1,000,000

Brucella spp. B. melitensis

Natural Host

suppressed

References [1]

[2] [3] [4] [5] [6] [7]

Timoney, J.F., J.H. Gillespie, F.W. Scott, and J.E. Barlough. 1988. The Genus Brucella. Hagan and Brunner's Microbiology and Infectious Diseases of Domestic Animals. 8th edition. Cornell Univ. Press, Comstock Publishing Assoc., Ithaca, New York. Burrows, W. 1968. Brucella. In Textbook of Microbiology. 19th edition. W.B. Saunders Co.,Philadelphia, Pennsylvania. Nicoletti, P. 1980. The epidemiology of bovine brucellosis. Adv. Vet. Sci. Compar. Med.24:70. Huxsoll, D.L., W.C. Patrick III, and C.D. Parrott. 1987. Veterinary services in biological disasters. J. Am. Vet. Med. Assoc. 190:714-722. Young, E.J. 1989. Clinical manifestations of human brucellosis, p.97-126.In:E.J. Young and M.J. Corbel (Ed.), Brucellosis: Clinical and laboratory aspects. CRC Press, Boca Raton, FL. Suspected Brucellosis Case Prompts Investigation of Possible Bioterrorism-Related Activity—New Hampshire and Massachusetts, 1999. MMWR 49 (23); 509-512. Acha, P.N., and B. Szyfres. 1980. Zoonoses and communicable diseases common to man and animals, p. 28-45. Pan American Health Organization, Washington, D.C.

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Medical Countermeasures Research at Dstl Porton Down, UK Petra C. F. OYSTON CBS Porton Down, Salisbury, Wiltshire, SP4 OJQ, United Kingdom Abstract. For almost a century Porton Down has been the site of defence research to provide protective measures for British Service personnel against chemical and biological warfare (BW). A wide variety of BW agents potentially pose a threat and Countermeasures are being devised and evaluated at Dstl Porton Down to combat this threat. Two approaches to provide effective medical Countermeasures are being undertaken. The first approach is to use prophylaxis or post-exposure therapies, such as antibiotics and anti-toxins. The second approach is to use vaccines, which are given before exposure to the agent and have the potential to provide very high levels of protection from disease. Many of the vaccines available against BW pathogens are not suitable for use. We are therefore undertaking research to produce effective, licensed vaccines for protection against BW agents, such as plague and tularemia.

1. A Brief History of Porton Down On the 22nd April 1915, the Algerian Division of the French Army was attacked with 150 tons of chlorine gas released by the German Army over a front of about 4 miles. The effect was devastating. More attacks followed, with the British Army first experiencing an attack on 1st May. This led to calls for retaliation in kind and for the development of a defensive capability to protect the troops. As a result, the research establishment was set up at Porton Down, to research an offensive chemical warfare (CW) capability and to provide defensive measures for the Armed Forces. This research continued for several decades, and biological warfare (BW) agents were added to the research programme. However, the moral and political climate was changing rapidly in the second part of the last century. Thus, by the late 1960s the UK renounced the development of BW weapons and became a signatory to the 1972 Convention on the Prohibition of the Development, Production and Stockpiling of Biological and Toxin Weapons, and the Chemical Weapons Convention. All research now undertaken at Porton Down is defensive in nature, with the aim of protecting the Armed Forces in the event of chemical or biological attack. The history of Porton Down can be found elsewhere [1, 2] for those seeking more detail.

2. Medical Countermesures against BW agents Most biological defence is subsumed in chemical defence, in that the respirator will prevent inhalation of aerosolised agents. However, a critical aspect of such a strategy is knowing when to don the respirator in the event of an attack, or when it is safe to remove it. It would be

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expected that significant numbers of personnel would be affected in the event of a BW attack, and for this reason there has been a programme of work at Porton Down evaluating various approaches to protect the Armed Forces should such a scenario arise. 3. Antivirals, Antibiotics and Antitoxins Biopharmaceutical companies spend millions of dollars a year developing new antimicrobial compounds, but the number of new antibiotics and antivirals appearing are few. At CBS Porton Down, we screen commercially available and new lead compounds against some of the pathogens of concern in order to ensure we have the most effective treatment possible, whether as a prophylactic or as a post-exposure therapy. Antibiotics are the best known example of antimicrobials, but are active only against bacteria. In principle, antibiotics could provide protection against all bacterial BW agents, but many bacterial BW agents are inherently resistant to antibiotics. For example the pathogenic Burkholderia are resistant to most available antibiotics, and possess multi-drug eflux pumps which render many compounds useless [3]. For some BW agents, antibiotics control the disease but the disease reappears when the antibiotic is withdrawn. This has been widely reported for melioidosis, where the period between therapy and recrudescence can be as long as over twenty years [4]. The use of antibiotics is also complicated by the fact that an aggressor may develop antibiotic-resistant strains of BW pathogens. There have been reports, for example, of a clinical isolate of plague which was multiply antibiotic resistant [5], showing how easily pathogens acquire resistance genes. Furthermore, Stepanov et al reported the production of a multiply antibiotic resistant strain of anthrax [6]. Such resistance, whether natural or engineered, would complicate therapy in the event of infection. Finally, the logistics of giving repeated doses of antibiotics to thousands of troops over an extended period of time, coupled with the side-effects this may induce in the soldiers, makes this an undesirable approach to prophylaxis in a BW theatre. There are few, if any, suitable commercial antivirals. The problem in devising antivirals lies with the way in which the virus uses the host cell to replicate, meaning that it is difficult to interfere with viral growth without also detrimentally affecting the host. In addition, most antivirals developed to date are virus-specific. An antitoxin is anything capable of inhibiting the activity of a toxin. This could be either a compound capable of inhibiting the activity of the toxin, or an antibody which binds to the toxin and thus blocks its action. Currently there are no inhibitory compounds available to treat intoxication, and all antitoxins at present are antibody preparations. The toxic doses of toxins such as botulinum toxin or ricin are quite high, so it is likely that an attack would trigger detectors on the battlefield warning of the hazard and possible exposure of personnel. In such a scenario antitoxins could be used for treatment of exposed individuals to prevent the onset of toxic symptoms. Antisera are generated by immunising animals, such a goats or sheep, with an inactivated form of the toxin (such as a toxoid) followed by purification of the antitoxin from repeated production bleeds. The plasma obtained is purified to IgG. Adverse affects such as analphylaxis are common when patients are given whole IgG products derived from a foreign host and at Porton Down we are looking at ways of producing effective antisera from fragments of the IgG, namely F(ab')2, Fab' and Fab. The rational behind these studies is that the reactogenic Fc portion of the molecule has been removed and thus the smaller molecules should result in fewer side effects. The smaller size of the fragments has the added advantage that they may reach their site of action more quickly than IgG and be more effective for post

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exposure therapy [7]. This approach is currently being investigated for antitoxins against botulinum toxin and ricin. A further point to note is that in general it is much faster to obtain a licence for use of antitoxins in humans than to licence a vaccine, permitting a usable product to be generated relatively quickly. 4. Vaccines Vaccines have the potential to provide high levels of protection against BW agents. They must be given prior to exposure, but once the course is complete then the individual requires only boosters at lengthy intervals. However, the pathogens of concern in a BW scenario are not usually those of concern to every day public health. As such, there are either BW agents against which we have no vaccine, or the vaccines are less than ideal. For example, the painful reaction many soldiers suffered following the initial dose of the anthrax vaccine in the Gulf War dissuaded many of them from returning to complete the course. Also, the vaccine must be able to induce an immune response capable of protecting against pneumonic infection. For example, the current plague vaccine was shown not to protect against pneumonic plague [8]. To reduce the risk of side-effects and to ensure effective immunity, the UK vaccination policy is to use only licensed vaccines to protect the Armed Forces. Research is underway at CBS Porton Down to develop licensed vaccines against the BW agents of concern, some of which is described in detail below.

4.1. Plague Two killed whole-cell vaccines are currently available for use. The first (Commonwealth Serum Laboratories Ltd., Australia) consists of heat-killed cells of fully virulent Y. pestis in saline. The second (Miles Inc., Canada) contains formaldehyde-killed Y. pestis cells. The regime consists of three primary doses administered intra-muscularly, followed by annual boosters. The protection induced by these killed whole cell vaccines has been shown to be variable and short-lived. Since the protection induced is highly variable and high levels of adverse reactions occur, the killed vaccines are unsuitable for general use, so are restricted to individuals who work with the pathogen or are potentially exposed to virulent strains. The killed vaccine fails to induce secretory IgA [9] and may not protect against pneumonic transmission [8]. A live vaccine is also available, derived from strain EV76. Strain EV76 is poorly defined, and resulted in excessive adverse reactions and highly variable responses between individuals [10]. In addition, EV76 was lethal for non-human primates [11] and resulted in hospitalisation of a proportion of human recipients [10]. Thus this strain is not suitable for general use. A second-generation vaccine of recombinant proteins developed at CBS Porton Down is currently undergoing clinical trials. The antigens in the formulation are the capsular Fl antigen and the V antigen, a component of the Type III secretion system [12]. These had been identified in 1970 as the essential antigens which must be produced by an attenuated Y. pestis strain for induction of an effective immune response [10]. The first challenge in developing this second generation vaccine was obtaining a source of antigens which could be produced on a large scale. The caf operon encoding Fl antigen is carried on one of the three Y. pestis virulence plasmids [13]. The monomeric unit of the Fl antigen has a molecular mass of 15.5

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kDa, but there is aggregation of the monomers into large complexes with masses in excess of 3 Mda [14]. This aggregation occurs spontaneously in solution. Cloning the cafoperon allowed high level expression of recombinant Fl antigen, a much safer source than isolating the antigen from plague cultures. The V antigen has a key structural and regulatory role in the type III secretion system of Y. pestis [15]. The protein has a molecular mass of 37 kDa, and similarly to Fl antigen it spontaneously aggregates [12]. The folding of V antigen is important to immunogenicity, as studies have shown that B-cell epitopes in the protein are conformational [16, 17]. V antigen is more difficult to produce than Fl antigen due to inherent instability of the protein. It is produced as a GST fusion, which is purified and subsequently cleaved to give pure V antigen. The Fl and V antigens have been shown to be able to induce a protective immune response individually, but a combination of the proteins had an additive effect. The optimum ratio for immunisation was shown to be 2:1 (F1:V) [18]. The interdependency of immunogenicity and conformation led us to deduce that combined free sub-unit was the approach to follow, rather than the production of a genetic fusion of the two antigens, an approach undertaken by other workers. At present the antigens are being taken into clinical trials in a formulation with alhydrogel as an adjuvant, given intramuscularly. Although this vaccine was shown to induce protection in mice against aerosol challenge, work is now underway to develop a vaccine which is mucosally delivered. In addition to inducing immune responses in the respiratory tract, mucosal delivery has the advantage of being needle-free. Much work has been undertaken to deliver the antigens using biodegradable polymeric microencapsulation. A preparation suitable for nasal administration has been produced which is fully protective against aerosol challenge in the mouse model after just two doses [19]. 4.2. Tularemia Tularemia in humans can occur in several forms depending to a large extent on the route of entry of the bacterium into the body. Tularemia can be a severely debilitating disease, especially when caused by F. tularensis subspecies tularensis. The most acute form of disease is associated with the inhalation of bacteria, although pneumonic disease can also occur as a complication of other forms of tularemia. The Live Vaccine Strain (LVS) vaccine remains the only effective vaccine against tularemia developed to date. However, this vaccine is not currently available, though work to licence it is underway in the USA. That this attenuated mutant of F. tularensis can induce protective immunity suggests that this approach to vaccine development is feasible. In a range of other pathogens, the introduction of defined mutations into genes required for growth of the pathogen in vivo has yielded safe and effective vaccines. The construction of a defined attenuated mutant of F. tularensis could provide a safe, effective and licensable tularemia vaccine. The aromatic amino acid and purine biosynthetic pathways have already been identified from genome sequence information as targets for the construction of a defined attenuated mutant [20]. However, the utility of this approach is limited because there are as yet no methods for the construction of allelic replacement mutants of F. tularensis. Work is currently underway at CBS Porton Down to develop methods to create isogenic allelic replacement mutants. Mutants of F. novocida compromised in their ability to replicate inside macrophages have been produced at the University of Victoria [21]. We are now examining these mutants for attenuation in the mouse infection model, and for their ability to induce a protective immune response. Interestingly, in one of these mutants the transposon interrupts a

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gene required for purine biosynthesis, and as stated above, purine mutants are of interest because in other bacterial species they have been used as attenuated vaccine strains. A second approach we are following is the evaluation of subunit vaccine antigens. One such potential protective subunit is lipopolysaccharide. Immunisation with lipopolysaccharide was able to protect mice against challenge with the LVS strain, but not against the more virulent Schu4 [22]. Therefore, this may form one part of a vaccine, but would require additional antigens for full protection. Other antigens would probably be proteins expressed by the pathogen. A crude outer membrane preparation of F. tularensis was able to protect mice, but so far individual proteins have been unable to induce a protective immune response [23]. Future progress towards identifying suitable antigens should be accelerated by the availability of the F. tularensis strain Schu4 genome sequence data. Analysis of the immune response to the LVS vaccine has shown a heterogeneity of immunogenic epitopes recognised in humans and this indicates it is likely that a sub-unit vaccine will need to be composed of a number of protective antigens to provide protection against virulent strains.

5. Summary In order to protect the UK Armed Forces, medical countermeasures such as antibiotics and vaccines against BW agents are being evaluated at CBS Porton Down. A second generation plague vaccine is currently undergoing clinical trials, and further work to develop a needlefree, mucosally delivered vaccine is on-going. For some of the pathogens, for example F. tularensis, nothing is known about the basis of virulence of the pathogen or suitable antigens for vaccine development. Genome sequence data will underpin all research efforts on these pathogens, but a priority remains the development of good genetic tools for manipulation of some of these organisms.

6. Acknowledgements Crown Copyright 2003 Dstl References [1] [2] [3]

[4] [5] [6] [7]

Carter GB. Porton Down: 75 years of Chemical and Biological Research. London: HMSO; 1992. Carter GB. Chemical and Biological Defence at Porton Down 1916-2000. London: The Stationery Office; 2000. Moore RA, DeShazer D, Reckseidler S, Weissman A, Woods DE. Efflux-mediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei. Antimicrobial Agents and Chemotherapy 1999; 43: 465-470. Sanford JP. Melioidosis: forgotten but not gone. Trans. Am. Clin.Climat. Assoc. 1977; 89: 201-205. Guiyoule A, Gerbaud G, Buchrieser C, Galimand M, Rahalison L, Chanteau S, Courvalin P, Carniel E. Transferable plasmid-mediated resistance to streptomycin in a clinical isolate of Yersinia pestis. Emerging Infectious Diseases 2001; 7: 43-48. Stepanov AV, Marinin LI, Pomerantsev AP, Staritsin NA. Development of novel vaccines against anthrax in man. Journal of Biotechnology 1996; 44: 155-160. Mayers CN, Hoiley JL, Brooks T. Antitoxin Therapy for botulinum intoxication. Reviews in Medical Microbiology 2001; 12: 1-9.

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[10] [11] [12] [13] [14]

[15] [16] [17] [18] [19] [20]

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Russell P, Eley SM, Hibbs SE, Manchee RJ, Stagg AJ, Titball RW. A comparison of plague vaccine, USP and EV76 vaccine induced protection against Yersiniapestis in a murine model. Vaccine 1995; 13: 1551-1556. Oyston PCF, Williamson ED, Leary SEC, Eley SM, Griffin KF, Titball RW. Immunization with live recombinant Salmonella typhimurium aroA producing Fl antigen protects against plague. Infection and Immunity 1995; 63: 563-568. Meyer KF. Effectiveness of live of killed plague vaccines in man. Bull. Wld. Hlth. Org. 1970; 42: 653666. Meyer KF, Cavanaugh DC, Bartelloni PJ, Marshall JD. Plague immunization. I. Past and present trends. J. Inf. Dis. 1974; 129: S13-S18. Titball RW, Williamson ED. Vaccination against bubonic and pneumonic plague. Vaccine 2001; 19: 4175-4184. Protsenko OA, Anisimov AP, Mosarov OT, Donnov NP, Popov YA, Kokushkin AM. Detection and characterization of Yersinia pestis plasmids determining pesticic I, fraction I and mouse toxin synthesis. Genetika 1983; 19: 1081-1090. Miller J, Williamson ED, Lakey JH, Pearce MJ, Jones SM, Titball RW. Macromolecular organisation of recombinant Yersinia pestis Fl antigen and the effect of structure on immunogenicity. Ferns Immunology and Medical Microbiology 1998; 21: 213-221. Price SB, Leung KY, Barve SS, Straley SC. Molecular analysis oflcrGVH, the V antigen operon of Yersinia pestis. J. Bacterial. 1989; 171: 5646-5653. Hill J, Leary SEC, Griffin KF, Williamson ED, Titball RW. Regions of Yersinia pestis V antigen that contribute to protection against plague identified by passive and active immunization. Infection and Immunity 1997; 65: 4476-4482. Pullen JK, Andersen GL, Welkos SL, Friedlander AM. Analysis of the Yersinia pestis V protein for the presence of linear antibody epitopes. Infection and Immunity 1998; 66: 521-527. Williamson ED, Vesey PM, Gillhespy KJ, Eley SM, Green M, Titball RW. An IgGl titre to the Fl and V antigens correlates with protection against plague in the mouse model. Clinical and Experimental Immunology 1999; 116: 107-114. Eyles JE, Sharp GJE, Williamson ED, Spiers ID, Alpar HO. Intra nasal administration of poly-lactic acid microsphere co- encapsulated Yersinia pestis subunits confers protection from pneumonic plague in the mouse. Vaccine 1998; 16: 698-707. Prior RG, Klasson L, Larsson P, Williams K, Lindler L, Sjostedt A, Svensson T, Tamas I, Wren BW, Oyston PCF, Andersson SGE, Titball RW. Preliminary analysis and annotation of the partial genome sequence of Francisella tularensis strain Schu 4. Journal of Applied Microbiology 2001; 91: 614-620. Gray CG, Cowley SC, Cheung KKM, Nano FE. The identification of five genetic loci of Francisella novicida associated with intracellular growth. FEMS Microbiology Letters 2002; 215:53-56. Fulop M, Mastroeni P, Green M, Titball RW. Role of antibody to lipopolysaccharide in protection against low- and high-virulence strains of Francisella tularensis. Vaccine 2001; 19: 4465-4472. Fulop M, Manchee R, Titball R. Role of Lipopolysaccharide and a Major Outer-Membrane Protein from Francisella-Tularensis in the Induction of Immunity against Tularemia. Vaccine 1995; 13: 1220-1225.

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Current Problems Regarding Detection and Identification of Biological Threats Michal BARTOSZCZE Military Institute of Hygiene and Epidemiology, Lubelska 2, 24-100 Pulawy, Poland 1. Introduction Bioterroristic attacks with B.anthracis spores in USA visualized to world opinion the scale of biological factor threats. On 22 persons infected by B.anthracis spores 5 died. Antibiotic therapy was applied in 32000 peoples (non-infected). The attack was accompanied with panic and disorganization: ransoming the medicines in drugstores, purchasing the protective equipment and also change in human behavior (limitation in people movement and isolation in houses). The economic losses as a result of biological attack are very high (buildings, persons and environment disinfection). Protection against unconventional biological attack is very difficult and demands unusual fancy for foreseeing the possible scenario of attack. In preparedness for attack consider for the indirect target of attack many substances (cosmetics, drugs even the chewing gums). In preparedness for protection against biological attack essential will be intellectual efforts (prejudice the enemy intentions). Proper prepared and efficiently acting defensive infrastructure will influence on the effectiveness of the protection against biological factors. Development of the molecular biology methods opened the great chances for using them for all men goods, but also caused the fear for using them for military offensive purposes. Thanks to the genetic engineering methods obtaining the biological factors (highly virulent and highly resistance on antibiotics) are become possible. The efficiency of production of the pathogens and theirs toxins was also enlarged. Attainable is transfer of virulence factors from one pathogen to another. Obtained in this method the " wolf in sheep's skin " can procure considerable difficulties in microbiological diagnostics. Accessible is also the modern technologies enlarging the pathogen resistance on environmental factors how as: temperature, light, drying (biopolymers). We should also mentioned about possibilities to "programming" the length of pathogen lives, which after "fulfilling the assignment" decay, giving the chance of safe return for winners after extermination of enemy. Among many decisive factors, which can have the influence on the possibility of decreases the results of use biological agents should be mentioned obligatory: rapid detection and identification of biological factor used, the proper preventive treatment and the medical management.

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The aims of identification • To identify the factor • To estimate the area of contamination • To evaluate the possible countermeasure efforts (antibiotics, disinfectants) • To assess the effectiveness of the decontamination efforts (decontamination of the persons, equipment, buildings, environment etc.) The objects of identification • Bacteria and bacteria's spores • Viruses • Toxines • Genetically modified factors • Etc. The list of factors used in the NATO SIBCA exercise • B. anthracis • Coxiella burnetii • Burkholderia mallei • Francisella tularensis • Brucella melitensis • V. cholerae, • Y. pestis • Vaccinia • VEE • Yellow fever virus The nature of the samples • Aerosol • Soil • Water • Food • Swabs from surfaces • Unconventional (envelope, chocolate) • Human and animal, insect etc. Requirement • The rapidity of the tests • The preciseness of the results • The generality (the possibility of uses for identification purposes the different samples: water, soil etc.) • Possibility of identification of different factors • The simplicity of the tests • The possibility of conducting the tests in field conditions • Equipment adaptation to different field conditions e.g. shock resistance

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2. Present technologies 2. 1. Detection methods There are technologies (1), which are assigned for aerosol detection, coming from low distance. The LRBSDS (Long Distance Biological Standoff Detection System), assigned for aerosol cloud detection within the radius of 30 km is an example. Equipped with laser transmitter for infrared, receiving telescope and detector. However, this system is not fully automatic. The problems with signals detection were reported, which were often disturbed, what was the significant detection non-suitability. The system was improved, and the system JBSDS (The Joint Biological Standoff Detection System) was created - the completely automatic system, able to monitor the aerosol cloud movement. It possesses abilities to differ the biological and non-biological clouds, gives possibilities of early warning and report. Following projects regarded detection elements, but identification of biological agents, too. The IBADS (The Interim Biological Agent Detection System) is one of them; it is semi automatic system, which possesses the aerosol concentrator and dynamic particle measuring device. The immunochromatographic tests are applied for micro-organisms identification. The technology of this system is cheap and allows to execute the initial microbe identification only. JPS (The Joint Portal Shields) is the first highly automatized system, improved because of sensor net increasing the detection sensitivity. All operations of this system are controlled by central computer unit. After concentration and physical characterisation, aerosol is next taken for automatic analysis with immunochromatographic methods, with the ability of 8 biological agents identification within 25 minutes. The JBPDS (The Joint Biological Point Detection System) is the next detection and identification system, build with two modules. Comparing with the previous one, it is characterised by significantly higher sensitivity and specificity. It creates the possibility of bioparticle presence detection within 60 seconds, with the ability of 10 organisms identification within 20 minutes. JBAIDS (The Joint Biological Agent Identification and Diagnostic System) - it is the portable device, prepared for simultaneous pathogen identification, as well from environmental or from clinical samples. The ability of fluorescence induction with the laser light beam, analysed thanks to sensitive photo-detectors, was used for biological agents detection, among others. This method allows to determine the living biological agents from the dead ones, and to determine the size and shape of particles. The technologically advanced systems are capable to analyse data fast, together with the alarm signal generation, if the critical level are exceeded, and transfer the obtained data to the commanding centres. The modern FLAPS (Fluorescence Aerodynamic Particle Seizers) system, executing fast analysis of the aerosol type, the shape and concentration, is the sample of this type technology. This type devices were used for examination of parcels suspected to contain the B. anthracis spores in some post offices in the USA (2). The common feature of mentioned above systems is, that they are mainly assigned for biological aerosols detection. However, the bioterrorist attack may be executed with not only aerosol way, but with the use of food and water and non-conventional methods (3, 4). Concerning the above, the mentioned above systems secure only the part of defensive necessities for bio-terrorist attack. Later, the technology allowing for biological agents detection in different environments shall be described.

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2.2. Luminometry It is the method allowing to detect the ATP (adenosino- triphosphate) content in living cells. The method based on the principle of emitted light detection, created at enzymatic decomposition of ATP. Emitted energy is proportional to the ATP content in the sample and is determined with the use of sensitive photo - detector - luminometer (5). The luminometric method is suitable for i.e. fast detection of bacteria presence in fluids, powders, lyophilizates. The PROFILE system, which allows to distinguish eucariotic and priocariotic cells, is one of few technologies assigned for ATP measurement. Because of adequate additional equipment, it allows to execute the examination of the contaminated surfaces, food, water, air, etc. It makes the fast detection of bacterial spores presence in samples possible, too, what may be used for screening examination of samples, suspected of having B. anthracis spores (6). As newest researches show, because of gammalisine application the luminometric method is capable to identify B. anthracis (7). The own research shown the possibility of Salmonella spp., rods identification with the luminometric method (8). Thinking about the versatility of bioluminometry in the aspect of bioterrorist threats, the WIHiE (Military Institute for Hygiene and Epidemiology) prepared and introduced for application the Field System of Bacterial Contaminations Detection (PSWZB).

2.3. Biological agents identification methods Conventional, classic microbiological methods, though they allow to detect and identify the biological agents, have many disadvantages from the military point of view. They are, i.e.: long time necessary for results obtaining, the need of having laboratory subsidiaries, trained personnel and significant material consumption.

2.4. Bio-sensor technologies In bio-sensor techniques, at the searching for i.e. antigens, light-pipe sensors covered with antibody are frequently used. The complex created after the antibody binding with the searched antigen, is detected with the use of antibody marked with fluorescent dye. Thanks to laser light beam , the strong induction of the marker in the reaction place takes place, producing the signal registered by the detector. Thanks to that, the determination on "dirty" samples are accessible to be done. The system Analyte 2000, based on this conception, may identify with the use of four probes, four samples at the same time. The test sensitivity amounts from 3 to 30 bacteria /ml, and the time of analysis equals 20 minutes (9). System RAPTOR (10) presents the development of the method mentioned above, completely automatised, portable, where the sensitivity of determinations is increased and the time of identification is reduced. Exchangeable multi-use blocks, where the tested material flows, were applied. The system allows to detect bacteria as well as their toxins and viruses, what is the significant advantage. The device may work in the permanent system, in the connection with air sample collector. Methods described above did not allow, mostly, to execute genetic tests. RAPID system - field automised system, which allows to identify pathogens with the "Real time" PCR method (11) became the brake-though. Thanks to adequate fluorescent dyes and marking

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probes and use of FRET phenomenon (Fluorescence Resonance Energy Transfer), the amplification process is observed constantly on the monitor screen. The apparatus is easy to run, requires the precision at the test sample preparing, only. We should mention about its connection with the early warning system "Leaders" (Lightweight Epidemiology Advanced Detection and Emergency Response). The RAPID system application for FMD virus identification on the cattle farm was important success.

2.5. Field diagnostic tests Contrary to more or less complicated and usually expensive technologies described before, there are some simple immunochromatographic tests, practically used, allowing to obtain the result within 10-15 minutes (3). Mono - and polyclonal antibodies, marked with colloidal gold are used in this method. The positive reaction is observed optically as a coloured thin line. The test sensitiveness is quite high and amounts from 10 3 to 10 4 CFU/ml. In the case of bioterrorist attack, the used agent concentration may be high (from 10 8 to 10 11). To increase the reading sensitivity, the bar readers are used, what eliminates the reading subjectivity. The Guardian reader is an example of this solution. The immunochromatographic method sensitiveness may be increased, using the UCP (Upconverted Phosphor) instead of colloidal gold. Thanks the induction of these particles with the light, similar to infrared rays, the visible light is emitted , registered by detector (UCPRS - Upconverting Phosphore Reporting System). Comparing to the method with the colloidal gold use, the UCPRS method is 10 - times more sensitive, with the efficient elimination of background "shining". Thanks to different substrate application, it is possible to detect with the use of this method several biological agents simultaneously (12). It should be pointed, that all described technologies allow to execute the more or less precise , but initial diagnostics. The initial identification confirmation is executed by reference laboratories with the adequate level of biological safety, equipped with unique devices and employing high class specialists.

2.6. Molecular biology methods in biological agents identification The basic method applied in genetics diagnostic is the PCR - polimerase chain reaction which allows to amplify selectively the chosen DNA fragments. Thanks to specific starters (primers), it is possible to detect bacterial genes localised on plasmids or chromosome (13). In the case of detection of agents containing RNA, the RT - PCR method is used. Classic PCR method allows to obtain good results, at clean colony and clinical material examination, especially. At the environmental samples examination, because of many PCR reaction inhibitors occurrence, much better identification results are obtained with the Nested PCR method, where two pairs of external and internal starters are used. The amplification reaction occurs with two stages. Products, created in the first phase (pre - existing amplification products) become the matrix for internal pair of primers, what, as a result, significantly improves the reaction sensitivity. Because of the genetic material exchange between bacteria in natural conditions, several, and even more than ten different primers should be used. The PCR - ELISA test, which is development of method mentioned above, is the connection of Nested PCR and ELISA technique. It consists on introduction of dUTP marked with digoxygenine into dNTP mix ("master mix"), what makes the creation of marked

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amplicones in the amplification process. In the next step, the hybridisation of created products with biotinilic probe of internal primers is executed. Obtained hybrid is placed on polystyrene plates, coated with streptavidine, where it is caught because of strong streptavidine - biotine binding. The hybrid detection is executed with the use of antibodies for digoxygenine, marked with enzyme. Thanks to enzyme and suitable substrate, the colour reaction occurs, marked on ELISA reader. It was shown that this test is from 10 times to 100 times more sensitive than classic PCR method. Multiplex PCR - allows for simultaneous amplification of several genome regions with the use of different pairs of primers. It has its advantages, because it allows detection of several agents in the same time, causing the decrease of examination time and decrease of reagents use. During the own examinations (14) Multiplex PCR was used for B. anthracis, Francisella tularensis and Vibrio cholerae detection. Other genetic methods used for pathogens identification were described in one of works (13). Nowadays, intensive research works take place over elaboration of simple tests "hand handle tickets", adjusted for genetic examinations execution (stripe method) in field conditions. Their sensitivity is similar to classic PCR method. 2.7. Mass spectrometry Mass spectrometry is one of more promising identification methods. The aerosol particles undergo the process of sonification for proteins liberation. Obtained products are purified, concentrated and separated with chromatography method, and then undergo with UV ionisation and are analysed in mass spectrometer. The method allows to identify bacteria, including the inter-strain differences. Further progress of this method was observed after the use of infrared rays (UR) for sample ionisation, instead of UV light, thanks to what the more specific signal and higher sensitivity were obtained. Moreover, what is worth of underlying, the method requires no special sample elaboration (15). PGCIMS (Pyrolisis - gas chromatography - ion mobility spectrometry). Allows to execute the chemical - biological identification (16). After concentration, the sample is treated with the high temperature, what causes the markers liberation, then analysed with the apparatus. That way, for instance, it is easy to detect the picoline acid of B. anthracis with this method. Produced apparatuses are not bigger than shoe-box, nowadays. 2.8. Flow cytometry This method found its application for detection pathogens as well as bacterial toxins. This method value is directly depending on reagents quality (antibodies, antigens). Except antibodies and antigens application, the possibility of use of dyes themselves for diagnostic purposes is worth of attention, which may selectively bind itself with one - or double-stranded nucleinic acids. The vast data concerning the topic of flow cytometry in microbiological diagnostics are contained in works of Stopa (17). The research on UCPRP use for biological agents identification with flow cytometry method seems to be promising (18). The suitability of the method for nucleic acids fragments was proved, what may be used for identification purposes (19).

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2.9. DNA "CHIP" technology It gives the possibility to execute the significant amount of hybridise experiments in the same time (15). The reaction takes place on extremely small plate, where oligonucleotides were placed. The tested DNA is marked with fluorescent marker and observed with conphocal fluorescent microscope. Point emitting fluorescent signal indicate the occurred hybridisation. Different types of mentioned technology create wide range of research and application possibilities (15). Last time, the significant progress of new biological agents detection and identification technologies took place (19). Part of them, after laboratory and field tests, shall be introduced to microbiological practice soon, giving their contribution for more efficient fight against biological threats.

3. General problems and challenges Rapid detection of biological threats gives us the possibility to usage (in time) the non-specific protective methods: usage the HEPA filter masks, individual protective clothes and the collective protection. Ideal will be the situation in which biological factors become detected very quickly, so the introduction of preventing undertakings is highly possible. Nowadays exists many different detection technologies of biological threats, but they need time to distinguish between the real biological factor from the artificial - simulants. Should be also mentioned, that purposeful use of simulants can create, at least at the beginning, the considerable complications (panic, chaos, disorganization) analogous to situation, when the real biological factor were used. Regarding to this only the quick identification of biological factor can prevent the dangerous consequences of spurious the attack. Nowadays exist many technological tendencies, which appear very promising. For example the bio-sensor technique (the immunosensors, optic sensors, taste chips.) also the bioanalysis technologies (flow cytometry, bioluminescence, Volatile Organic compound analysis, DNA analysis for forensic identification etc.). But in the majority they are still in test stage. Among quick identification methods immunochromatographic techniques should be mentioned. However the results of this methods also require the confirmation by the genetic tests. In turn, the genetic tests can create the incorrect results especially, when the environmental samples were tested (the phenomena of naturally exchange of genetic material between bacteria's). Should be brought to the attention that in special cases, even in field conditions, there are needs of usage the classical microbiology methods e.g. culturing the suspicious material for obtaining the precise results. Sometimes the results obtained by the classical microbiology methods are the final confirmatory tests for the results obtained by different methods. According to this the microbiology techniques should be improved and adapt to the field conditions. The limitation of wider implementation to practice the different diagnostic methods the mass spectrometry is e.g. the high price of the device. Apart from the costs of apparatus considerable limitation is also the weight and size of equipment - flow cytometer. The modern identification methods e.g.: the chip and microarray techniques are still not implemented for field diagnosis needs.

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The diagnostic possibilities of the NATO members' countries have the crucial meaning in fighting with world bioterrorism. The problem in protection against bioterrorism in regional and world scale is lack of synergistic laboratories. Necessary it is also the standardization and unification of identification methods for comparing results. Indispensable is the necessity of organization by medical service the international training concentrated on identifications of biological factors. In last years we observed considerable progress in microbiological diagnostic, especially on the genetic level, but however such techniques are not in common use (high cost of modern technology). This cause, that fast reaction on local level will not be possible in case of attack. The limitations are also: weak laboratories infrastructure, lack of experienced staff, impediment to laboratory standards, lack of unification diagnostic procedures. We should also mentioned, that many technologies are designated for laboratory conditions. The technologies, which possess the highest value, are designated and were tested in field conditions. Also unusually essential is continues training of the diagnostic personnel, continues stand by the system, possession of unalterable reserve the reagents designated for identification purposes in case of bioterroristic attack. The most important elements in field of protection are possession of the diagnostic potential for detection genetically modified biological factors. Using the genetically modified biological factors is challenging for Health Service Units, which have to undertake the challenge concerning the public health.

References [1] [2] [3] [4] [5] [6]

[7] [8] [9] [10] [11]

Walt D R, Franz D R. Biological warfare detection. Anal Biochem 2000; 72:738A-46A. Leoma G A. Detecting Biological Agents in Mail Using Fluorescence Particle Sizing. Detection of Bioterrorism Agents. Biodetection Technologies. Furthering Science through Information. Knowledge Press 2003 ;9-21. Bartoszcze M, Niemcewicz M, Malinski M. Recognition of Biological Threats. AAVDM Newsletter 2001; 6:113-4. Bartoszcze M, Niemcewicz M., Chomiczewski K. Biological threats (aerosol, water, unconventional). Joint Service Scientific Conference on Chemical and Biological Defense Research 2002 Nov 19-21; Hunt Valley, Maryland; 21. Bartoszcze M, Bielawska A. The past, present and future of luminometric methods in Biological Detection. Rapid methods of Analysis of Biological Materials in the Environment. Ed. P. Stopa and M.Bartoszcze. Kluwer Academic Publisher Dordrecht/Boston/London 2000;73-7. Bartoszcze M, Arciuch H, Chomiczewski K, Matras J. Some Problems Concerning Application of the Luminometric Methods in the Detection of Bacillus anthracis Spores. Proc.of the 1996 ERDEC Conference on Chemical and Biological Defense Research 1997 Nov 19-22; Aberdeen Proving Ground; 711-2. Nelson D, Loomis L, Fischatti V A. Using Bacterial Enzymes for Rapid Identification of Bacteria. Laboratory of Bacterial Pathogenesis. The Rockefeller University, NY and New Horizons Diagnostics Corporation, Columbia, MD; Information leaflet; 2002. Lidacki A, Bartoszcze M, Arciuch H, Skoczek A, Mierzejewski J. The evaluation of IMS method for biological detection. Proc. of the ERDEC Scientific Conference on Chemical and Biological Defence Research 1995 Nov. 14-17; Aberdeen Proving Ground;775-7. Tempelman L A, King K, Anderson G P, Liglers F S. Quantitating Staphylococcal Enterotoxin B in Diverse Media Using a Portable Fiber Optic Biosensor. Anal Biochem 1996;233:50-7. Lim D. Real time/near real time biosensor. Detection of Bioterrorism Agents. Biodetection Technologies. Furthering Science through Information. Knowledge Press; 2003;1 17-36. Ritter T. Fighting Germs on the Front Lines: an Integrated Laboratory Approach to Field and Lab Analysis and Surveillance. Detection of Bioterrorism Agents. Biodetection Technologies. Furthering Science through Information. Knowledge Press; 2003;95-103.

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Cooper D E. Upconverting Phosphor Technology Overview. Biological Agent Detection and Identification. DARPA, 1999April 27-30; Santa Fe, New Mexico; 76-100. Matras J, Bartoszcze M: Bacillus anthracis. Post.Mikrobiol 2002;41:3-19 Niemcewicz M, Osiak B, Gawel J, Bartoszcze M. Zastosowanie PFGE i PCR w roznicowaniu niechorobotworczych (bezplazmidowych) szczep6w B. Anthracis, B.spp.813. II Konferencja naukowa. Ochrona przed Zagrozeniami Biologicznymi; 2002 listopad 19; Pulawy;27. Donlon M. Biosensors - The tool for fast detection. NATO ARW. 2003 Jan 15-18 Warsaw;31. Spangler G E. Miniaturizing Gas Chromatography in Combination with Ion Mobility Spectrometry. DARPA, 1999 April 27-30; Santa Fe, New Mexico; 302 Stopa P J. The Application of Flow Cytometry For the Detection and Identification of Microbiological Agents. Ph.D. Dissertation. Military Institute of Hygiene and Epidemiology Warsaw; 1999. Wright B. Compact Flow Cytometer. Biological Agent Detection and Identification. DARPA Santa Fe, New Mexico 1999 April 27-3 ;94-100 CBNSP-Annual Report Technology Development. Chemical &Biological National Security Program. http//www.nn.doe.gov/cbnp/tech-dev.sht

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Rediscovering Remote Sensing: Improving Infectious Disease Surveillance Debra M. NIEMEYER, MS, MA, Ph.D., MT (ASCP)1 Lt Col, USAF, BSC Program Executive Office for Chemical and Biological Defense 5109 Leesburg Pike, Skyline #6, Suite 401B, Falls Church VA 22041 Abstract Satellites have been used over the last 30 years to examine and map terrestrial features, and to study properties of the earth's surface. Recently, researchers have explored using remotely sensed data to evaluate associations between vector- and waterborne diseases, habitat changes, human health and transmission risks. With the wealth of archival sensor data, and on-going efforts to launch improved sensor systems, we need to devote our efforts and resources to exploring remote sensing applications to better assess environmental hazards and prevent disease. A new application of an existing technology, remote sensing will improve force protection by enhancing our ability to prevent, monitor and record untoward exposure of our Armed Forces to "chemical, biological and similar hazards" (Public Law 105-85 (10 USC 1074f), Section 765) [1]. Through assessment of archival, baseline and documented changes of environmental factors, we can improve epidemiological surveillance for a variety of hazards, man-made and naturally occurring, prior to and during deployments. Through linkage of satellite imaging and other disparate data with electronic medical surveillance systems, we will be able to use smaller, "expeditionary" personnel packages and equipment footprints to provide near- to real-time assessment in garrison and in theater. Discussion will focus on the application of remotely sensed data for monitoring distribution of communicable diseases.

Disclaimer. The conclusions and opinions expressed in this document are those of the author. They do not reflect the official position of the United States Government, Department of Defense, Joint Program Executive Office for Chemical & Biological Defense, United States Army or Air Force.

1. "Take the High Ground" In 1972, the launch of Lansat-1 initiated the collection and analysis of remotely sensed data for mapping the earth's surface [2]. Since the dawn of time, man has sought the "high ground" for improved observation. Especially important in warfare mountaintops, balloons, and then airplanes provided a better vantage, enhancing surveillance. During the FrancoPrussian War (1870-1871), the first use of balloons for military observation was documented [3]. Aerial reconnaissance and photography was used during the American Civil War to follow enemy troop movements, direct artillery barrages, map gun emplacements and chart harbor defenses and coast lines [4,5,6]. In 1909, Wilber Wright first used the aircraft as an aerial surveillance platform, taking motion pictures over Italy [6]. During World War I, the British

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conducted aerial photography using balloons and aircraft to document large regions for operations planning [7]. Following that war, unmanned craft such as kites and balloons were used for a variety of peaceful surveillance applications ranging from forestry conservation to topographical mapping, and charting of sea coasts [8]. The application of aerial photography for environmental monitoring and meteorological research followed and continues to be used for many functions to include the management of forests and waters, in pollution and vegetation damage control, and in urban planning [8]. Scientists obtain snapshots thousands of feet up to assess environmental changes. As early as 1970, scientists recognized the value of and applied "remote" satellite techniques to improve epidemiological surveillance [9]. The first published remote sensing application for disease surveillance was part of a dracunculiasis eradication campaign in the Republic of Benin [2,10]. Waterborne disease surveillance is also feasible as evidenced by the tracking of global climatic changes associated with cholera outbreaks [11,12].

2. Future Applications For some time, experts have urged measures be taken to more effectively deal with emerging infectious diseases. Recommendations followed for development of a global infectious disease surveillance system [13-15]. Numerous electronic medical surveillance systems are under development and being used by federal, state and Department of Defense (DoD) agencies (Table 12). The systems work well as electronic medical records or for enhancing syndromic surveillance, and several provide data mining tools for the epidemiologist. Lacking, however, is the ability to integrate disparate environmental assessment data, such as Global Information Systems (GIS) to include satellite data [15], and Global Positioning Systems (GPS) to mark and track environmental anomalies and provide prospective analysis for outbreak prediction. As an example, one system being developed by a U SAP-corporate consortium and tested by DoD, the Lightweight Epidemiological Advanced Detection and Emergency Response System (LEADERS), will incorporate disparate data for outbreak prediction [23]. Another system being evaluated by the Air Force Force Protection Battlelab at Lackland Air Force Base, the Deployed Environmental Surveillance System (DESS) will pull data from a variety of sources, to include tactical chemical and biological sensors to enhance the commander's situational awareness [26]. A recent concepts deployment to Honduras resulted in the successful demonstration of rapid screening for Dengue virus and strain specific identification for the vector, Aedes, using realtime PCR [27,28]. Studies are underway to evaluate system linkage with LEADERS and other electronic patient encounter systems, rapid testing technologies, and a Combat Command and Control System (CSC2) capable of GPS and secured (SIPRNET) data receipt [29]. The next step will include incorporation of remote sensing data feeds of select overseas locations for epidemiological analysis [30]. The goal is to concepts test the analysis of remote sensing data to predict occurrence of and track vector-borne diseases and diseases associated with refugee migration and poor sanitation, and to integrate this information onto a common medical surveillance platform. An excellent model of a stand-alone system that employs climate and satellite data for disease surveillance is the collaborative project by DoD Global Emerging Infections Surveillance and Response System (GEIS) and the National Aeronautics and Space Administration (NASA) to monitor Rift Valley Fever (RFV) epidemics in Eastern Africa [31]. Climate and satellite data, regional maps of areas at risk will be available on the Web to

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facilitate a better understanding of the environmental factors associated with disease outbreak and spread, thereby improving opportunities for disease control and prevention. Table 12. Examples of DoD Electronic Medical Surveillance Systems' System

Sponsor2

Employed3

DMSS Defense Medical Surveillance System

DoD / USACHPPM

Retrospective DoD patient database maintained by AMSA.

ESSENCE Electronic Surveillance for the Early Notification of Community Based Epidemics

DoD GEIS developed in response to PDD NSTC-7 on emerging infections (18)

In use by DoD; all MTF data visible to all users

GEMS Global Expeditionary Medical System

AF / HQ ACC/SG

Used in CENTAF, USAFE, being evaluated for PACAF and DoD use, i.e., DEPMEDS and Navy Fleet (SAMS linkage); integration with TMIP under evaluation

LEADERS Lightweight Epidemiological Advanced and Emergency Response System

AF/ HQ USAF/SGX

Utility assessment done at WHMC Fall 01, and being evaluated at WRAMC, Navy Medical Center, Bethesda as DoD initiative. Started as 1year COSSI initiative - AFIndustry partnering to leverage development in the commercial IM/IT sector.

SAMS Shipboard NonTactical ADP Program (SNAP) Automated Medical System

Navy / BUMED

On-board ships

Features4 Primary data repository containing up-to-date and historical data on diseases and medical events (e.g., hospitalizations, ambulatory visits, reportable diseases, HIV tests, acute respiratory diseases, and health risk appraisals) and longitudinal data on personnel and deployment (16). AMSA routinely publishes summaries of notifiable diseases, trends of illnesses of special surveillance interest and field reports describing outbreaks and case occurrences in the Medical Surveillance Monthly Report (MSMR) [16,17]. A sensitive, specific, standardized, timely and flexible health indicator surveillance system (syndromic surveillance). Transparent acquisition, analysis, and dissemination via secure web site of aggregated daily Ambulatory Data System (ADS). Post 9/11 ESSENCE expanded to include ADS data from 413 DoD MTFs in addition to NCR primary care clinics and ERs. ADS data from 121 Army, 110 Navy, 80 Air Force, and 2 Coast Guard installations are processed daily [19,20]. Three part system: 1) PEM - Patient Encounter Module (electronic medical record), 2) TEM - Theater Epidemiological Module (multiple MTF status visible at theater level, i.e., number of cases, bed status, etc.), 3) TOM - Theater Occupational Module - for occupational health assessments (under development) [21,22]. Application Service Provider (ASP)-based; electronic patient encounter, epidemiological data mining tools with automatic alerts and reporting, incident and events management, links for "Labs, Rads, Pharm" data. Evaluation underway to link, disparate databases for nonconventional data feeds (i.e., Veterinary/Public Health, meteorological weather, community OTC pharmaceutical sales); evaluation for TMIP, CHCS/2, GEMS interface on-going. Post 9-11 collaborated with CDC to bring 250+ NY hospitals on-line [23,24]. An automated medical administrative management system designed to address the requirements of shipboard Medical Departments. Included features for the management of health care, administration functions, monitoring functions, medical supplies and health protection programs [25].

Originally published in the Society of Armed Forces Medical Laboratory Scientists (SAFMLS) Newsletter, Society Scope Winter 2003; 6(1):1. Reprint coordinated through Editor; modified for the NATO Conference Series Book.

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3) 4)

Listing is not intended to be all-inclusive USACHPPM--U.S. Army Center for Health Promotion and Preventive Medicine; GEIS--DoD Global Emerging Infections Surveillance and Response System; HQ ACC/SG--Office of the Command Surgeon, Air Combat Command, Langley AFB VA; HQ USAF/SGX-Directorate of Expeditionary Operations, Science & Technology, Office of the Air Force Surgeon General, Boiling AFB DC; BUMED-Navy Bureau of Medicine and Surgery, Washington DC. AMSA-Army Medical Surveillance Agency; MTF-Medical Treatment Facility; DEPMEDS-Deployable Medical System; WHMC-Wilford Hall Medical Center, Lackland AFB, TX; WRAMC-Walter Reed Army Medical Center, Washington DC; National Navy Medical Center, Bethesda, MD; COSSI--Commercial Operations & Support Savings Initiative. NCR-National Capital Region; CDC--Center for Disease Control & Prevention

3. Conclusion An ever-expanding global mission pushes deployments to and establishment of fixed facilities in regions with endemic infectious diseases and substandard sanitary conditions. Coupled with natural disaster, refugees, eroding infrastructure, and possibility of attack by overt or covert release of biological agents, the need for an integrated multifaceted medical surveillance system is pressing. Evaluating how to use remote sensing to enhance disease surveillance is not only prudent, but it is necessary. Tools to see and monitor "over the horizon" are important to accurately assess the environment. These tools provide early hazard warning to enhance the commander's situational awareness of the battlespace. Laboratory specialists contribute important data for medical surveillance thereby improving situational awareness and enhancing battlespace management. Electronic laboratory reporting can markedly improve surveillance, but it is not without it's challenges [32]. Therefore, key is a team approach with laboratory specialists working alongside preventive medicine, infectious disease and public health specialists, civil and environmental engineers, industrial hygienists, informatics and systems experts to ensure provision of accurate and timely biohazard data. Critical laboratory data includes biological warfare agent and pathogen identification, antimicrobial resistance, therapeutic drug monitoring and nonspecific infectious disease marker results. The on-going challenge remains the search for and understanding of emerging and re-assessment of current technologies for novel applications to enhance force protection in the new expeditionary environment. References [1] [2] [3] [4] [5] [6] [7] [8] [9]

Public Law 105-85 (10 USC 1074f), SECTION 765. Improved Medical Tracking System for Members Deployed Overseas in Contingency or Combat Operations; retrieved at: http://www4.law.cornell.edU/uscode/l 0/1074f.html. Beck, LR, BM Lobitz, BL Wood. Remote sensing and human health: new sensors and new opportunities. Emerg Infect Dis 2000 May-Jun;6(3):217-27; retrieved at: http://www.cdc.gov/ncidod/eid/vol6no3/beck.htm. Balloon; retrieved at: http://encarta.msn.com/FinaVConcise.asp?ti=060E3000. Civil War "Firsts;" retrieved at: http://www.civilwarhome.com/civilwarfirsts.htm. Lowe, TSC, Personal Observation, The Balloons with the Army of the Potomac; retrieved at: http://www.civilwarhome.com/balloons.htm. Center for Remote Sensing and Spatial Analysis, Rutgers University. History of Aerial Photography, retrieved at: http://deathstar.rutgers.edu/courses/airphoto/airphoto 1 /sld023 .htm. History of Ballooning; retrieved at: http://airtime.hvpermart.net/history.htm. Aerial photography in Canada: A brief history; retrieved at: http://airphotos.nrcan.gc.ca/history.html. Cline, BL. New eyes for epidemiologists: aerial photography and other remote sensing techniques. Am J Epidemiol 1970 Aug;92(2):85-9; abstract retrieved at:

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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&listuids=5464318&dopt= Abstract. Clark KC, JR Osleeb, JM Sheery, JP Meert, RW Larsson. The use of remote sensing and geographic infomation systems in UNICEF's dracunculiasis (Guinea Worm) eradication effort. Prev Vet Med 1990;229-235. Colwell, RR. Global climate and infectious disease: the cholera paradigm. Science 1996 Dec 20;274 (5295):2025-31; abstract retrieved at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list uids=8953025 &dopt=Abstract. Lobitz, B, Beck L, Huq A, Wood B, Fuchs G, Faruque AS, Colwell R. Climate and infectious disease: use of remote sensing for detection of Vibrio cholerae by indirect measurement. Proc Natl Acad Sci USA, 2000 Feb 15;97(4):1438-43; retrieved at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool-pubmed&punmedid=l-677480. Morse, SS, B Rosenburg, J Woodall. Global Monitoring of Emerging Diseases: Design for a Demonstration Program; retrieved from: http://www.fas.org/promed/papers/proposal.html. Shalala, D. Collaboration in the Fight Against Infectious Diseases, Emerg Infect Dis 1998 Jul-Sep;4(3); 354-7; retrieved at: http://www.cdc.gov/ncidod/eid/vol4no3/shalala.htm. Nuttall, I., DW Rumisha, TRK Pilatwe, et, al., GIS Management Tools for the Control of Tropical Diseases: Applications in Botswana, Senegal, and Morocco; The International Development Research Centre Web site: http://www.idrc.ca/books/focus/766/nutta.html. Defense Medical Surveillance System (DMSS), Army Medical Surveillance Agency (AMSA); retrieved at: http://www.amsa.army.mil/AMSA/amsa_home.htm. Medical Surveillance Monthly Report (MSMR), AMSA. National Science and Technology Council Presidential Decision Directive 7 (NSTC-7, 1996) documented the lack of U.S. readiness to identify and respond to emerging infectious diseases and ordered DoD and other agencies to improve surveillance, response, and prevention capabilities; retrieved at: http://www.fas.org/irp/offdocs/pdd ntsc7.htm. Electronic Surveillance for the Early Notification of Community Based Epidemics (ESSENSE) Back ground Paper, Department of the Army, Office of The Surgeon General. DoD Global Emerging Infections Surveillance and Response System, retrieved at: http://www.geis.ha.osd.mil/. Morrow R.C., KO Schafer, RL Williams. Quality of deployment surveillance data in Southwest Asia. Mil, Mil Med 2001 Jun:166(6):475-9; abstract: http://www.ncbi.nlm.nih/gov/entrez/query.fcgi?cmd+Retrieve&db=PubMed&list uids=11413722&dopt =Abstract. Humphrey, E, presentation entitled, "Global Expeditionary Medical System." Headquarters Air Combat Command Office of the Command Surgeon, 5 Mar 2002. Niemeyer, D, E Hanson, R, R. Rowley, R Munson, K Schafer. Real-time Medical Surveillance for Early Warning and Mitigation of Environmental Hazards. Proceedings of the International Conference on Protection Against Biological Threats, Sponsored by DARPA and the General Karol Kaczkowski Military Institute of Hygiene and Epidemiology, Warsaw, Poland, June 2001. Bunk, S. Early Warning. The Scientist 16(9): 14, April 29, 2002. Virtual Navy Hospital. Military Sealift Command Medical Manual; Appendix A, Glossary. Shipboard Non-Tactical ADP Program (SNAP) Automated Medical System (SAMS); retrived at: http://www.vnh.org/Admin/MSCMedManual/AppendixA.htmlffdefinitions. Niemeyer, D. Kenny Battlelab Initiative, Deployed Environmental Surveillance System (DESS), Air Force, Force Protection Battlelab, May 2000. Swaby, J, D Lowe. Deployed Environmental Surveillance System (DESS) Initiative Briefing Joint Services Integration Group, Mar 2001. Personal Correspondence, Col J Swaby, Air Force Institute of Operational Health, Brooks City-Base TX, Sep 2002. Combat Support Command and Control System (CSC2) Joint Expeditionary Experiment 2000 (JEFX 2000) After Initiative Report. Niemeyer, D, J Swaby. Proposed Kenny Battlelab Initiative, Remote Sensing for Infectious Disease Surveillance from a Common Expeditionary Platform (draft). Climate and Disease Connections—Rift Valley Fever Monitor. DoD GEIS; retrieved at: http://www.geis.ha.osd.mil/RVFWeb/index.htm.

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Overhage JM, J Suico, CJ McDonald. Electronic laboratory reporting: barriers, solutions and findings. J Public Health Manag Pract 2001 Nov;7(6):60-6; abstract retrieved at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd±=Retrieve&db=PubMed&list uids=11713754&dopt=Abstract

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The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Debra M. NIEMEYER, MS, MA, PhD, MT (ASCP)1,2,3 Joint Program Executive Office for Chemical and Biological Defense 5109 Leesburg Pike, Skyline#6, Suite 401B, Falls Church VA 2204 Abstract. The Joint Biological Agent Identification and Diagnostic System (JBAIDS) program will field a reusable, portable, modified commercial system for military use. The system will be capable of simultaneous, reliable identification of multiple Biological Warfare Agents and pathogens of military significance. JBAIDS will enhance force protection by providing medical personnel and commanders an expedient means to reliably and quickly identify biological threats in the operational environment, whether naturally occurring or man-made. Information derived from JBAIDS will improve situational awareness and facilitate the application of effective preventive measures, prophylaxis, and appropriate treatment. Medical personnel will use the JBAIDS to quickly identify exposure to or infection by biological agents and screen for endemic diseases. System employment will be at multiple levels of health service support to include deployed medical units, fixed medical facilities, ground vehicles, aircraft, and aboard ship for contingency and humanitarian mission, and homeland security medical support. The acquisition, production and fielding of JBAIDS will occur in tree increments or blocks. Block I will be a nucleic acid amplification system that will identify a limited number or biological agents. Block II includes the capabilities of Block I, plus the capability to identify a limited number of biological toxins. Block III will identify Block I and Block II agents, plus additional bacterial, viruses, fungi, and toxins referenced from standard threat lists, region-tailored based on the Combatant Commander guidance. The Block III system will be lightweight, handheld device employed by the medic for point of care testing. With automated sample preparation, testing and results interpretation, first responders will use it for biohazard assessments, recon and surveillance activities. Block III will interface with existing medical surveillance networks and feed pertinent information into Chemical, Biological, Radiological, and Nuclear Warning and reporting systems. Each block will incrementally improve decision-making through enhancement of the Commander's situational awareness of biological hazards in the battle space. A review of the program, to include the near-term acquisition strategy and technical requirements will be discussed.

Disclaimer. The conclusions and opinions expressed in this document are those of the author. They do not reflect the official position of the U.S. Government, Department of Defense, Joint 1 JBAIDS Team: Bakeer B, Belrose B, Bentley C, Craig P, Korte K, Melling D, Wilson S, Joint Program Executive Office for Chemical and Biological Defense (JPEO-CBD), Falls Church VA; Bayless b, JPEO-CBD, 311th HSW, Brooks City-Base TX; Allen k, U.S. Army Medical Material Command, Legal Office, Fort Detrick MD, Potter W, Selfridge L, U.S. Army Space and Missile Defense command (USASMDC), JPEO-CBD, Chemical Bioloical Medical Systems (CBMS), Frederick MD; 2 Other/Support: Danley D, Ranhofer R, JPEOCBD, CBMS, Frederick, MD, Fanelli W, House K, JPEO-CBD, Falls Church VA. 3 Submitted to the Society of Armed Forces Medical Laboratory Scientists (SAFMLS) Newsletter, Society Scope, for publication. Reprint coordinated through Editor, modified for the NATO Conference Series Book. Note: Cleared for Open Publication.

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Program Executive Office for Chemical and Biological Defense, United States Army or United States Air Force. 1. Introduction The Joint Biological Agent Identification and Diagnostic System (JBAIDS) exemplifies the new United States (U.S.) Department of Defense (DoD) approach to the acquisition of products and materials under the streamlined acquisition process instituted by DoD and the U.S. Congress, August 2002. This program constitutes DoD's first effort to develop and field a common medical test platform that will identify biological agents - Biological Warfare (BW) agents and pathogens of operational concern. The system will be configured to support reliable, fast and specific identification of biological agents from a variety of clinical specimens and environmental sample sources. JBAIDS will enhance force protection by providing commanders and medical personnel the capability to determine appropriate treatment, effective preventive medical measures and medical prophylaxis in response to the presence of biological agents [1]. "Lessons learned" from the Persian Gulf War pointed to the need for BW agent detectors and subsequent material solutions alleviated capability gaps. However, another deficiency was identified in response to "Gulf War Syndrome" and other ailments suffered by military personnel - medical personnel needed a portable diagnostic tool to quickly identify disease-causing biologic agents. Thus, the concept for JBAIDS was proposed [2]. Its need is reinforced by the threat of biological attack faced by our forces deployed around the world. Furthermore, on-going operations in war-torn locations regularly present with infectious disease challenges that demand far-forward agent identification capabilities to retain troop readiness, and quickly access specific patient treatment options to include medical evacuation [3]As a result of the September 11th attack on the World Trade Center in New York City, and the subsequent release of Anthrax spores in the U.S. postal system, the U.S. Military moved forward to purchase and field medical BW agent identification equipment for installation and carrier battle group defense. However, still lacking is a common "workhorse system", a complete package with support equipment, supplies, and an extensive array of validated agent assays, sample protocols, and standardized training. Additionally, a consolidated DoD plan is needed to obtain system clearance from the U.S. Food and Drug Administration (FDA) for diagnostic use. The JBAIDS program encompasses that complete system package and FDA plan [1,2].

2. Incremental Capabilities Development JBAIDS is a competitive development and production program designed to meet military end user requirements. The JBAIDS Office is housed within Chemical-Biological Medical Systems (CBMS) of the Joint Program Executive Office for Chemical and Biological Defense (JPEOCBD). Different from other medical products such as vaccines, protective skin lotions, and anticonvulsants, a multi-block configuration, spiral development and fielding approach is proposed to obtain a licensed multi-agent identification system. The JBAIDS Block I program consists of the development and testing of BW agent identification hardware, along with Production Block I options for manufacture, integration,

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and fielding of 1KB AIDS systems late 2004 to early 2005. System clearance by FDA will be initiated in Block I and carried throughout for each Block upgrade. The system will comprise platform test equipment, assay test kits with controls, protocols for sample preparation and system operation, ancillary equipment and consumable supplies, maintenance manuals, and training materials (Table 13). Block I will provide a capability to identify ten biological agents (Table 14). The JBAIDS Office will procure a modified Commercial-Off-The-Shelve (COTS) or modify a Non-Development-Item (NDI) Table 13. Components of JBAIDS

JBAIDS System Protocols • •

Sample Preparation System Operation

Platform Test Equipment

Expendable Supplies

• • • •

• •

Assay Analysis Hardware Laptop Computer Associated Software Storage/Shipping Case

•

Assay Test Kits Shelf-Stable Reagent Kits Other Laboratory Consumables

Table 14. Select Block I and II Agents1 AGENT OR DISEASE

ORGANISM

Block 1 Anthrax Brucellosis Ebola-Marburg Plague Q-Fever Salmonellosis Smallpox Tularemia Typhus Fever

Bacillus anthracis Brucellae Filoviridae Yersinia pestis Coxiella bumetii Salmonellae Orthopox viruses Francisella tularensis Rickettsia typhi

Block II Clostridium botulinum Botulium Ricin Ricinus communis Staphylococcus Enterotoxins (i.e., SEB) Staphyloccus aureus Notes: 1) Representative agents for which assays will evaluated for development 2) Does not provide a complete list 3) Agents are not listed in any priority or importance

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system design to meet the requirement. The COTS/NDI system will be used by medical laboratory specialists configured to support forward deployed operations for force health protection. The Block I system will undergo environmental, reliability, performance and suitability testing in late 2003 and early 2004. FDA testing for 510(k) submissions will be performed in parallel. The Block II increment is an evolutionary enhancement to field a toxin identification capability by expanding the modular design of the Block I hardware, software, and addition of test reagents to handle toxin identification. Additional tests will be added to the Block I assay complement (Table 14), expanding test panel options for the laboratory operator. Block III will fully integrate microorganism and toxin identification technologies into a single, lightweight, handheld or smaller unit that can correctly identify at least 50 different biological agents (bacteria, viruses, and toxins) in less than 15 minutes. Block III requirements will be fine-tuned when mature technologies surface in the future that have potential to meet JBAIDS out year requirements, to include automated sample processing and linkage to theaterlevel electronic medical information systems that will tie into a lager early warning and reporting systems. Emerging candidate technologies range from nucleic acid and antigen microarray platforms, to nanotechnology for single cell analysis - the "lab on a chip" concept [4,5]. The Government's acquisition plan allowed for commercial companies to bid any technology in response to the Pre-solicitation announcing the star of a Market Survey for suitable off-the-shelf solutions [6]. Only relatively mature technology was evaluated, having to meet the identified end user screening requirements, for example, the system must already exist either in production or functioning prototypes. Additionally, proposed technology had to pass a two-week long performance-based "Fly-Off Laboratory Test and be suitable for accelerated Developmental and Operational testing and evaluation with rapid fielding of requirementscompliant systems by December 2004. The down-selected contractor is to posses the capacity to manufacture in accordance with Good Manufacturing Practices (cGMP) and be compliant with new biological surety requirements [7]. Following contract award, summer-fall 2003, the contractor needs to be ready to submit the platform, with sample protocols, and key assays (i.e., Anthrax and Smallpox) to the FDA for clearance as a diagnostic device. Assays for the remaining Block I biological agents will be incrementally submitted as the reagent kits are produced. 3. Streamlined Acquisition JBAIDS is a unique program that combines a streamlined acquisition process with an evolutionary block, spiral development strategy by leveraging available commercial biotechnology and manufacturing capabilities, and utilizing the research strength of government laboratories. This new process provides a template by which to evaluate and down-select against established end user requirements, mature, off-the-shelf technologies for inclusion into current and future Joint Service Chemical and Biological Defense programs. Representatives from across the DoD other U.S. federal agencies (i.e., Departments of Health and Human Services, Agriculture, Energy, and the Environmental Protection Agency) and the North American Treaty Organization participate on Integrated Product Teams. These different agencies have banded together to select promising candidate systems to be further developed to provide a much-needed capability, setting a precedent for resource-sharing. This streamlined approach ultimately benefits the user community by dramatically reducing acquisition time and

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coasts. It serves as a model depicting for Program Managers and Material Developers how to take advantage of off-the-shelf technologies to rapidly field much needed multi-use products. JBAIDS will be a flexible, multi-use technology employed on the conventional battlefield and in far-forward locations around the globe to combat terrorism, improve force health protection and patient care, and enhance Homeland Defense. 4. Summary JBAIDS, the first program in which FDA clearance will be sought for rapid biological agent analysis, will serve as the benchmark for. future Government and Industry partnering. Furthermore, an innovative fielding plan employs parallel processing, and will reduce delivery time by delivering a Block I capability for environmental sample testing and surveillance while the system moves through FDA review. Unlike medical devices currently employed for biological defense, a complete system package will be delivered, ready for immediate use. Additionally, the JBAIDS Office is currently working with JPEO-CBD Critical Reagents Program, the Armed Forces Institute of Pathology, and other Centers of Excellence, such as the Air Force Institute For Operational Medicine, the U.S. Army Medical Research Institute of Infectious Diseases, and the U.S. Army Soldier Biological Chemical Command, to set up an overarching Quality Assurance and Proficiency Program to assess operator performance in testing samples containing biological agents. Operator certification standards will be established along with re-certification requirements. Furthermore, a New Equipment Training course will be designed utilizing the best of existing military and civilian training programs. This process will also identify course updates in military schools necessary for successful system fielding. The JBAIDS program long-term acquisition strategy ensures continuous improvement by leveraging off of the "latest and greatest" technological solution Industry has to offer. The same acquisition strategy will be used for Block II, toxin identification, with a market survey projected late 2003 following Block I contract award. A DoD first, this strategy will ensure that the best, most modern and readily available capability is quickly and efficiency evaluated against approved end user requirements to rapidly field a material solution to the warfighter.

References [1]

Joint Biological Agent Identification and Diagnostic System (JBAIDS) Operational Requirements Document, 6 Sep 2002.

[2] [3]

Annual Report to Congress and Performance Plan, Washington DC, DoD Program, 2003. Niemeyer D. Improved Public Health Surveillance: Advanced Technologies and Techniques (invited manuscript submitted to JCLA for Summer 2003 publication). Niemeyer D. Genomics and Bioinformatics: The Next Technological Leap. Society Scope, Summer; 5(2) and Fall 02;5(3):2 (Editor's Page). Rudert F. genomic and Proteomics Tools for the Clinic. Curr Opin Mol Ther 2000 Dec;2(6):633-42; abstract retrieved at:

[4] [5]

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=l 1249740&dopt=Abstract

[6]

JBAIDS Pre Solicitation Notice, Federal Business Opportunities Solicitation Number DASG60-02-R-0008 (closed 28 Mar 2002). FBO Archived document, on-line, Internet, available at: http://www.eps.eov/servlet/Solicitation/R/USA/SMDC/DASG60/DASG60-02-R-0008.

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[7]

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Possession, use and transfer of select agents and toxins, in 42 Code of federal Regulations (CFR) Part 73, Interim Final Rule, on-line, Internet, available at: http://www.cdc.gov/od/sap.

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Biosensors - The tool for fast detection Mildred A. DONLON Program Manager, Biological Warfare Defense DARPA/SPO 3701 North Fairfax Drive,Arlington, VA 22203-1714, USA Abstract. The threat of attack on military and civilian targets with chemical and biological weapons is a growing national concern. The Defense Advanced Research Projects Agency (DARPA) is developing technologies for detecting biological materials in the natural environment. While several technologies show promise as broadband detectors, there is no "silver bullet" that detects all chemical and biological materials at the requisite levels of sensitivity and specificity. DARPA is developing enabling components for use in biological detectors.

The threat of attack on military and civilian targets employing chemical and biological weapons is a growing national concern. Technologies for detection of these materials in the natural environment are being developed by DARPA. While several technologies show great promise as broadband detectors, there is no "silver bullet" that detects all chemical and biological materials at the requisite levels of sensitivity and specificity. DARPA is developing biosensor technologies to identify and develop new component technologies which will enable the development of fast, highly sensitive, and highly specific biosensor systems that will reduce false alarm rates and increase the ability to detect and identify multiple biological warfare agents. Most current biosensor systems use antibody-based components with fluorescent reporters to detect the BW agent and the Polymerase Chain Reaction (PCR) to amplify the DNA genome. This multistep approach is both time consuming and complex, and limits the development of both rapid and unattended detection and identification systems. Biosensor technologies are currently under development to support this goal: the enhancement/replacement of antibodies, mass spectrometry technologies, and a phylogenic microchip. Enhancement/Replacement of Antibodies is being approached by developing small molecular weight compounds with high affinities and specificities that will enhance current antibody identification protocols, with the ultimate objective of using these kinds of moieties to replace the antibody as the principal detection/identification molecule in biosensors. The mass spectrometer is known as the most powerful laboratory analytical tool for analysis of a broad spectrum of chemical and biological materials. A Phylogenic Microchip, containing an expanded hierarchical set of more than 100 oligonucleotide probes, is being developed which will enable the parallel detection and identification of a variety of species of organisms allowing rapid determination in unknown samples.

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Figure 33. FACS analysis showing specific binding of fluorescently-labeled mAbs to B. anthracis spores

This project has prepared anii-Bacillus anthracis spore and anti-exosporium monoclonal antibodies (mAbs) and showed that many of the mAbs were species specific for B. Anthracis spores (Figure 33). The mAbs were used to produce smaller polypeptides for example Fab fragments, single-chain antibodies (scFvs), variable and complementary determining regions. These fragments were tested as ligands for spore binding. Fab fragments bound cognate spores nearly as well as intact mAbs. Many of these mAbs appear to bind a single exosporium-associated glycoprotein, which is part of the exosporium hair-like nap. Commercial phage display peptide libraries were also screened (5 peptides/phage) to identify short peptides (5-6 amino acids) that bind B. anthracis spores. The purified peptides are being modified to increase valence and improve specificity. Smaller fragments are presently being tested. Infra-Red Mass Spectrometer Mass spectrometry is a technique for the determination of the masses of molecules and specific fragmentation products formed following vaporization and ionization. Detailed analysis of the mass distribution of the molecule and its fragments leads to molecular identification. These molecular measurements can be carried out at the attomole (10-18 mole) level of material using specialized laboratory-based instruments. The combination of specific molecular identification and extreme sensitivity makes mass spectrometry one of the most powerful analytical laboratory tools. While such capability has existed in the laboratory for many years, the development of a small, portable mass spectrometer for potential field detection of chemical and biological substances remains under development. Usually such instruments have employed UV lasers at 337nm. However, a number of laboratories have reported that IR lasers offer various advantages for desorption of proteins and especially for desorption of proteins from thick samples. MALDI mass spectra were obtained on a prototype Mantis time-of-flight instrument (Science and Engineering Services, Inc., Burtonsville, MD, USA). The instrument has a fourinch end-cap reflection and was operated in the positive mode, at 20kV accelerating voltage with a 0.3 us delay time. A PTI (London, Ontario, Canada) model PL2300 nitrogen laser was used as a UV laser source and a prototype of an SESI (Burtonsville, MD) model TMIR-1000

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all-solid-state optical parametric oscillator (OPO) based tunable laser source was used at 2.94 um in IR experiments. Tablel5. Mass units associated with various bacterial spores and treatments

Organism

IR MALDI (1)

UV MALDI (1)

B. cereus

4833

4833

7930 9534 (4,5) 12453 B. subtilis

B. globigii

6847 (4,5) 6949 (4,5) 7779 (4) 8064 (4) 8651 (4) 9136 (4,5)

6949 7779 8064 8651

6910 7082 7346 8898

7082 7346

UV MALDI and 5% TFA (2) 4833 6712 6834 7082 7930 (3) 9534 (3) 12453 6847 6949 (3) 7779 9130 (3) 6910 7082 7346 8898 (3)

Spectra obtained from B. cereus, B. subtilis and B. globigii spores using IR- and UVMALDI were obtained. Respectively Table 15 summarizes the biomarker ions that were observed from these microorganisms. The number of biomarker peaks desorbed in the mass range 4000-14000 Da is greater when the IR laser is used. In the analysis of B. cereus spores, the peak at m/z 4833 dominates in both IR and UV spectra. However, IR-MALDI desorbs several additional biomarker ions above 6000 Da.

Phylogenic Microchip The most characteristic molecules for identification of an organism are the genetic materials DNA and RNA. Researchers working at Argonne National Laboratory (ANL), from the Englehardt Institute in Moscow, have developed a tool for rapid identification of microorganisms using a three dimensional gel pad chip. The target moiety in their investigation is ribosomal RNA (rRNA). Since rRNA is present in many thousand copies per cell, the selection of this target obviates the need for signal amplification using PCR-based methods. Therefore, analysis of RNA dramatically speeds the analysis as compared to other nucleic acid identification approaches. In the evolutionary history of life, the RNA molecule and the ribosome structure were among the first objects developed. In particular, the 16S fraction of rRNA is intriguing to study as it appears to be one of the genetic elements which encodes the roadmap of evolutionary progression and divergence from simple to complex organisms. The 16S rRNA consists of 1 500nucleotides in a characteristic structure. As life has evolved, this

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molecule has evolved with it. Every living creature from bacteria to humans has rRNA materials within its cells. In the RNA structure there are highly conserved regions along with regions that are show considerable variability. By judicious selection of RNA complementary probes, regions of the 16S rRNA can be used to detect and phylogenetically distinguish organisms at the genus and species level-using the RNA chip. The scientists working at ANL have been able to develop a two dimensional array of complementary nucleic acid probes immobilized within a 10 X 60 micron gel pad. The gel pads act as a very small test tubes for carrying out the annealing reactions between the complementary probes and the rRNA oligonucleotides. Detection is carried out by breaking open the cells of interest, cleaving the rRNA, allowing the resulting oligonucleotides to flow over the chip, and measuring the fluorescence of the individual pads using a fluorescent microscope. From a knowledge of the complementary sequence, the sequence of a particular section of RNA can be determined and identification of the organism can be obtained. This type of chip is capable of distinguishing organisms that differ in only a single base pair at the target site. The first application of this technology has been the development of a "Bacillus Chip." The genus Bacillus is quite important for biological detection applications because it includes the organism responsible for anthrax. However, the surface structure of spores of the genus Bacillus is such that highly specific antibodies for anthrax are not as yet available. The "Bacillus Chip" is a technology that allows the specific identification of anthrax from closely related organisms. One caveat of the rRNA based chip is that it is of little use in detecting viruses which do not contain an amplified target such as rRNA and proteinaceous toxins which may only contain small variable amounts of contaminating nucleic acids. These caveats may be overcome by the amplifying the signal through PCR based methods or enhancing the detection of low copy signals using other technologies such as the mass spectrometer for readout.

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A flexible approach to biomanufacturing K.P. O'CONNELL, P. E. ANDERSON, D.C. LUKENS, M. H. KIM, A.S. KHAN, R. G. THOMPSON, J.T. PARK, J. J. VALDES U.S. Army Edgewood Chemical Biological Center, APG, USA N. BEEK, T. CHASE, .W. BENTLEY Chesapeake PERL, Inc., 387 Technology Drive, College Park, USA Jordan Kostov and Govind Rao University of Maryland Baltimore County, USA Abstract. A flexible, creative, and rapidly responsive biomanufacturing infrastructure is an essential part of an effective overall strategy for bioterrorism preparedness and biological defense. A variety of approaches and technologies are evolving to provide the capacity to bring innovations in biological threat detection, prophylaxis, and therapeutics from the laboratory bench to advanced development and ultimately to the end user and/or the marketplace. Biotechnology products, including affinity reagents, real time PCR probes and primers, molecular elements for microarray design and manufacture, therapeutic peptides, and vaccines, each have unique requirements for their production at useful scales. Another critical feature of a flexible bio-manufacturing facility is the ability to archive a variety of biological materials in a secure fashion. We will describe in this paper the multifaceted approach to biological manufacturing being advanced by the U.S. Army Edgewood Chemical Biological Center and its partners in government, academia and industry. State-of-the-art biological manufacturing methods (efficient cell culture reactors, cost analysis studies) as well as traditional methods (fermentation) and an advanced cryorepository are being used to solve problems in biological agent detection, agent simulation, environmental decontamination, and the production of biologicals for human clinical trials. Results from research on real-time optical monitoring of in vivo production of recombinant proteins are also described.

Introduction All immunologically based and DNA/RNA hybridization-based sensors that detect biological warfare agents require agent-specific molecules to form a specific and sensitive interface between the sensor apparatus and the threat agent. For immunosensors, those reagents are commonly polyclonal or monoclonal antibodies. Antibodies are proteins produced by the immune system that selectively recognize and bind target molecules or organisms with high affinity and selectivity. As such, they are the essential component in immunosensors that detect biological warfare agents and are the source of their sensitivity and selectivity. The biological defense research establishments of most countries incorporate into their sensor platforms antibodies produced either in whole animals (polyclonal) or in mammalian cell cultures (monoclonal). There is considerable lot-to-lot variability in the current production of antibodies. This is especially true for polyclonal antibodies, which require the injection of a disarmed B W agent into animals. The individual response of each animal to an agent can vary dramatically. The process of developing antibodies in animals or in mammalian cell culture is also time-consuming, which limits the capacity for "just-in-time" or surge production in time

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of conflict. We discuss here our efforts towards providing flexible, rapid, and scalable manufacturing solutions for the production of biological defense reagents. We have established (or are establishing) these capabilities with the additional goal of providing broader biotechnology manufacturing services to researchers in government, academic, and biotechnology industry laboratories. 1. Reagent Development 1. 1. Recombinant Antibodies For Simulant Detection In addition to antibodies that bind BW agents, there is a need for antibodies that bind and detect organisms and substances that simulate BW agents. A panel of BW agent simulants that are non-toxic and non-pathogenic is widely used for the development and testing of biosensors and environmental samplers, in a work setting without the need for high levels of biological containment. Two such simulants are bacteriophage MS2 (a non-pathogenic virus of the bacterium Escherichia coli, which is used to simulate viruses) and ovalbumin (a benign protein which is used to simulate protein toxins, such as ricin). To meet the need for highquality, inexpensive antibody reagents that bind these BW simulants, we have used a powerful technique called phage display [1,2] to isolate antibody genes from immunized mice. The resulting antibody molecules are called Fabs, indicating that they are comprised of heavy and light chain antibody sequences, which form the antigen-binding variable region, but do not contain the IgG constant region. We have cloned and characterized three new reagents, two that bind ovalbumin and one that binds MS2. Briefly, recombinant antibodies are cloned from a library of heavy and light chain genes cloned into a DNA expression vector appropriate for the organism in which the antibodies will be expressed. In our case, the vector also created a fusion between each antibody in the library and one of the coat proteins of the filamentous bacteriophage Ml3 (Figure 34). Expression of the antibody genes in E. coli cells in the presence of helper phage creates a population of Ml3 particles each displaying a different antibody. The resulting particles each contain the gene that encodes the antibody displayed on its surface. The display of the recombinant antibodies on the surface of the phage allowed us to obtain clones of antigen-specific antibodies through binding recombinant MS2 coat protein immobilized in the wells of microtiter plates (a form of affinity purification called "biopanning"). To minimize the possibility of contaminating bacterial cultures with intact MS2 and to obtain the purest antigen possible for affinity screening, we expressed and purified a recombinant version of the MS2 coat protein. Figure 34. Schematic of antibody displaying filamentous phage particle. A phage particle in the display library contains DNA encoding an antibody from the immunized mouse, and displays that same antibody on its surface. The entire repertoire of antibodies cloned from the mouse is thereby converted to a recombinant form that can be screened by applying the population to a surface coated in the antigen of interest.

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After purification, the recombinant coat protein was used to screen the antibody library for clones that bind MS2. Ovalbumin for both immunization and biopanning was obtained commercially (Pierce Co, Rockford, IL). Recombinant anti-MS2 and anti-ovalbumin antibodies were used in enzyme-linked immunosorbent assays (ELISAs) to detect their corresponding antigens. Purified MS2 was detectable down to a level of 250 ng (protein equivalent) in this experiment (Figure 35). The recombinant coat protein used for biopanning and screening, however, was detected to a level of approximately 20 ng [3]. This apparent greater sensitivity for the recombinant coat protein over the intact virus may reflect the isolation of the antibody using the recombinant protein as the biopanning target. It may also indicate that the epitope bound by the anti-MS2 Fab is partly obscured when the coat protein is in the three-dimensional context of the intact viral coat. Use of intact virions as the target, as well as performing the affinity capture of anti-body-displaying phage in solution will allow the isolation of Fabs with even greater affinity for intact MS2. Anti-MS2 did not bind phage Ml3, BSA, or ovalbumin (data not shown). Using partially purified recombinant antibodies OVA-3 and OVA-4, ovalbumin was detectable down to a level of 1 microgram (data not shown). 1.2. Peptides as Affinity Reagents To complement the development of recombinant antibodies, we have employed other combinatorial/genetic approaches to derive novel reagents for affinity-based biosensors. Antibodies, being relatively large molecules of moderate stability, require storage conditions not always available to persons in the field, outside highly controlled environments. The traditional hybridoma approach for developing antigen-specific antibodies is also costly, labor intensive and may access only a fraction of the variants of antibodies thought to be encoded within the immunological genome [4]. The understanding that only a relatively short stretch of the antibody polypeptide actually comprises the antigen-binding site makes searching for short peptides with antigen-binding capacity a reasonable undertaking. [5-7].

Figure 35. ELISA detection of (A) MS2 virions and (B) recombinant MS2 coat protein using purified anti-MS2 Fab antibody. Values represent the mean of duplicate measurements, adjusted by subtracting the absorbance measured in the no antigen control. The antibody appears to be more sensitive for the detection of the recombinant coat protein.

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We have employed a combinatorial random peptide display library expressed in E. coli to identify short, linear peptide sequences that bind to ricin and staphylococcal enterotoxin B. Kinetic analysis of peptide binding to the toxins shows lower equilibrium binding constants for the peptides than monoclonal antibodies. This is attributed to both slower association rate constants and faster dissociation rates for the peptides. The ricin peptides bound to ricin with a KD of luM versus the antibody's KD of 14 nM. The SEB peptide bound to SEB with an affinity of 31 nM compared to the antibody's affinity of 0.35 nM. Further characterization, testing and refining of these reagents is currently underway. 2. Antibody production 2.1. General Remarks As mentioned above, antibodies form the molecular interface between immunosensors and the target molecules or organisms they detect. Many of the reagents used in these sensor systems are monoclonal antibodies (mAbs) that are specific for target organisms of concern. The use of mAb specific for biological warfare (BW) agents in fielded assays requires the maintenance of a high-quality and economical supply of these reagents. A common method for producing monoclonal antibodies from an existing hybridoma cell line is to inject a laboratory animal (usually a mouse) with a population of the cell line. The hybridomas grow in the peritoneum of the mouse and produce antibody in a fluid that accumulates there, called ascites. However, occasionally hybridoma cells produce abdominal tumors rather than ascites fluid, necessitating the growth of the cell line in vitro to obtain antibodies [8]. In addition, there is a growing ethical concern about the humaneness of producing mAb using this method. An increased emphasis on development of better in vitro methods has generated commercially available methods for production of mAb without the use of live animal hosts [8]. We have worked with and evaluated three in-vitro cell culture methods for the production of monoclonal antibodies (mAb): gas-permeable bags, Integra CELLine membrane flasks, and a hollow fiber bioreactor. 2.2. Production of Monoclonal Antibodies in Three Types of Bioreactors In developing and optimizing methods for producing 0.1 to 10 grams of monoclonal antibodies, we examined three in vitro technologies. In vitro methods were chosen to avoid the regulatory issues encountered when establishing animal colonies, and to develop more controlled, defined manufacturing processes. The methods/devices examined were: • A Cell-Pharm® System 1500™ Hollow Fiber Bioreactor (Unisyn, Hopkinton MA, USA) in this study. In a hollow fiber bioreactor system, nutrient medium is separated from cells and secreted mAb by semi-permeable capillaries. Because most of the cells were attached in the capillary surface, the cell density in the bioreactor could not be determined during the culture. The antibody produced in the Unisyn CP1500 bioreactor (a hollow-fiber cartridge with a l0kd molecular weight cutoff) was approximately 450 mg in a 3-month continuous incubation period. • Integra CELLine flasks (Integra Biosciences Inc., Liamsville MD, USA). These units provided easy access to both the nutrient medium compartment and the cell

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compartment to allow frequent monitoring of cultures [9, 10]. Maintaining producing cultures required periodic splitting of cells and exchange of the nutrient medium. Cells and spent medium were removed easily for counting cells and harvesting antibodies, and the number and viability of cells in the cell compartment of the flask were able to be tightly controlled. The nutrient medium was poured easily from the reservoir, allowing for quick medium exchange while permitting viable cells to remain in the cell compartment. Total cell density reached approximately 7xl07 cells/ml within 4 weeks of inoculation and remained at that level thereafter. The viability of cells in the cell compartment decreased steadily and then remained steady at approximately 20%. The high cell concentrations in the small volume cell compartment of Integra CELLine allowed us to achieve high concentrations of mAb, routinely as high as 0.6 mg/ml as measured by analytical protein-A HPLC. Single one-liter flasks produced as much as 110 mg of mAb in a 3-month period. Higher antibody concentrations are probably possible with further optimization of the frequency of antibody harvests and nutrient medium exchange, and reactor cell population. Gas-permeable bags (TC Technologies, Minneapolis MM, USA) are simply that; polymer bags into which cells and medium are placed and incubated in a 5% CO2 atmosphere at 37 °C. Initially the viability of cells is high, but diminishes with time; the production of mAb is usually inversely correlated with cell viability. We found that hybridoma cells cultured in 2-liter gas-permeable bags produced the most antibody per unit of medium consumed, in the least amount of time. In a representative bag culture, total cell concentration increased to approximately 6.4x106 cells/ml within 2 weeks of inoculation. Viable cell concentration increased within the first week of the culture and diminished slowly to approximately 5.8xl05 cells/ml. The viability of cells in the bag peaked at 1 week and decreased steadily thereafter. The concentration of antibodies in gas-permeable bags increased most rapidly during the second week of the culture after inoculation, reaching and maintaining a maximum concentration of 0.15 mg/ml after 15 days. A typical single 2-liter gas permeable bag produced approximately 240 mg of mAb from our test cell line in a 1-month period. 2.3. Results and Summary Remarks Antibodies were harvested from media drawn from each of these three types of reactors using single-step preparative chromatography [11-13]. Purified mAbs produced all three types of reactors were found to be active against the target antigen (in this case, cells of Francisella tularensis) (data not shown). Naturally, the viability of cells and antibody yield vary considerably among cell lines. The performance of each method was evaluated by considering productivity, cost, ease of handling and risk of contamination (Tables 16 and 17). Based on this work, we have chosen gas-permeable bags as our preferred cell culture method for routine production of mAbs on both research (< 1 gram) and production (> 1 gram) scales. The frequent need to open the flask bioreactors, remove spent culture medium and feed with fresh medium created opportunities for contamination. Unlike the flask bioreactors, gas-permeable bags require essentially a single access (depositing the initial load of cells and medium). Subsequent samples to monitor viability and mAb production are taken with a syringe through a septum, greatly reducing the opportunity for contamination.

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Table 16. Operational characteristics of three in vitro hybridoma culture methods

Variable

Integra CELLine

Hollow Fiber Bioreactor

Gas-Permeable Bag

Cell Density

High

High

Low

Principle of Operation

Perfusion

Perfusion

Stationary

Capital Equipment

No

Yes

No

CO2 Incubator Required?

Yes

No

Yes

Contamination Risk

Significant

Significant

Low

Table 17. Comparison of the three different cell culture methods examined in this study Gas Permeable Bag (2-Liter)

Variable

Integra CELLine: CL1000

Hollow Fiber Bioreactor: CP1500

Productivity (mg MAb/unit/mo)

40

130

240

Antibody concentration (mg/ml)

0.6

0.5

0.12

Media harvested (1/g MAb)

1.7

2.0

8.3

Total media consumed (1/g MAb)

50

150

8.3

Cost for culturing (materials only)

Moderate

High

Low

Recovery of purification process

85%

92%

78%

Purity (by gel filtration chromatography)

98.4%

99.4%

97.1%

Activity (by ELISA)

Active

Active

Active

3. Other Biotechnology Products 3.1. Organophosphate-Cleaving Enzymes Neurotoxic chemical warfare (CW) agents in the hands of extremist terrorist organizations and rogue nations in recent years have significantly increased the prospect of intentional exposure

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of civilian populations and military personnel. Currently available decontaminants are corrosive to equipment, and pose environmental concerns. The use of catalytic enzymes in place of highly reactive decontaminants offers obvious advantages, i.e. they are non-toxic, noncorrosive, and environmentally benign. Two CW-degrading enzymes, OPH (organophosphorus hydrolase) from Pseudomonas diminuta and Flavobacterium sp. for highly toxic V-type [14-16] and OPAA (organophosphorus acid anhydrolase) from Alteromonas JD6.5 for G-agents such as Soman and Sarin [17,18] have been cloned and expressed in Escherichia coli strains. In addition, a modified OPAA, Q-OPAA designed for a long-term protection of exposed skin to chemical Gagents has been expressed in E. coli cells. In single-batch incubation of cells in a 1-L shaker flask, typical OPH and Q-OPAA yields ranged 10-20 and 30-40 mg/L, respectively. We have also developed larger-scale fermentations to explore the mass production of these enzymes. We are currently focused on scaling up the production of these enzymes in bacterial fermentations. Several 20 liter-scale fermentation runs in Luria broth were conducted to optimize the production of OPH, prior to attempting a 1000-liter fermentation. Culture growth characteristics (growth rates, pH, dissolved oxygen consumption profile, and final cell density) among the four runs were reproducible; however, OPH activity varied considerably. Only the 20-L fermentation run #4 that had been added with 1 mM CoC12 showed a drastic increase in OPH enzyme activity (from 238 to 2012 U/ml in crude cell lysate). In the 1000 liter run, adding 1 mM CoCl2 prior to cell harvest resulted in obtaining 20.7 mg OPH per liter. 3.2. Production of Biologicals Under current Good Manufacturing Practices (cGMP) Recently, a 1400 square feet clean room facility has been designed and currently under construction at our facility. The clean room facility provides a controlled single-pass HEPA (High Efficiency Particulate Air) filtered suites to meet environmental air quality according to the current Good Manufacturing Practices (cGMP) set forth by the U. S. Food and Drug Administration (21 CFR). Our manufacturing facility has been segregated into flexible GMP suites for microbial fermentation, mammalian cell culture, recovery, and downstream purification. The design of GMP suites also allows two simultaneous production campaigns: cell culture and fermentation. Separated from the production space will be a quality control suite and associated support facilities for archival storage of strains and cell lines, and controlled storage for the holding and release of raw materials during the quality control (QC) process. The QC suite is planned to include two areas: a QC analytical laboratory for routine analysis of raw materials, samples from utilities, and finished products, and a QC microbiology laboratory for biological testing (to ensure sterility and absence of contaminants, endotoxins, etc.). Once commissioned and fully validated according to the cGMP requirements for product safety and quality, we will be able to support a broad spectrum of research and product testing (for example, phase I and II clinical trials) and customers (federal and state research agencies, small- to medium biotechnology firms).

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4. Novel Manufacturing Methods 4.1. The Glycosylation Problem As mentioned above, several groups have described bacteriophage-based systems for the selection and cloning of specific antibody gene sequences. We described previously the advantages of recombinant antibodies [3]. They are genetically completely defined, in that their sequences are fully known; malleable, in that their sequences can be manipulated to alter their properties; and producible in a variety of organisms into which the antibody genes can be inserted. These systems include bacteria (e.g., E. coli). However, a drawback to the production of antibodies in bacteria is their lack of ability to properly modify the expressed antibodies with sugar molecules, a process called glycosylation. Glycosylation of antibodies is desirable when the antibodies are to be used as human therapeutics, as these sugar moieties reduce the antigenicity of the antibodies, and therefore reduce the body's tendency to recognize them as foreign. Glycosylation also affects how the immune system interacts with recombinant antibodies to facilitate binding to an antigen. To obtain glycosylation of recombinant proteins, worker can express them in eukaryotic cells, such as yeast or cultured mammalian cells, or in whole transgenic higher organisms including mammals and plants. The time required to develop expression systems in transgenic animals or plants can be costly, making these approaches economically feasible only for very large-scale expression. The use of mammals in the production of antibodies and other proteins is becoming increasingly controversial for ethical reasons [19].

4.2. Insect Cells and Larvae as Bioreactors for Recombinant Protein Products An attractive alternative to transgenic animal or plant expression for antibodies or indeed, any other proteins, is to express the desired genes in transformed insect larvae. Insects are sufficiently molecularly sophisticated to glycosylate antibodies [20, 21]. Certain species, such as Trichoplusia ni, are also inexpensive to raise and can be grown in very large numbers in automated facilities. Most importantly, insect viruses (most harmless to humans) can be used to infect insects via ingestion and can be used as delivery vehicles for the genes of interest [22]. Transfected cells of infected larvae can produce the desired protein product in large quantities; however, gene expression in transformed insects is not foolproof, and not every larva expresses the desired gene at the same level. To reduce the amount of "background" insect protein in a preparation relative to the recombinant product, it is desirable to identify larvae that are the highest producers, and to extract the recombinant product from those larvae preferentially. To this end, we established a protein expression system for use in larvae that would allow a manufacturer to readily identify larvae producing large amounts of a recombinant protein. The method involves the fusion of the gene encoding the desired product to a reporter gene encoding a colored protein, in this case DsRed, an auto-fluorescing protein derived from Discosoma coral.

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4.3. Cloning Strategy and Results The DsRed gene was cloned into the vector in a manner permitting the fusion of the DsRed gene to any gene cloned between it and the promoter region. Accumulation of the product would therefore be tied directly to the accumulation of DsRed, which can be monitored by illuminating the larvae with light in the visible portion of the EM spectrum (excitation 545nm, emission = 620nm). In this way larvae producing a desired recombinant protein can be easily identified and selectively harvested. Such larvae could be selected either by hand, or by an automated larva "picker" guided by machine vision. A similar technology was described by Cha et al. [23] in which green fluorescent protein (GFP) was used as the reporter. We next demonstrated the expression of a useful protein using the DsRed fusion vector. This required the engineering of a set of antibody genes and their cloning into the DsRed fusion vector so that one of the antibody's light chain gene and the DsRed reporter protein would be expressed as a fusion protein. This fusion allowed the determination of expression levels of the antibody in cells or larvae before beginning the process of antibody extraction and purification. The fusion protein was engineered in a way that allows the selective removal of the DsRed fluorescent marker after purification. To accomplish these goals, an existing gene set encoding the heavy and light chains of a recombinant anti-botulinum toxin Fab was modified for expression in insect cells and cloned into both the original vector (without DsRed) and the DsRed-modified vector. The vector chosen for the initial proof-of-principle experiments was the insect baculovirus expression vector pAcAB3, and our modified version of pAcAB3 containing the DsRed gene. Preliminary experiments examining the expression of DsRed protein alone gave striking results: larvae expressing quantities of DsRed so great that the larvae turn bright pink (data not shown). DsRed expression was also noted to be strong in cultured Sf9 cells (an insect cell line; N. van Beek, unpublished results). When the antibody-encoding genes were cloned into the DsRed fusion vector and introduced into larvae, some larvae were estimated to produce as much as 1-3 mg antibody per larva (data not shown). Samples of the antibody are currently being purified and will be tested with control lots of the Botfab to compare the affinities and specific activities. The results obtained here are preliminary to the incorporation of the antibody geneDsRed construct into baculoviruses and the introduction of the baculoviruses into T. ni larvae. This work will be a proof of concept experiment to demonstrate that the insect gene expression system. A complete demonstration of the system will include the expression of a desired recombinant protein in larvae fused to DsRed, the identification of high producers visually using DsRed as the marker, the purification of the recombinant protein, and determination that the recombinant protein has the desired activity. Features designed to test all these aspects of the system have been engineered into the vectors. 4.4. Initial Studies in Machine Vision A long-term goal of this work is the rapid identification of larvae that are producing large quantities of recombinant product relative to the overall transfected population. The dynamics of protein production and its balance with the endogeneous protease activities create a very narrow window of opportunity to achieve high protein yield. This window can be defined by frequent sampling of larval tissues; however this could kill the larvae. Ultimately, we intend to accomplish this goal using robotics guided by machine vision that is capable of identifying

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high-producing larvae using a fluorometer to detect the reporter protein-product fusion. The resulting concentrations were visualized and measured non-invasively through its green fluorescence under UV excitation. Thus, physical sampling of the product can be avoided, and the decrease of the protein concentration eliminated. Additional advantage of the protein fusion is the substantial increase in production of proteolytically sensitive proteins. In preliminary studies, a portable fluorometer was constructed (ref SPIE) and used to detect DsRed protein in expressed in larvae for various durations after infection. Larvae were grown, harvested, and frozen prior to observation. DsRed was expressed to different extent in the tissues. As the infection begins in the gut, usually the belly is the brightest. The sides of the larvae are of similar brightness, and the back is the dimmest. In the sensor designed for this study, larvae were positioned beneath the device in a laying position, simulating real conditions. A frozen larva usually has an S-shape, thus the measurements were taken from the sides of the larvae. Despite the fact that only two side measurements were taken, the data for each larvae batch showed good correlation with the amount of DsRed, determined from the fluorescence intensity of the total soluble protein (TSP) (data not shown). In most of the cases the correlation between the amount of DsRed and the device readings was lower than the correlation between DsRed and the average of the fourside fluorescence. This suggests that a better estimate of the DsRed amount could be attained if more than one sensor is used, i.e. positioned at different angles above the larvae so different portions of its circumference are observed. It is interesting to note that while measured fluorescence was a strong predictor of total DsRed in larvae, it did not correlate with the percentage of the DsRed in TSP at 92 and 120 hours after infection. This is likely a result of bioproduction dynamics. From average of 12 mg per larvae at 67 hours post infection (SD=6.7 mg), TSP dropped to 4.2 mg at 92 hours (SD=2.6 mg) and later recovered to 11.1 mg at 120 hours (SD=5.9). However, the percentage of DsRed remained essentially unchanged. This might suggest that there is some upper level of the DsRed concentration in the cell and the further increase of the DsRed amount is achieved only in parallel with the production of other proteins. The poor correlation between total larval fluorescence and the percent DsRed in TSP could also be the result of the defrosting of frozen larvae, during which some melanization was observed. The fluorescence from strongly melanized larvae was anomalously high, possibly because of the high light scattering. However, protein gel analysis suggested that the DsRed quantity is similar to the amount in the non-melanized larvae. This suggests that after freezing, the larvae should be measured before thawing, or the melanin formation could introduce a significant error in the production estimate. Our work to date indicates that the concentration of the fluorescent recombinant protein can be successfully measured in frozen larvae with a high degree of correlation with the protein concentration determined by traditional methods. The method is applicable both for tracking the in-vivo larvae production of protein (by freezing samples for later measurements) and for sorting. The results suggest that a single reading from the side of the larvae may not be enough to perform reliable measurements. It is preferable to have multisided measurements from at least three sides. Given the low cost of the fluorometer sensors, the method is readily applicable for industrial purposes.

5. Secure Repositories Edgewood Chemical Biological Center (ECBC) operates the Critical Reagent Repository Program (CRP). The Critical Reagent Repository program works closely with laboratories

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across DoD, industry, academia, other government agencies, and the defense research establishments in allied countries to ensure that the best biodefense reagents, methods, and information are available to our customers. To support the antibody development program, a number of different antibodies are produced from hybridomas sent here by program participants. Antibodies are produced and distributed to DoD and allied laboratories for evaluation and study. This task often requires that several cell lines be grown simultaneously for production of antibodies on a research scale (< 1 gram) in the CRP production laboratories at ECBC. 6. Concluding Remarks This work is part of an overall strategy to develop an improved suite of biodetection reagents by establishing the methods for cloning, isolation and large-scale production of recombinant polypeptides. The antibodies being developed in this program complement another ongoing effort in our group to isolate and characterize small peptide aptamers that bind and detect BW agents. Our work in reagent development is, in turn, a subset of our larger effort to provide flexible and scalable biotechnology solutions to customers in government, academia, and the biotechnology industry. Recombinant protein production in bacteria, cell culture, or lower eukaryotes is in step with a growing consensus that current methods of producing recombinant products in mammals (especially the use of ascites culture for monoclonal antibodies) in quantity are inhumane. Traditionally, monoclonal antibodies are produced by injecting hybridoma cells into animal hosts, and subsequently collecting abdominal fluids (ascites) that contain the essentially pure antibody. This method produces great discomfort in animals and is strongly discouraged for large-scale antibody production (National Research Council, 1999). Phage display library construction and subsequent cloning of antibody genes, minimizes animal use by using only those animals initially immunized in the process. Gene expression in insect cells should provide a useful means for the production of materials for human therapeutics. The manufacture of products for use in humans requires a strict adherence to cGMP (current Good Manufacturing Practices) to ensure the safety, quality and purity of product. An economic analysis will need to be performed once data on product yield is obtained. Preliminary data indicate that, in the case of Botfab, approximately 300-1000 larvae may be required to produce one gram of antibody. Other proteins will need to be expressed, and the cost of raising, infecting, growing/harvesting larvae, and subsequent purification of product will have to be determined. Since several technological obstacles have already been overcome for the automation of a larval production line, it seems likely that only minor molecular modifications will be required to obtain efficient recombinant protein manufacturing. Market analysis, including the going rates for recombinant proteins made by other methods, and the overall demand for recombinant proteins, will ultimately determine the economic viability of this and other protein production systems.

References [1]

Parmley, SF, Smith GP. 1988. Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73:305-318.

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Hogrefe H, Shopes R. 1994. Construction of Phagemid Display Libraries with PCR-amplified Lmmunoglobulin Sequences. PCR Methods Appl. S109-S122. O'Connell KP, Anderson PE, Khan AS, Valdes JJ, Stinchcombe TJ, Shopes R, Khalil M, Eldefrawi ME. Recombinant antibodies for the detection of bacteriophage MS2 and ovalbumin. Proceedings of the 21st Army Science Conference, Baltimore MD, December 2000. Winter G,MiIsteinC. 1991. Man-made antibodies. Nature 349:293-99. Devlin JJ, Panganiban LC, Devlin PE. 1990. Peptide libraries: a source of specific protein binding molecules. Science. 249:404-6. Davies J, Riechmann L. 1995. Antibody VH domains as small recognition units. Biotechnology 13:4759. Horwell DC. 1995. The'peptoid' approach to the design of non-peptide, small molecule agonists and antagonists of neuropeptides. Trends Biotechnol. 13:132-4. Jackson LR, Trudel LJ, Lipman NS. 1999. Small-scale monoclonal antibody production in vitro: methods and resources. Lab Animal (autumn issue), pp 20-30. Trebak M, Chong JM, Herlyn D, Speicher DW. 1999. Efficient laboratory-scale production of monoclonal antibodies using membrane based high-density cell culture technology. J. Immunol. Methods 230:59-70. Wolf ML, DeSutter T. 1999. High-density cell cultures in a new passive membrane-based bioreactor. Pages 293-301 in Biotechnology International II (Connor and Fox, eds.) San Francisco: Universal Medical Press, Inc. Gagnon, P. 1996. Protein A affinity chromatography. Chapter 9 in: Purification Tools for Monoclonal Antibodies Tucson, AZ: Validated Biosystems, Inc. Fulton SP, Meys M, Varady L, Jansen R, Afeyan NB. 1991. Antibody quantification in seconds using affinity perfusion chromatography. BioChromatography 11:226-231. Blank GS, Vetterlein, D. 1990. Quantification of monoclonal antibodies in complex mixtures by protein G high-performance liquid affinity chromatography. Anal. Biochem. 190: 317-320. Lai K, Stolowich NJ, Wild JR. 1995. Characterization of P-S bond hydrolysis in organophosphorothioate pesticides by organophosphorus hydrolase. Arch. Biochem. Biophys. 318: 5964. Kolakowski JE, DeFrank JJ, Harvey SP, Szafraniec LL, Beaudry WT, Lai K, Wild JR. 1997. Enzymatic hydrolysis of chemical warfare agent, VX and its neurotoxic analogues by organophosphorus hydrolase. Biocatalysis and Biotransformation 15: 297-312. Rastogi VK, DeFrank J J, Cheng TC, Wild JR. 1997. Enzymatic hydrolysis of Russian-VX by organophosphorus hydrolase. Biochem. Biophys. Res. Commun. 241: 294-296. Cheng TC, Harvey SP, Chen GL. 1996. Cloning and expression of a gene encoding a bacterial enzyme for decontamination of organophosphorus nerve agents and nucleotide sequence of the enzyme. Appl. Environ. Microbiol. 62: 1636-1641. Cheng TC, Liu L, Wang B, Wu J, DeFrank, JJ, Anderson DM, Rastogi VK, Hamilton AB. 1997. Nucleotide sequence of a gene encoding an organophosphorus nerve agent-degrading enzyme from Alteromonas haloplanktis. J. Ind. Microbiol. & Biotech. 18: 49-55. Committee on Methods of Producing Monoclonal Antibodies, Institute for laboratory Animal Research, National Research Council. 1999. Pages 45-47 in Monoclonal Antibody Production. Washington, D.C.: National Academy Press. Hsu TA, Takahashi N, Tsukamoto Y, Kato K, Shimada I, Masuda K, Whiteley EM, Fan JQ, Lee YC, Betenbaugh MJ. 1997. Differential N-glycan patterns of secreted and intracellular IgG produced in Trichoplusia ni cells. J Biol Chem. 272:9062-70. Davis TR, Wood HA. 1995. Intrinsic glycosylation potentials of insect cell cultures and insect larvae. In Vitro Cell Dev Biol Anim. 31:659-63. Pham MQ, Naggie S, Wier M, Cha HJ, Bentley WE. 1999. Human interleukin-2 production in insect (Trichoplusia ni) larvae: effects and partial control of proteolysis. Biotechnol Bioeng. 62:175-82. Cha HJ, Dalai NG, Pham MQ, Bentley WE. 1999. Purification of human interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with green fluorescent protein and metal affinity ligand. Biotechnol Prog. 15:283-6. Kostov, Y., Tolosa, L., O'Connell, K., Anderson, P., Liu, Y., van Beek, N., Rao, G. 2003. Monitoring of DsRed protein concentration in frozen insect larvae. SPIE Proceedings, Vol. #4967, Genetically engineered and optical probes for biomedical applications.

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Development of Rapid Fingerprinting and Detection Assays for Biological Agents of Mass Destruction Vito G. DelVecchio Director of Research, Institute of Molecular Biology and Medicine, University of Scranton, Scranton, PA, USA Abstract. Recent advances in genomics and proteomics have generated new strategies for the development of nucleic acid- and immunochemical-based detection assays as well as rapid fingerprinting systems for biological warfare agents (BWA). The increasing list of sequenced and annotated microorganisms has enhanced the data obtained from comparative genomics and proteomics. In silico studies have facilitated the identification of putative signature sequences for various BWA and provided repetitive sequences that may be exploited in future rapid fingerprinting methods. Suppressive subtractive hybridization investigations of closely related strains and species of BWA have also pinpointed signature sequences for use in probe assays. Proteomics studies using 2 D-gels and MALDI-TOF of spores, secretomes under simulated host cell conditions, as well as vegetative cells have been used in determining differences that can be applied to probe assays. Protein chip SELDI-TOF can be used to observe differences of BWA outer membrane and exosporium proteins. All of these strategies are now being applied to technical platforms that will allow rapid detection and fingerprinting.

Recent world events have heightened the need for rapid, user-friendly, and field-worthy assays for biological agents of mass destruction (BWMD). The genomic era has opened the way for new strategies for detection and diagnosis of BWMD. The increasing list of sequenced and annotated microorganisms will certainly impact on the specificity of such assays and help devise new fingerprinting systems. Post or functional genomic will also generate new methodologies that will not only be applied to BWMD but to the identification of genes found in a diverse pathogens and a vast range of disease conditions. Our laboratory has employed several genomic strategies to identify biomarkers or target sequences that can be used in probing assays for BWMD. These include the use of bioinfomatics, rep-PCR polymorphisms, and suppressive subtractive hybridization. Proteomic techniques such as comparisons of secreted proteins, exosporia, and global analysis of closely related species have been used in biomarker discovery. Certain criteria must be adhered to in the development of molecular probing assays. A specific nucleic acid or antigen target should be unique for the BWA in question, the target must be conserved or found in all isolates of the BWA, and the target should have no polymorphisms or areas of plasticity in the nucleotide composition. Molecular fingerprinting is used to discriminate between closely related strains or isolates of a microorganism. Fingerprinting can determine if an infectious outbreak is the result of

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several or one strain of a pathogen or are the different isolate epidemiologically related. It can also determine the source of a BWMD by comparison of the fingerprint of the strain in question with a database of fingerprints of strains from many geographic locations. Thus it may be able to determine if a certain BWMD originated from a particular terrorist group or hostile state. Traditionally scientists have utilized both molecular and non-molecular methods to type microorganisms. These have included RFLP, ribotyping, PFGE, phage typing, and plasmid profiling. These methods have advantages and disadvantages such as being labor-intensive, taking long periods of time to accomplish, requiring Southern blotting, or needing to liberate DNA from cell embedded in agarose blocks, or can take days or even weeks to complete. The ideal system should be easy to perform, rapid, cost effective, discriminatory and reproducible, serve as a bar code or signature for strain identification, and enable determination of the source of an outbreak. One method that has involved a minimum of time and work expenditure has been repetitive sequenced-based PCR (Rep-PCR) [3]. Takes advantage of the fact that both prokaryote an eukaryote genomes contain dispersed repetitive sequences separating longer single-copy DNA sequences. The repetitive elements vary in position and number on the chromosome of different strains of a pathogen. PCR primers, directed outward from the rep elements, amplify the regions of long single copy DNA sequences situated between the rep elements. The areas between the repetitive sequences are the only part of the genome that is amplified. Since these vary in size, they generate PCR products of different sizes. Electrophoresis of these fragments yields a fingerprints that act as barcodes. Advantages of this method are: DNA is easily and quickly liberated from cells, PCR is so sensitive that only small amounts of template DNA are needed, does not require expensive equipment, and these procedures are relatively rapid—being completed in less than 24 hours.

Figure 36. Rep-PCR fingerprint of different Bacilli. The bands enclosed in green were digitally enhanced to differentiate minor differences. Bands unique to one species can be excised from the gel, cloned, sequenced, and used as a putative candidate for probe development.

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The proteome is defined as the sum total of all proteins produced by an organism under a defined set of conditions or at a particular time in the life of a cell. Proteomics deals with the study of multiprotein systems in which the focus is on the interplay of multiple distinct proteins and their roles as part of a larger network [6]. Protein identification using proteomic technology is achieved by partial sequence analysis with the aid of database matching tools. Traditional proteomics investigations use two-dimensional SDS-PAGE, in which the proteins are first separated by isoelectric focusing (IEF) in the first dimension and by SDS-PAGE in the second dimension. This is a powerful technique for resolving complex mixtures of proteins and permitting the simultaneous analysis of hundreds or thousands of proteins. IEF separates proteins on the basis of their isoelectric point (pI). The pI of a protein is the pH at which the protein has a zero net charge. When a protein mixture is applied to an IEF gel, each protein migrates until it reaches the pH that matches its pI. At this point the protein cannot migrate. Proteins with different pIs are therefore align at different points throughout the gel. IEF offers the greatest resolving power in separating proteins according to net charge. Proteins on IEF strips are then separated in the second dimension by polyacrlyamide gel electrophoresis in the presence of the detergent SDS. SDS binds to most proteins by hydrophobic interactions in amounts proportional to the molecular mass of the protein, about one molecule of SDS for every two amino acid residues. The bound SDS contributes a large negative charge, rendering the intrinsic charge of the protein insignificant. Furthermore, when SDS is bound, most proteins assume an almond shape, resulting in a similar ratio of charge to mass. Protein separation therefore is based almost exclusively on the basis of mass, with smaller polypeptides migrating most rapidly. After electrophoresis, the proteins are visualized by staining with SYPRO® Ruby, a ruthenium-based fluorescent stain shown to have several advantages over other commonly used protein stains with respect to sensitivity and linear dynamic range [7]. The stained gels are imaged and the spots are picked for protein identification. The protein spots are subjected to protease (e.g. trypsin) digestion to yield smaller peptide fragments, the number and size of which are characteristic for an individual protein. The digested protein is then spotted on a plate and identified by mass spectrometry (MS). In general, there are 3 essential components in a mass spectrometer, i.e., an ion source, a mass analyzer, and a detector [1]. One commonly used instrument for most MS work is Matrix-Assisted Laser Desorption lonization Time of Flight (MALDI-TOF) Mass Spectrometer. In this instrument, the ion source, equipped with a laser produces ions from the sample. The sample to be analyzed is mixed with an aromatic matrix that absorbs light at a specific wavelength. Typical matrix compounds include 2,5 dihydroxybenzoic acid, 3,5dimethoxy-4-hydroxycinnamic acid and a-cyano-4-hydroxycinnamic acid. The mixture of sample and matrix is spotted onto a metal plate and allowed to evaporate in air. After drying, the target plate is inserted into the vacuum chamber of the MS. A laser directed at the sample is fired, resulting in the ionization and desorption events. A high voltage applied to the target plate provides potential to push the ions into the flight tube. The ions formed in the MALDI source are then extracted and directed into the TOF mass analyzer. The mass analyzer resolves ions based on their mass/charge (m/z) ratio that is proportional to their velocity. This is dependent upon the time required for the ions to travel the length of the flight tube, i.e., the time between application of voltage to the plate and the registration of signal by the detector. The smaller the m/z value, the shorter is the flight time

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and the faster the ions reach the detector. The detector then converts the kinetic energy of the arriving particles into electrical signals. The mass spectrum generated from MALDI-TOF-MS is called a mass fingerprint that is characteristic of a particular protein. The identity of an unknown protein is determined by comparing its peptide mass fingerprint with the theoretical spectrum generated by digestion of each of the proteins in a database by using a search engine like Mascot from Matrix Science Ltd. (http://www.matrixscience.com). Protein identification from enzymatically-derived peptides depends on the frequency of specific cleavage sites within a protein. The cleavage sites yield a set of potential peptide masses that are unique to that sequence entry when compared to all other entries in the database. If a significant number of the experimentally determined peptide molecular weights match the m/z values in the theoretical mass spectrum, a match is obtained and the protein is identified. Some of the databases commonly used for protein identification include NCBInr, SWISS-PROT, TrEMBL and OWL. Thus, a rapid and automated protein characterization is achieved. Alternatively and more advantageous in terms of speed and accuracy of identification, is matching of MS spectra with translations of the nucleotide sequence from an annotated genome of the same organism, allowing the investigator to match expressed proteins to their corresponding ORFs. For instance, the availability of a completely sequenced and annotated B. melitensis genome has paved the way for a highly comprehensive and rapid analysis of its proteome.

Figure 37. Method of protein identification using the Mascot search engine

The results of proteome analysis indicate which genes are expressed under a given set of conditions, how protein products are modified and how they might interact. Unlike the genome, the proteome is not static. It changes with the state of development, under conditions of environmental stress and during disease states of a tissue. There are many more proteins in a

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proteome (mainly due to posttranslational modifications) than genes in a genome. Fig.37 summarizes the steps that we used in the global analysis of B. melitensis proteome. The utilization of fully automated and robotized instruments from Genomic Solutions, Inc. (Ann Arbor, MI, USA) for staining and imaging of gels, protein spot picking, protein digestion, and spotting onto MALDI target plates. For details regarding the capabilities of these instruments and the ongoing collection of proteomic data please see the IMBM Proteomics website at http://www.proteome.scranton.edu. SYPRO® Ruby protein gel stain was used for a more sensitive fluorescence detection of proteins since it has a linear dynamic range that exceeds that of silver by a factor of 5 to 10. Further, SYPRO® Ruby staining is fully compatible with Edman sequencing and mass spectrometry. To date, a total of 937 B. melitensis 16M protein spots have been identified using twodimensional gel electrophoresis and peptide mass fingerprinting. These proteins corresponded to 269 discrete open reading frames in the B. melitensis genome and were classified under appropriate metabolic categories [2]. A majority of these proteins were hydrophilic and only a few were hydrophobic, with transmembrane domain-containing proteins being underrepresented. Proteome analysis has shown that the two circular chromosomes of B. melitensis are functionally active [12]. The expressed ORFs identified to date by proteomics analysis revealed that they are evenly distributed over the physical map of both chromosomes. These data suggest that the two chromosomes are indispensable for the survival of this organism. Proteins involved in membrane transport as well as in carbohydrate and protein metabolism composed the majority of proteins identified in B. melitensis. A significant number of proteins with unknown functions were included under the "hypothetical proteins" category. This category was assigned by Integrated Genomics based on putative genes predicted from the annotated sequence of the B. melitensis genome (10). Comparative proteomics is a powerful tool in identifying similarities and differences in the biochemical pathways that ultimately determine the metabolic state of an organism. By looking at the expression levels of hundreds or even thousands of proteins simultaneously, one gets a global picture of what pathways are blocked, upregulated, downregulated or unaffected. Ultimately, perturbations of affected pathways have predictive values in determining the final phenotype of an organism. Such information is extremely important in probe development. Our laboratory has also conducted a global comparative analysis of Brucella abortus and B. melitensis proteomes. Initial results have indicated that their proteomes have significant differences in both the number of protein spots and their qualitative and quantitative expression pattterns on 2-D gels. Focusing on these proteomic differences may contribute to our understanding of the metabolic differences that impart host preference and virulence in the different species of Brucella. Bacillus anthracis, the causative agent of the often-fatal disease anthrax, has attracted worldwide attention due to its recent use as an agent of bioterrorism. Fully virulent strains of B. anthracis carry two large plasmids, pXOl (181.7 kb) and pXO2 (96.2 kb) that encode for toxin production and capsule formation, respectively. The genes pagA, cya and lefon pXO 1 code for the synthesis of protective antigen (PA), lethal factor (LF), and edema factor (EF), respectively [4; 8]. Systemic infection following the inhalation of B. anthracis spores usually results in shock and death within a few days following the onset of symptoms. Treatment with antimicrobials is effective only if initiated in the early stages of infection during the incubation period, which indicates the importance of rapid, sensitive and accurate diagnosis of anthrax infection [4].

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Knowledge of proteins that are induced in the host during infection (i.e., stress factors) and contribute to pathogenicity would aide in the design of safe, efficient vaccines against B. anthracis, lead to the discovery of a new generation of effective anti-anthrax drugs [8] and will greatly impact on probe development. B. anthracis is encapsulated in vivo, but produces a capsule in vitro only in the presence of bicarbonate and CO2 by growing B. anthracis on CAP plates in an atmosphere of 5% CO2 95% air[10]. It has been described that bicarbonate influences the transcription of the toxin genes, in addition to capB. Since bicarbonate is present in all body fluids in vivo (CO2 + H+ + OH" <=> H+ + HCO3 at physiological pH), it may act as a signal for the expression of all B. anthracis virulence factors. In this scenario, bicarbonate would signal the vegetative cells that they are no longer in a soil environment but in a warm-blooded organism [10; 5; 11]. Bacteria, particularly those of the Bacillus genus, are known to secrete proteins into their growth medium in large quantity. The secretome of a microorganism is the sum total of proteins secreted by the organism under a defined set of conditions. Thus it feasible to identify proteins secreted by B. anthracis, which elucidate virulence and stress factors that have not yet been characterized. A synthetic, minimal medium is available that induces B. anthracis to produce a toxins for use in anthrax vaccine development [9]. By growing B. anthracis in a protein-free medium the yield of toxin production increased two to five-fold above previously reported values. It has been shown that the transcription of the toxin genes, pagA, lefand cya are "coordinately regulated," (i.e. "induced" by bicarbonate and enhanced at 37°C), [8]. The effect of temperature on toxin induction supports the hypothesis that these conditions correspond to an in vivo environment surrogate [10]. Protein identification is possible due to advances in two-dimensional gel electrophoresis (2D-E), improved mass spectrometry (MS) techniques and the availability of complete genomic sequences of various bacilli (http://www.tigr.org/tdb/mdb/mdbcomplete.html: http://www.ncbi.nlm.nih.gov/) The identification and characterization of proteins expressed in an environment that simulates the in vivo condition for B. anthracis yielded valuable information associated with the virulence and pathogenicity of this organism, especially concerning the role of the chromosome. The proteins that were identified under each set of growth conditions were compared. Forty-one spots (open circle) were matched between the average gels of the two conditions tested (Fig. 38). Thirty-five spots were induction-independent (i.e. expressed at the same level under both sets of conditions), (Fig. 38, category A). The proteins that were CO2-HCO3inducible (Fig. 38, category B) or CO2-HCO3 -repressible (Fig.38, category C) were also examined. There were at least five proteins that appeared to be CO2-HCO3~-inducible. The category D represents spots which need further resolution (i.e. pI vs MW).

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Figure 38. Super-imposed average gels non-induced (red) versus induced (green). Category A (CO2-indifferent), Category B (CO2-inducible), Category C (CO2repressible), Category D (spot zooming needed).

In this study 27 proteins isolated under induction were assigned probable identifications; eighteen without-induction proteins were identified. One protein was correlated with an immunoreactive protein in a different organism; this could lead to a development of new methods of nucleic-acid based and immunochemical biomarkers for B. anthracis. This investigation could also lead to the identification of virulence factors not yet elucidated. A comparative proteomic study of the exosporium of B. anthracis, B. cereus, and B. thuringeinsis is presently in progress. This could also generate future probes.

References [1]

Corthals, G.L., S.P. Gygi, R. Aebersold and S.D. Patterson. 1999. Identification of proteins by mass spectrometry. p. 197-231. In Rabilloud, T. (ed), Proteome research: 2-D gel electrophoresis and detection methods, Springer-Verlag, NY.

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[2]

[3] [4] [5] [6] [7] [8] [9] [10] [11] [12]

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DelVecchio, V.G., V. Kapatral, R. J. Redkar, G. Patra, C. Mujer, T. Los, N. Ivanova, I. Anderson, A. Bhattacharyya, A. Lykidis, G. Reznik, L. Jablonski, N. Larsen, M. D'Souza, A. Bernal, M. Mazur, E. Goltsman, E. Selkov, P. H. Elzer, S. Hagius, D. O'Callaghan, J. J. Letesson, R. Haselkorn, N. Kyrpides, and R. Overbeek. 2002. The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc. Natl. Acad. Sci. USA. 99: 443-448. DelVecchio, V.G., J.M. Petroziello, M.J. Gress, F.K. McCleskey, G.P. Melcher, H.K. Crouch, and J.R. Lupski. 1995. Molecular Genotyping of methicillin-resistant Staphylococcus aureus via fluorophore enhanced repetitive-sequence PCR. J. Clin Microbiol. 33:2141. Dixon, T.C., M. Meselson, J.Guillemin, and P.C. Hanna. 1999. Anthrax N.Engl.J.Med. 341:815-826. Fouet, A., M. Mock. 1996. Differential influence of the two Bacillus anthracis plasmids on regulation of virulence gene expression Infect.Immun. 64: 4928-4932. Liebler, D.C. 2002. Introduction to Proteomics: Tools for the New Biology. Humana Press Inc., Totowa, NJ, 198 pages. Lopez, M.F., K. Berggren, E. Chernokalskaya, A. Lazarev, M. Robinson and W.F. Patton. 2000. A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in twodimensional gels and identification by peptide mass profiling. Electrophoresis. 21: 3673-3683. Mock, M., A. Fouet. 2001. Anthrax Annu.Rev.Microbiol. 55: 647-671. Ristroph, J.D., B.E. Ivins. 1983. Elaboration of Bacillus anthracis antigens in a new, defined culture medium Infect.Immun. 39: 483-486. Sirard, J.C., M. Mock, and A. Fouet. 1994. The three Bacillus anthracis toxin genes are coordinately regulated by bicarbonate and temperature J.Bacteriol. 176: 5188-5192. Stretton, S., A.E. Goodman. 1998. Carbon dioxide as a regulator of gene expression in microorganisms Antonie Van Leeuwenhoek 73: 79-85. Wagner, M. A., M. Eschenbrenner, T.A. Horn, J.A. Kraycer, C.V. Mujer, S. Hagius, P. Elzer and V. G. DelVecchio. 2002. Global analysis of the Brucella melitensis proteome: identification of proteins expressed in laboratory-grown culture. Proteomics. 2: 1047-1060.

Preparedness Against Bioterrorism and Re-Emerging Infectious Diseases J. Kocik et al. (Eds.) IOS Press, 2004

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Decontamination of Drinking Water and Liquid Media by Cold Plasma in the Special Periods Alexander A. PIVOVAROV Ukrainian State Chemical-Technolgy University 8, Gagarin Ave., Dnepropetrovsk, Ukraine 49005, [email protected] Abstract. The results of researching to decontamination of drinking water and waste water under action glow dischargeplasma are considered. Perspective of application of such way of decontamination of liquid media colonised by pathogenic and potentially pathogenic micro organisms and viruses in real conditions and in the specific of time is shown. As a result decontamination drinking water can meet the existing standards and be suitable for the subsequent use.

1. Introduction Ukraine is a problem country as to sanitary quality of drinking water and waste water discharged in to the rivers and reservoirs. The existing enterprises for preparation of drinking water do not provide its due decontamination at the final stage of preparation, which is the reason of often mass epidemics of the population. On the other hand, being on the ways of penetration of illegal immigrants from the countries of Near East and Southeast Asia in to Western Europe, Ukraine is under constant and growing threat of possible terrorist act of biological or any other character. No less acute is problem of maintenance by quality drinking water of the population during natural calamities, such as droughts, floods, high waters and floodings of the basic sources of water supply. Plentiful atmospheric precipitation and floods caused by them in Europe, Ukraine and in the south of Russia in the summer of 2002 testify to the necessity of taking emergency measures connected, first of all, with maintenance by highquality water for the inhabitants of cities, countryside and hardly accessible areas. Besides in the recent years growing tendency of universal deterioration of ecological conditions not only in Ukraine, but also in the countries around it and as a result the reducing of general immune status of the population is registered. The number of diseases caused by pathogenic microorganisms and viruses continuously grows. The increase of water consumption connected with the growth of cities, low degree of purification and growth of volume of industrial and household waste water promotes more intensive microbial pollution of the reservoirs used as sources of centralised industrial-drinking water supply. As there is no reliable technology of clearing of water purification against of virus and bacterial pollution, drinking water continues to remain one of the main factors of transfer of many infectious diseases [1]. There is a significant amount of works showing that pollution by enteroviruses of objects of the environment, including drinking water, causes a number of complex problems.

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First of all, it is caused by a long survival of enteroviruses outside human body and their ability to cause serious pathologies like polio and related diseases, encephalitis, hepatitis, gastroeteritis, aphtosa syndrome, acute respiratory enteroviruses diseases, miocarditis in newborn babies, acute haemorrhoids conjunctivitis etc. [2,3]. The level of contamination largely depends on the quality of drinking water. Presence of serovariety of enteroviruses (more than 68), absence of cross immunity to them are the reasons that during lifetime a person can be infected repeatedly by enteroviruses. A convincing proof of the possibility by enteroviruses through water are the outbreaks of various enteroviruses infections, including polio, which took place in the different countries in various years with the sickness rate from several tens up to several thousands [4,5]. Methods of drinking water decontamination used during its preparation are ineffective in relation to enteroviruses [6]. According to the statistical data in a city with the population of one million through the use of water which does not meet sanitary requirements, these arises daily 600 clinical and subclinical forms of diseases with enteroviruses aetiology [7]. Content of 0.3-0.5 mg/1 of free chlorine in water does not provide its complete purification from enteroviruses [8,9]. In research [7] a decrease of a titre of poliovirus by 104 times was observed at the content of chlorine 5-20 mg/1. During outbreaks of cholera, which took place on the territory of Ukraine and other NIS, the basic preventive measure was the increase of concentration of chlorine in drinking water 4-5 times in comparison with usual water. Thus, natural or artificial infringement in the system water preparation at any moment can result in epidemiologie complications [10]. Now there are no official normative documents determining the admissible content of enteroviruses in drinking water. According to the recommendations of WHO it should not to exceed one virus particle per 100-1000 1. Some authors [7] believe the virus agents in it should not be present at all. In least decades in Ukraine from water supply there were isolated 2109 strains of various representatives of vibriogenus, wich makes up 1.2 % of the number of the investigated tests. Even more widely (up to 20 % of positive results) they are distributed in reservoirs, water from which goes to purification facilities. Unfortunately, the isolation of vibrios from water is hampered because of the absence of reliable ways to concentrate them [11]. Among all decontamination methods used chlorination despite of is now in widest use. So, by this method more than 500 km3 of natural waters in the world are decontaminate using about 2 million tons of chlorine annually [12]. It is a fact that for some geographical areas the application of chemical methods of water decontamination is connected with significant difficulties. In conditions of low temperatures decontamination action of chlorine is not effected and at high temperatures its unproductive losses take place. The transportation of reagents to remote or scarcely populated areas costs dearly besides for reagent processing capital purification facilities are necessary. Under such conditions purification methods based on use of electrical energy are most perspective. The advantages of methods of electroprocessing of liquids in comparison with traditional ways of purification are obvious. Most evident are simplicity of the technological scheme, absence of necessity in premises for a storing reagents and equipment for their preparation, simplicity of process automation and opportunity to improve working conditions, a wide spectrum of the improved characteristics of liquids and smaller specific volumes, weight and dimensions of facilities [13-15]. The application of new methods of water decontamination up to sanitary norms and above makes possible a steep decrease in enteroviral infections incidence. Most perspective are the methods based on application of nonequilibrium low-temperature plasma (otherwise known as glow discharge plasma (GDP)) [16,17]. They have by a number of advantages: smaller size of the equipment, opportunity of automation both of the process and quality control of processed

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environment, low engagement of human resources, opportunity of the use of new solutions, though poorly investigated, but having useful potential and properties. The basis for the process is the contact plasma discharge on the surface of a liquid phase formed between an electrode in a gas phase and a surface of a liquid, in which the second electrode is immersed. GDP serves a source of chemically active atoms and molecules in a liquid causing set of chemical reactions of redox character, rendering disinfection action on pathogenic and potentially pathogenic microorganisms and viruses contained in drinking water or other liquid media [18,19]. 2. Experimental Sanitary - microbiological and hygienic evaluation of processing of water by GDP is has been done in Research Institute of Hygiene of Academy of Medicine, Research Institute Practical Toxicology and Disinfection, Moscow, Russia and Scientific-Research Institute of Hygiene, Kiev, Ukraine. The processes of decontamination of drinking water in the terms pathogenic and potentially pathogenic microorganisms were experimentally studied: Esherichia Coli, Staphylococcus aureus, Staphylococcus epidermidis, Proteus vulgaris, Candida albicans, Clostridium, Salmonella typhimurium, Salmonella munhen, Salmonella infantis, Salmonella derbi, Pseudomonas pneumoniae, Vibrio cholerae, Cornebacterium diphtheria gravis and also Poliomyelitius virus, Hepatitis virus B In the experiments to study decontaminating of GDP effect water was infected by cultures of the mentioned above bacteria in concentration, 24-hour to real conditions of possible pollution of drinking water (from 103 up to 106 microorganisms per litre). To specify a degree of GDP effect on the spore forms of bacteria having the greatest stability to any sort of adverse influences of physical, chemical and biological nature for this purpose strains of Clostridium were taken. It is known, that their vegetative form are less resistant, therefore in the experiment their culture was previously warmed up within 20 minutes at 70 °C, which resulted in destruction of vegetative forms and preservation of spore ones. Infecting of water was carried out by spore forms of Clostridium and vegetative ones separately. For bacteriological research of drinking water before and after its processing by glow discharge plasma dense and liquid nutritious medium were used. In experiments with waste water dechlorinated drinking water was used with addition 0.1 % and 1.0 % of a waste liquid from sewage network (before purification and decontamination) and also waste liquid, to which pathogenic, enterobacteria (Salmonella of 4 strains): Salmonella typhimurium, Salmonella munchen, Salmonella infantis, Salmonella derby were added. Initial concentration of Salmonella (control) and the results of GDP effect processed media were determined by inoculation of medium of dense bismuth sulphite 0.1-1.0 ml medium by inactivation of the processed waste water on liquid nutritious medium (magnesium broth of double concentration), which made it possible to determine the amount of bacteria water under study. All other bacteria indicated in the study of drinking water were determined in waste water without preliminary infection. Besides taken into account were such resistant kind of microorganisms as Enterococcus. Their isolation was done in the SlanecBertley medium. After 24-hour incubation of crops in a thermostat at 37 °C carried out calculation of GMN (general microbial number of colonies), identification and calculation of the rest of micro flora was done. To define biochemical activity of investigated cultures isolated from water differential Kigler media were used. In case of necessity (for Salmonella first of all) serological diagnosis of "suspicious" cultures was carried out. For modelling virus pollution in

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the experiment a phage of E.coly and virus of polio were used. Coliphages are widely used now in experimental research as virus models. Besides, on coliphage control of drinking water purification is carried out in sanitary - epidemiological stations of various countries to determine viral contamination [36]. Attenuated strain of polio virus 1 is close in its resistance to physical and chemical factors to wild strains of various enteroviruses and is applied in experimental conditions to estimate efficiency of purification and decontamination in terms of viruses in the most countries of the world. The technique of experimental research consists in the following. In certain volume (from 0.5 up to 3.0 1) of dechlorinated drinking water and active filtered waste water coliphage and polio virus were introduced up to final concentrations from ones up to hundreds PFU in one litre and from tens up to thousands PFU in a ml. Microorganisms suspensors obtained in researched waters were placed in special reactor with volume 30 ml and subjected to GDP effect for 1, 3, 5 and 10 minutes. In case of the minimal content of microorganisms (ones and hundreds in litre) as well as under action of plasma for a long time (5 and 10 minutes) the volume of each research test equalled to 30-1000 ml. The isolation of coliphages in tests of water was carried out by two methods: by direct inoculation and method of additional growth according to the technique given in work [20]. While studying tests with volume of 30 ml all liquid was inoculated on firm nutritious media. After cultivation in a thermostat a recalculation of PFU (pateh forming units) of coliphages for 1 ml or for 1 1 of researched liquid was done. A polio virus for isolated on the culture inoculated of Hep-2. For each test cultivation 4 test tube with culture of cells were used. The registration of results was done by cytopathic effect in 24 hours registration on the third, fifth, seventh and tenth day. On the tenth day the titre of the polio virus in TCD5o/ml was calculated according to [21]. In the absence of cytopathic effect in the test an additional passage for culture of tissue was done. The research assess the quality of water also included a study organoleptic and phisicochemical properties of water, content of organic substances and basic anthropogenic pollutants, metals and non-metal elements. In several series of research the influence of processing of drinking and river water with a various level of pollution by glow discharge plasma investigated. Preparation of model waters was done by the organic pollutants: chlorine and introducing phosphororganic pesticides, chloroform, benzapirene, carcinogenic substances, phenols as well as salts of metals: A12(SO4)318H2O, Fe2(SO4)3 9H2O, CuCl2, Cd(NO3)2 4H2O, CoCl26H2O, Pb(NO3)2, NiCl 2 6H 2 O, KMnO4, K2Cr2O7, Ba(NO3)2, Zn(NO3)2 6H2O, (NH4)6Mo7O244H2O. 3. Results and Discussion GDP effect on inactivation of coliphages and viruses in drinking water. At the first stage of experimental research dynamics of inactivation of various concentration coliphages in drinking water was studied for various time duration of GDP influence. The results of research are submitted in Tab. 18. The given data testify that the degree of inactivation of coliphages at a high level of pollution of drinking water (tens of thousands PFU/ml) after GDP treatment for 1 minute equalled 95.71 %, which is caused by the presence in the suspension of highly resistant and less resistant populations of microorganisms. Further increase of time of GDP influence resulted in the growth of a degree of inactivation. At maximal time of processing (10 min.) the amount of coliphages was reduced in water supply to 99.92 %.

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Table 18. Inactivation of coliphages in water supply in dependence on time duration of GDP effect (average date) Time of treatment, min.

PFU/ml.

Control 1.0 3.0 5.0 10.0

1.2-104 5.0-102 1.7-102 64 8.8

Inactivation, %

95.71 98.53 99.41 99.92

Time of treatment, mm. Control 1.0 3.0 5.0 10.0

PFU/ml.

1,2-102 46 0.4 0 0

Inactivation, %

61.67 99.66 100.0 100.0

Time of treatment, mm. Control 1.0 3.0 5.0 10.0

PFU/ml.

Inactivation, %

64 37 4.1 1.0 0

42.46 93.60 93.44 100.0

At initial pollution of drinking water at the level of hundreds and tens PFU/ml after one minute processing the amount coliphages was reduced on the average by 61.67 % and 42.46 %, correspondingly and only the increase of time of GDP effect on water for 3 minutes resulted in one more essential inactivation of coliphages (99.66 and 93.6 %). The analysis of the received data has shown that irrespective of the initial pollution of drinking water, after a 3-minute GDP influence was observed appraise equal decrease of the content coliphages in drinking water. Further the time necessary for complete inactivation of coliphages depended on initial concentration of microorganisms in the water. Of significant interest is the study of GDP effect to inactivate microorganisms contained in drinking water at the level of ones, tens and hundreds PFU in 1 litre, which is most frequent in natural conditions in existing methods of purification of water or under nonobservance of modes of its treatment. In this connection a series of studies to define the degree of coliphages inactivation was done to assess GDP effect under conditions of their presence in drinking water in the concentration given above. The results of research are given in Tab. 19. Table 19. Inactivation of coliphages in drinking water depending on duration of GDP affect (average date) Time of treatment, min. Control 1.0 3.0

PFU/1

Inactivation, %

9.4- 102 4.6-102 0

51.10 100.0

Time of treatment, min. Control 1.0 3.0

PFU/1

Inactivation, %

30 0 0

100.0 100.0

The data sited show that in drinking water pollution at the level of hundreds PFU/1 at one-minute GDP influence the degree of inactivation makes 51.10 % and after a 3-minute treatment of such water the degree of inactivation achieves 100 %. In a lower initial pollution (at the level of tens PFU/1) 1 min. of treatment is enough for complete coliphages inactivation. Along with the above research the GDP effect on inactivation of polio virus was studied. The results of this research are submitted in Tab. 20. Seen from the tab. 18 the polio virus contained in drinking water at the level of thousands of virions in 1 ml is inactivated by 45.1 % after oneminutes GDP treatment and by 99.9 % after the effect of glow discharge plasma after 3 minutes. Complete inactivation of the virus was observed after a 5-minute GDP treatment. At the content of the virus in drinking water at the level of hundreds PFU/ml after one-minute treatment it is inactivation by 38.1 % and is not found in tests of water after 3 minutes of GDP treatment. At the lowest concentration of the virus in drinking water (at the level of a few) it is not found in tests of water after one-minutes GDP influence.

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Table 20. Dynamics of inactivation of the polio virus in drinking water under GDP influence Time of treatment, min. Control 1.0 3.0 5.0

Titreofthe virus in TCD5o/ml

Inactivation, %

3.2±0,2 1.79±0,23 traces 0

45.1 99.9 100.0

Time of treatment, min. Control 1.0 3.0 5.0

Titre of the virus in TCDso/ml

Inactivation, %

2.19+0,23 1.34±0,2 0 0

38.1 100.0 100.0

Time of treatment, min. Control 1.0 3.0 5.0

Titre of the virus in TCD50/ml

Inactivation, %

0.84+0,2 0 0 0

100.0 100.0 100.0

It is necessary to note that at 5-minute of GDP treatment water renders toxic effect on cells of tissue culture and Esherichia Coli (strein HB2). This phenomenon was repeatedly observed in the research and was confirmed in passages. Thus, the given data of experimental research testify to high inactivation GDP effect as to indicators (coliphages) and viruses in drinking water. GDP effect on coliphages and viruses inactivation in waste water. The study into coliphages inactivation in waste water was carried out with use of imitate and filtrate of native waste water. The results of research are presented in Tab. 21 Table 21. Inactivation of coliphages in waste water in dependence on duration of GDP influence (average date) Time of treatment, min.

Control 2.0 3.0 5.0 10.0

Imitate of waste water

Filtrate native waste water

PFU/ml.

Inactivation, %

PFU/ml.

Inactivation, %

5.6-104 1.6-104 2.3-102 9.7 0

71.50 99.98 99.98 100.0

5.6-104 1.2.103 1.9-102 0.67 0

97.54 99.61 99.99 100.0

The presented data testify that after 1.0 min. treatment of waste water coliphages are inactivated in the imitate of waste water by 71.5 % and in the filtrate by 97.54 %. After an increase of time of GDP influence up to 3 min. the degree of inactivation practically remained the same equalled 99.58 and 99.61 % correspondingly. The greatest degree of inactivation of coliphages (more than 3-4 orders) in these waters was registered after 5 minutes of GDP treatment. After 5.0 min. of processing it was established that in nutritious media on the inoculated water tests grow of Esherichia Coli (strain HB2) was not registered. In the study of inactivation process of the polio virus in waste water it was established that after 1 minute of GDP treatment the degree of inactivation reaches 52.5 and 41.3 % depending on initial concentration of the virus in waste water (Tab. 22). The increase of time of GDP treatment up to 3 min. resulted in inactivation of the virus by 99.64 % (at higher initial concentration of the virus in waste water) and by 99.9 % at lower concentration of the virus. After 5 min. of GDP treatment the virus was found in tests in insignificant quantity (in 1 of 4 infected test tube) and was not found in tests of water in the second case. Thus received data testify to high inactivating effect of GDP as to the polio virus and coliphages contained in waste water. Practically 5-minute GDP treatment of such water results in practically complete inactivation of viruses contained in it.

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Table 22. Dynamics of inactivation of the polio virus in the filtrate of native waste water under GDP influence Time of treatment, min. Control 1.0 3.0 5.0

Titre of the virus in TCD5o/ml 3.28±0,2 1.55±0,23 0.84±0,2 traces

Inactivation, %

Time of treatment, min.

-

Control 1.0 3.0 5.0

52.8 99.64 99.99

Inactivation, %

Titre of the virus in TCD5o/ml 2.64±0,2 1.55±0,3 traces 0

41.3 99.9 100.0

Efficiency of decontamination of water containing spores of B.cereus was studied in term of initial concentration of the spores in water at various parameters of plant work. Antimicrobial effect of the GDP was found to shown in the itself relation of Staphylococcus aureus and Micobacterias B5 after 30 min., P.auruginosa - 15 min., yeast-like fungi Candida albicans - 5 min. Most resistant to GDP influence among vegetative forms of microorganisms are S.aureus and Micobacterias B5. Less resistant are yeast-like fungi Candida albicans and P.auruginosa. On the basis of the data obtained for the further research S.aureus and Micobacterias B5 were selected. The results of decontamination of water contaminated by S.aureus and Micobacterias B5 treated by GDP are presented in Tab. 23 Study of efficiency of decontamination of water containing test - virus (vaccine strain of the polio virus of type 1) used in the development of modes of disinfection of products of medical purpose carried out under GDP influence on water for 1, 3, 5, 10, 15 and 30 min. The control for the tests was effected through tests of waters containing the virus before its GDP treatment. The results of research are given in tab. 24. Table 23. Decontamination of water contaminated with S.aureus and Micobacterias under GDP influence Time of treatment, mm.

Microorganism.

Quantity of viable cells in 1 ml of water. Before treatment After treatment 3.86-105

Staphylococcus aureus

5.0 10.0 15.0 30.0

2.64-105 1.82-102 1.60-101 0

Micobacterias 15.0 30.0 45.0 The note: "+" - presence of growth; "-" - absence of growth.

+

+ -

Table 24. Decontamination of water containing of polio virus under GDP influence Time of treatment, min.

Quantity of the virus, Ig TCD5o/ml Before treatment

After treatment

5.26+0,07

1.0 3.0 5.0

3.26+0,07 0.43+0,08 0

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Organoleptic properties and phisico-chemical composition of water treated with glow discharge plasma. Drinking water. Estimation of the parallel phenomena in water as a result of GDP effect during decontamination of drinking water was done along the following basic guidelines: study of possible negative influence of the given treatment method on the quality of water obtained, study of the efficiency of additional purification of water from various kinds of chemical pollution. The analysis of results of sanitary - chemical tests has shown that under GDP effect (at the maximal GDP exposition found by the results of microbiological study to be 5 min.) organoleptic parameters of the treated water did not change greatly. The odour of water practically remained at the same level, colour intensity of water after treatment was reduced on the average by 35 %, at the initial level appropriate to the hygienic rules (20°). Turbidity of water after treatment was considerably reduced. Change of physical properties of water at the same time was registered. After treatment the pH shift to alkaline medium (from 7.5 to 9.4 units) was observed which exceeds the hygienic standards for drinking water; temperature of the water rose on the average by 10-15°C. Insignificant decrease of salt content in the treated water was found to meet optimum standards for drinking waters of hydrocarbonate class (250-500 mg/1). The treatment of running of water by GDP resulted in the change of the amount of nitrogen containing compounds; the content of ammonium nitrogen in the treated water decreased on the average by 22 %, and the concentration of nitrites grew approximately by 30 %, but did not exceed the hygienic norm; the content of nitrates practically did not change. As a result of study into the quality of water by the indices of salt compositions before and after GDP treatment it was fond that in the treated drinking water, in comparison with control, some decrease in general salt content is registered. It is explained by the basic reduction of concentration of bicarbonates as well as insignificant decrease of salt hardness (calcium and magnesium). The received data testify that indirect parameters namely content of organic substances in water under study (permanganate oxidability, the organic carbon) changes differently. Thus permanganate oxidability in GDP treated water grew and the organic compounds diminished which apparently, is caused by processes of oxidation of organic substances and their transition into easily oxidised forms. The concentration of organic carbon was reduced on the average by 60 % in comparison to the initial level. The analysis of quality of running water by other parameters of organic pollution of water, in particular by halogen containing compounds (GCC), formed during chlorination of water testifies to high efficiency of water purification from GCC as a result of its GDP treatment. The concentration of GCC in this case did not exceed the hygienic standards. The similar regularity was established for other widespread anthropogenic organic pollutants of water (surface - active substances, phenols etc.) at their initial content in the level of the ultimate concentrations. River water. The experimental study was done on the bases of model water solutions described by higher levels of chemical pollution. For this purpose river water was used with previously introduced various kinds of chemical pollutants (phenols, carcinogenic substance, polyform compounds, and salts of heavy metals). The analysis of results of organoleptic tests has shown that during GDP water treatment the odour of water practically did not vary at high initial colour intensity (38°), the treated of water had colour intensity far below the hygienic standards for sources of industrial and drinking water supply (10°). Alongside with it, as well as in the previous series of tests, the shift of the hydrogen parameter index from 7.5 down to 9.8 was registered. The oxidation of inorganic nitrogen containing substances was estimated by the change of the content of ammonia, nitrites and

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nitrates. In water after glow discharge treatment the content of ammonia was reduced by 1.3 times, nitrites remained at the same level as before treatment and nitrite content rose a little (0.7 times). By the change of the content of organic substances in river water it is possible to note some increase of the content of light organic compounds (by the permanganate oxidation index) after its GDP treatment (7.4 mg O2/l). The content of organic carbon after treatment water was reduced by 3 times (from 19.5 down to 6.5 mg/1). The analysis of research results has shown that at on GDP influence on river water containing phenols there is a decrease of their concentration practically by 2 times. The efficiency of purification is high enough, however the content of phenol in the initial and treated water exceeds maximum norms (admissible concentration limit (ACL) - 0.001 Mr ). The similar regularity is established also allowable for benzapirene. The obtained data testify that the decrease of concentration of benzapirene (82 %) is observed; after water treatment its content in the treated water does not exceed treatment maximum allowable concentration. The highest efficiency of purification is registered for halogen-containicy compounds in river water. The research has shown that during treatment of river water glow discharge plasma there occurs a considerable decrease of concentration of halogen-containicy compounds the contents GCC in the water is at the level of the hygienic norm (30 mkg/1). The result of the analysis of the data obtained makes it possible to make the conclusion that the use of the given method alongside with decontamination provides effective purification river water of a number of organic compounds and, first of all, from galoidform compounds, phenols and carcinogenic substances. The influence of GDP treatment on water and water solutions containing ions of various metals, was also investigated for two cases. In the first case selected for research was drinking water with a low content of compounds of metals polluting such water; in the second case the research object was river water containing compounds of metals of various concentrations characteristic of river water from reservoirs adjacent to industrial cities. As a result of research it is established that the concentration alkaline and alkaline-earth metals in drinking water before treatment varies insignificantly. The content of magnesium is reduced by 26 % and concentrations of calcium, potassium and sodium practically do not change. As regards to other researched elements a significant decrease of concentration of ions of metals after GDP treatment of drinking and river water is registered. Most effective is the purification of water of zinc, copper, manganese, iron, aluminium and other polyvalent metals. Especially it is necessary to note, that the GDP treatment of water with the high content of metal compounds is as effective as of the water with their low content. General result of the GDP treatment of drinking and river water is the decrease of metal compounds to the levels required by hygienic norms. In 1995-1997 JSC "Dneprovsky Machine-Building Factory", Dnepropetrovsk, Ukraine, under research supervision of the author of this article produced pilot plasmachemical plant (Fig. 39) with productivity up to 1.0 -1.6 m3/hour of treated liquid media.

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Figure:39. Pilot plasmachemical plant

Further production of such plant in Ukraine was suspended due to insufficient funding. However, the accumulated experience of development, designing, manufacture and operation of plasmachemical equipment provides for a possibility to produce similar plasmachemical complexes mainly for decontamination of local flows of drinking and waste water including special periods of decontamination of centres bacterial pollution or destruction of the accumulated stocks of the bacteriological weapons. Such complexes can be applied for purification of local flows with homogeneous chemical composition of the extracted in mechanical engineering, hydrometallurgy etc. The author expresses deep gratitude to the workers of the above-mentioned institutions and enterprises the help rendered in the course of the research.

References [1] [2] [3] [4] [5] [6] [7] [8]

Viruses and bacteria in drinking water /V.I. Zadorognjaa, V.A. Bondarenko, V.V. Alekseenko etc. // Chemistry and technology of water -1993, -15, Nol.-P. 66-71. Wiedenmann A., Eleger B., Botrenhart R. Enterobactericeae as a criterion for the quality of drinking and swimmng-podwater//Zbl.Bacteriol/-1987. -B184, No 5.- P. 426. Kooij D. Properties Aeromonas and their occurrence hygienic significance in drinking water //Zbl.Bacteriol.-1988.- B.107..N91.-P. 1-17. Edge I.,Finch P. Observations on bacterial aftergrowth in water supply distribution systems, implications for disinfection strategics// J.Inst. Water Environ. Manag.-1987.-l,.Nol.-P.104-l 10. Lloud B. J.,Wheeler D. C.,Pardon M. The relationship between water-related discard and water quality with particular reference to urban water supply in developing country //Water Sci. And Technol.-1989.21,No6/7.-P. 579-591. Akin E.W. Occurrence of viruses in treated drinking water in the United States //Water Sci. And Technol.-1985.-17,No24/5.-P.689-700. Goldberg M.B.,Dicita V.J., Caldorwood S.B. Identification of an ironregulated virulence determinant in vibrio cholerae, using Tupho a mutagenesis//Infest,Immun.-1990.-59,.Nol.-55-60. Slade J.S. Viruses and bacteria and bacteria in a chick well //Water Sci. And Technol.-1985.-17,No 10.P.lll-125.

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[9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]

[21] [22]

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Desinfection of advanced wastewater treatment influent by chlorine dioxide and ozone: Experiments using seeding poliovirus/ R.Warriner, K.D.Kostenbader, D.O.Cliver, W.-Ch. Ku// Water Res.-1985.19,No12. P.1515-1526. Lovzchevich E.L. Some aspects of inactivation enteroviruses process by chlorine // Hygiene and sanitation.-1973.- No 10.-P.11-15. Schulze E. Die virologische Uberwachung der Trink-wasserqualitat eine Ubersicht//Zbl.Microbiol.1990.-145,N22.-P.135-143. Magnetic immunesorption for selective concentrating of cholera embryos / S.A.Shaplo, E.I.Efimenko, N.I.Narbutovich etc. // Information of the problem-thematic of'Cholera' .-Rostov upon Don , 1989.-P.2728. Syrkina I.G., Uljankina G.S., Abramova V.I. Disinfectants. Review. Inform.series. Industry of chlorine. M. NIITECHIM -1986.-87 P. S.E., Speck M. L. Inactivation of microorganisms by electrohydraulic shock // Appl.microb.-1967.-l 5. P.1031-1037. Gilliland S.E., Speck M. L. Mechanism of the bactericidal action produced by electrohydraulic shock // Apple. Microb.- 1967.-15.P.1038-1044. Edebo L., Holme I., Selin I. Influence of the conductivity of the microbiological effect of transient electric arcs in aqueous systems .//Appl. Microb.-1969.- 17,.Nol.-P.59-62. Robinson J.W., Ham Mooyoung, Balaster A.N. Ultraviolet radiation from electrical discharges in water. // J .Appl. Phys. -1973. -44. P.73-75. Malik M.A., Chaffar A., Malik S.A. Water purification by electrical discharge. // Plasma Sources Sci. Technol. -2001,-10. P. 82-91. Pivovarov A.A., Sergejeva O.V. Physico-chemical transformations in water environments under action of non-equilibrium plasma of glow discharge. Problems of chemistry and chemical technology. Vol. 3, 1999. P. 48-50. Pivovarov A.A. The Advanced Technology of Extraction of Noble Metals from Industrial Waste in Circumstance of Market Economy. NATO Science Program. Advanced Research Workshop (ARW) "From Transitional Economy to Sustainable Development". Dnepropetrovsk, Ukraine, October 22-25, 2001. The instruction by definition of bactericide properties of new disinfectants./ Authorized by Ministry of Health of USSR No739-68. 06.05.1968. The methodical of recommendations by definition of preparations virulidic activity. /Authorised by Ministry of Health of USSR Nol 119-73. 06.09.1973.

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Antibacterial adsorption-filtering materials for individual protection means of organism 1. ANTIBACTERIAL ACTION OF METAL-CONTAINING CARBON ADSORBENTS N. KARTEL, A. GRIGORIEV, D. SHVETS, V. STRELKO Institute for Sorption and Problems of Endoecology, NAS of Ukraine, Kiev, Ukraine, e-mail: [email protected]

1. Introduction Questions of clearing of ecosystem objects (water, products of feed) from viruses, microorganisms and fungi, especially pathogenic for human organism, are exclusively important for today [1]. Their occurrence in products of feed and drinking water can be one of consequences of technogenic activity, infringement of sanitary norms in food industry, and also probable acts of bioterrorism directed on creation of complicated epidemiologic situations in concrete regions or objects. On the other hand, in medical practice the large importance is got by various materials with antibacterial activity for more effective treatment of contaminated wounds, ulcers and burns [2]. In this connection the search and creation of new perspective materials and technologies on their basis capable to keep and to render antibacterial (bactericidal, bacteriostatic) influence on microorganisms, is the major research problem of the experts of various areas - chemistry, material science, microbiology, medicine etc. To one of the fruitful approaches in creation of materials with antibacterial activity represents use of adsorption materials. Really, various adsorbents, having the developed porous structure and surface of interface are capable to keep crates of microorganisms at the expense of various intermolecular and ionic interactions of collective character with corresponding protein fragments of membrane structures of bacteria [3]. However the majority of adsorbents as a rule has no expressed antibacterial influence in relation to adsorbed on their surface microbial cells; that results in formation of conglomerates of activators with local infecting concentration [4, 5]. For maintenance bactericidal ability of adsorption materials the antibacterial means are introduced into their structure. The purpose of the present work was the study sorption-bactericidal action of carbon materials modified by ions of copper and zinc.

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2. Material and methods. In work it is used the 24-hour cultures of Staphylococcus aureus 209, Pseudomonas aeruginosa 103 and Escherichia coli 41, grown up on agarous nutritious media. The colonies of microorganisms were washed off from plates by isotonic solution of sodium chloride, twice washed and prepared a suspension, containing 109 microbial cells on 1 ml. The carbon adsorbents SCN and KAU represented accordingly spheres and grains of the size up to 0.5 mm. In experiments it was used carbons, having on a surface oxygen-containing functional groups, and also ions Cu2+ and Zn2+ with concentrations in the range of 2.5-0.025 meq per 1 g of a material. With the purpose of definition optimum of bactericidal concentration of metals included in adsorption material, previously by method of serial dilutions it was studied an action of various concentration (0.1-25 mg/ml) of copper and zinc salts (CuCl2, ZnCl2) in a physiological solution on viability of researched microorganisms. For definition of a level of microorganism adsorption on carbon materials the last were sterilized, packed on 0.5 cm3 in flasks, where 0.75 ml of microorganism suspension (109 microbial cells) was brought in. Adsorption was carried out at temperature 4 °C during I h at weak vibration of flasks. Further in flasks 10 ml of buffered physiological solution (pH 7.2) was brought in, shook up, and centrifugated during 10 min at 1000 cycles/min for deposition of adsorbent. Liquid phase was put on agarous nutritious media Endo and yolk-salt agar depending on a kind of microorganisms was taken in experiment. By practical consideration it was selected such concentration and volume of sowed material, that the number of evolved colonies did not exceed 200-300 on a plate. The control was served a similar volume of microorganism suspension without carbon material. The results were estimated, proceeding from differences of control and test sowings in recalculation on amount of adsorbed microbial cells in 1 cm3 of adsorption material by following formula: A = (K-O) / V where: A - sorption activity of carbon, (K-O) - amount of microbial cells adsorbed on carbon, V - volume of carbon material used in experiment.

Further the sorbents with keeping on it microbial cells were washed six-times by buffered physiological solution (pH 7.2), and elutes were sowed on agarous media. Bactericidal effect was observed at the following carrying washed adsorbent in meat-peptone broth (MPB). The infection ability of activators adsorbed on carbon materials was determined by a level of colonization of tissues on model of the isolated intestine fragment of white impure rats. The fragments were washed by a physiological solution, ligatured on the one side. Into a gleam it was quantitatively entered a suspension of carbon material SCN (size of particles up to 500 u) with definite amount of cells E. Coli 41 adsorbed on it. Then imposed second ligature. By control it was served the intestine fragments of similar length with entered in them suspensions E. coli of the same concentration as in experiment without adsorbent. To except the reproducing of microorganisms the experiments on adhesion were carried out at temperature 4 °C during 30 min. The ligature were removed, the fragments of intestine washed out by a buffered physiological solution (pH 7.2), homogenized, diluted to sowing concentration and sowed on plates of Endo' agar, differentiating thus adsorbed microorganisms

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from non-adsorbed ones on a surface of mucosa. The results were estimated on a difference between evolved colonies in test and control sowings in colony-formation units on 1 cm3 of a mucosa surface of intestine.

3. Results and Discussion Adhesion of microorganisms in metal-containing carbon sorbents. The study of influence of salts CuCl2 and ZnCb in decreasing concentration on growth of E. coli has shown that copper chloride in concentration of 0.4 mg/ml, and zinc chloride in concentration of 1.5 mg/ml do not render appreciable influence on reproducing broth culture E. coli 41. The appeared inhibition concentrations were: for salt of copper - 0.7-1.0 mg/ml, for salt of zinc - 4.0-15.0 mg/ml. The data of these experiments have formed the basis for introducing ions of copper and zinc in structure of adsorbents in a range of concentrations including threshold meanings for salt solutions - 80-0.6 mg/ml (2.5-0.25 meq per 1 g of sorbent). The study of adsorption of microorganisms has shown that oxidized carbon materials such as SCN, not modified by metals and widely used in medicine, substantially (from 5.4* 108 up to 7.1*108 microbial cells by 1 g of adsorbent) have adsorb E. coli and P. aeruginosa (Table 25). The level of adsorption S. aureus is rather low and makes 1.9*108 microbial cells by 1 g of adsorbent. It is necessary to note that carbons without oxygen-containing groups have greater adsorption activity concerning this type of microorganism - on the average 6.4* 108 microbial cells by 1 g of adsorbent. The introduction of copper ions in SCN in concentration 2.5 meq/g allows practically completely adsorb microbial cells from suspension (7.5*108 microbial cells by 1 g of adsorbent). It is possible to consider decreasing concentration of copper ions up to 0.025 meq/g proved for non-oxidized samples. On oxidized samples with the copper ions included in structure a level of adsorption of E. coli and P. aeruginosa is some less in comparison with samples of SCN, not containing copper ions. It is represented interesting that, as against oxidized copper-containing samples of SCN, having a rather low level of adsorption in the relation S. aureus, oxidized carbons with ions of zinc are characterized high adsorption activity concerning this microorganism (100% - at concentration Zn2+- 2.5 meq/g and 97.1% at concentration Zn2+- 0.25 meq/g). At the same time introduction in the carbon sample of SCN of zinc ions results in some decrease of a level of adsorption of E. coli and P. aeruginosa in comparison with a control sample, which has especially small concentration of metal (0.025 meq/g). Last years the increased attention is given to carbon sorption materials such as KAU (active carbon from fruit-stones), as to cheaper natural materials. The oxidized form of a sample of KAU, not modified by ions of metals, has quite high adsorption activity concerning researched microorganisms, reaching 7.5* 108 microbial cells by 1 g of adsorbent. Nonoxidized samples of KAU are characterized by a smaller level of adsorption in comparison with non-oxidized SCN and oxidized KAU. The updating fruit-stones carbons by copper ions provide high sorption-bactericidal effect at all samples taken in experiments. Thus as optimum concentration of copper ions it is possible to count 0.25 meq on 1 g of adsorbent, as the parameters of a level of adsorption in this case change in limits from 72 up to 100%. It is necessary to note that the copper ions in these samples are less effective in relation S. aureus, than ions of zinc. The sorption-bactericidal effect of fruit-stones carbons with concentration of ions of zinc 0.25 meq/g remains high for all taken in experiments microflora.

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The found out distinctions in levels of adsorption interaction at various samples with concrete cultures of microorganisms allow assuming an opportunity of the directed application of the modified carbon materials. All carbon materials containing ions of copper and zinc, as against unmodified, render more appeared destructive action on bacterial cells, and this proves to be true by absence of growth of microorganisms in sowings of elutes and at entering samples of carbons with adsorbed cells in meat-peptone broth. Table 25. Comparative sorption-bactericidal efficiency of carbon metal-containing adsorbents Sample of carbon materials SCN - initial - oxidized

Content of metal, meq/g

0

2.5 SCN-Cu2+ - initial - oxidized

0.25 0.025

2.5 SCN-Zn2+ - initial - oxidized

0.25 0.025

KAU - initial - oxidized

0.25 0.025

2.5 KAU-Zn2+ - initial - oxidized

(7.0+0.2)* 108 (7.1±0.4)*108 (7.4+0. 1)*108 (7.4+0.1)*108 (7.3+0.2)* 10s (7.0+0.2)* 108 (7.0+0.3)* 108 (4.6+0.4)* 108 (7.0+0.5)* 108 (6.5+0.2)* 108 (7.0+0.3)* 108 (5.9+0.1)*108 (5.0+0.7)*108 (2.8+0.7)* 10"

(6.4±0.6)*108 (1. 9+0.7)* 108 (7.5+0. 1)*108 (7.4+0. 1)*108 (5.7+0.3)* 108 (3.3+0.6)* 108 (5.6+0.6)*108 (2.9+0.2)* 108 (6.8+0.2)* 108 (7.5+0.1)*108 (6.5+0.6)* 108 (7.3+0.4)* 108 (5.1+0.6)*108 (7.2+0.3)* 108

(6.1+0.1)*108 (5.4±0.2)*108 (7.5+0. 1)*108 (7.5±0.1)*108 (7.5+0. 1)*108 (5. 1+0.5)* 108 (6.4+0. 1)*108 (3.4+0.4)* 108 (6.5+0.4)* 108 (6.1+0.1)*108 (6.2+0.3)* 108 (5.2+0.5)* 108 (3.8+0.9)* 108 (3.0±0.5)*108

(5.9±0.4)*108 (6.8±0.3)*108 (7.5+0.1)*108 (7.5+0.1)*108 (7.0±0.3)*108 (7.5+0. 1)*108 (5.8+0.5)* 108 (6.3+0.5)* 108 (7.2+0.3)* 108 (7.5+0.1)* 108 (6.4+0.4)* 108 (7.5+0. 1)*108 (3.5+0.7)* 108 (5.0+0.3)* 108

(4.9+0.2)* 108 (7.5+0.1)*108 (7.5+0. 1)*108 (7.5+0.1)*108 (5.4+0.3)* 108 (7.2+0.2)* 108 (5.5+0.4)* 108 (6.9+0.6)* 108 (7.5+0.1)* 108 (7.5+0. 1)*108 (7.5+0.1)*108 (7.2±0.3)*108 (5.3+0.1)* 108 (6.8+0.8)* 108

(5.9+0.2)* 108 (7.0+0.2)* 108 (7.5+0. 1)*108 (7.5+0. 1)*108 (7.4+0.2)* 108 (7.5+0. 1)*108 (5.7+0.8)* 108 (6.5+0.2)* 108 (7.5+0.1)*108 (7.5±0.1)*108 (6.6+0.2)* 108 (7.4+0.1)*108 (5.9+0.6)* 108 (5.2+0.4)* 108

0 2.5

KAU-Cu2+ - initial - oxidized

Sorption-bactericidal activity, microbial cells per 1 cm3 of adsorbents (M+m) P. aeruginosa S. aureus E. coli

0.25 0.025

Due to ability of carbon metal-containing adsorbents to destroy adsorbed microbial cells and simultaneously to adsorb their toxins, these materials can be used in medicine. For the proof of this thesis we carry out comparative study of a level of colonization of intestine mucosa of white rats by culture E. coli adsorbed on unmodified and copper-containing carbon such as SCN. The introduction of copper-containing carbon suspension with adsorbed cells of E. coli 41 in a cavity of living fragment of intestine strengthens a level of colonization in comparison with the control in 10-16 times. If to accept a level of colonization of intestine mucosa by bacteria E. coli, entered without adsorbent, for 1, the level of colonization for metal-containing carbons is equal 0, and for unmodified non-oxidized carbons SCN - 11+2. Thus, it is possible to assume that use of adsorption materials which are not having bactericidal properties, will promote adhesion process of activators on tissues are trope to them .

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Efficiency of adsorption of Cholera vibrionfrom water by copper-containing carbon fibrous adsorbents. Using the developed approaches on modification of carbon adsorbents by metal ions it was studied the efficiency of sorption removal of Cholera vibrion from water solutions. The structural - sorption characteristics of carbon materials and their modified by copper ions forms are illustrated in Table 26 It was found that the content of copper ions in sorbents in the range of 2-4 mg/g did not change essentially parameters of carbon fabric and felt but changed their surface chemistry. This is testified by the decreasing of pH zero of charge surface of carbons modified by copper ions (Table 26). The shape of the adsorption isotherm of trypsine on the carbon fabric in phosphate buffer testifies to the absence of influence at decreasing ratio of solid and liquid phases. The modification of carbon fabric by copper ions leads to increase the trypsine adsorption in 2.0-2.5 times. By analogy with this the influence of the modification was shown at study of the sorption activity of carbon materials to Cholera vibrion (Table 27). The influence of the nature of carbon materials is displayed at absence of microorganism on the carbon felt, and this correlated with its low structural-sorption characteristics and lower kinetic of the Cholera vibrion extraction in compare with the carbon fabric. It was established that using the ratio of solid and liquid phase 1:50, time of contact 5 min and contain of copper ions in the sorption material 2.3 mg/g it was observed 100% extraction of Cholera vibrion from sea water solution. With increase of the Cholera vibrion content to 106 microbial bodies was also founded 100% extraction of them from solutions by the modificated carbon fabric. So the modification of carbon materials by copper ions does not only increase their bactericidal properties but perhaps forms the copper (II) ion complexes with nitrogen- and oxygen groups of trypsine besides electrostatic interaction of carbon with polar molecules of albumen and the formation of H-bonds with hydroxyl and carboxyl carbon groups. Besides that, ions of copper on the surface of carbon sorbents can be able interact with spiral albumens and neutralize charge of membranes. Table 26. The structural - sorption characteristics of carbon materials Sorbents

Content of Cu2+; mg/g

Carbon fabric Carbon felt

2.3 3.9

Volume of sorption pores; cm3/g H2O C6H6 0.06 0.01 0.01 0.05 0.01 1.21 0.02 1.15

Adsorption of methylene blue, mg/g

pH zero of charge of surface

135 75 430 360

6.8 6.3 7.4 6.6

Table 27. Sorption ability of carbon materials to Cholera vibrion (the concentration of 104 m. b./ml). Sorbent

Content of Cu2+; mg/g

S:L

Time of contact; min

Sorption ability, %

Carbon fabric

1.0 1.3 2.3 4.0 -

1:50

5

1:100

5 60 5 60

60 70 95 100 100 0 0 75 100

Carbon felt

3.9

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Figure 40. The influence of ratio Solid: Liquid on the adsorption of the trypsine by carbon fabric

4. Conclusions The carried out researches have shown that the copper and zinc ions are perspective at updating carbon materials with the purpose of giving them bactericidal properties. The received preliminary results of application of local sorption for treatment of an experimental purulent wound of white impure rats with use of metal-containing adsorbents have shown absence any general resorbtive toxic action of copper and zinc ions on organism of animals. As now there are various forms carbon adsorbents (from grains of various forms and thin powders up to fibrous structure), there is a real prospect of application of metal-containing adsorbents as for local adsorption in surgical practice, and for oral sorption in infectious intestine pathology. Besides the modifying of carbon materials is necessary and useful in case of their use for additional purification or additional disinfections of drinking water, which can be carried out even in household conditions.

References [1] [2] [3] [4] [5]

Grigor'eva, L.V., Kas'yanenko, A.M., Korchak, G.I. et al (1985) Sanitary microbiology of evtrofed water basins, Zdorov'ya, Kiev. Enterosorption/EA. Belyakov N.A. (1991), Tsentr Sorbtsionnyh Tekhnologiy, Leningrad. Iljin, L.A. (1987). Antimicrobial materials, Medicina, Moscow. Grigoriev, A.V., Zemskov, V.S., Shor-Chudnovsky, M.E., et al (1990) Interaction of metal-comtaining adsorbents with pathogenic microflora, Clinic Surgery, N3, pp. 42-44. Grigoriev, A.V., Znamensky, V.A., Bugaev, V.I., et al (1991) Adhesion of microorganisms in metalcontaining carbon sorbents, Microbiol. J., V. 53, N2, pp. 98-103.

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Antibacterial adsorption-filtering materials for individual protection means of organism 2. FINE FILTERING MATERIALS ON THE BASE OF POLYPROPYLENE MICROFIBRES AND THEIR ANTIBACTERIAL PROPERTY M. TSEBRENKO, V. REZANOVA, I. TSEBRENKO, M. MAYBORODA, N. KARTEL* Kiev National University of Technologies and Design, Kiev, Ukraine, *Institute for Sorption and Problems of Endoecology ofNAS of Ukraine, 1. Introduction One of methods of effective protection of the human organism from harmful influence of colloid particles of air and water media, including crates of microorganisms and their toxins, is the microfiltration through fine fiber filters. In the Problem Laboratory of Synthetic Fibres at the Kiev National University of Technologies and Design the fundamental researches are executed in the field of physical chemistry of melts of polymer mixes allowed to create scientific bases of reception of ultrathin synthetic fibres [1,2]. The principal scheme of preparing ultrathin synthetic fibres as well as microphotographies of obtained bi-component product and texture of filtering material (after extraction of one of the component) are shown on Fig. 41.

X 1OOO

Figure 41. Scheme of ultrathin fibres formation and SEM microphotos of obtained product

By practical result of these researches was the development of the technology to prepare essentially new fine fibre filtering materials (FM) and filters, which are already

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produced by JSC "UKRFILTR" (Chernigov, Ukraine). Basic structural unit of developed FM are the ultra thin fibres with unique structure of a surface: each fibre of micron size is covered on all surface with most thin microfibrilles, departing from the basic fibre (see Fig. 42).

Figure 42. Microfibrillar structure of ultrathin polypropylene fibres

In result the extremely advanced surface turns out and it provids high adsorption ability and capacity. Such fibres are not present in a nature, and they can not be prepared on traditional technologies. The comparison of various filtering materials and membranes on selectivity testifies that we have created principally new FM of volume action. Its selectivity is higher, than traditional deep ones, and in the range of particles near 1 micron it separates as a membrane of a set type. This is possible to explain by several physico-chemical processes, which take place at fine filtration: adsorption, contacting effects, Brown diffision. The important role is played also an electrostatic interaction. Developed fine fibre polypropylene filtering materials, as against all other synthetic fibres, have a negative charge, which superiors a charge of a natural leather. It enables to catch microparticles and microdrops considerably smaller, than nominal size of pores of FM. By main characteristics the developed FM are at a level or surpass the filtering materials of such well-known producers, as "Millipor", "Pall", "Gelman" etc. To the present time the filters from polypropylene (PP) microfibres with a subtlety of clearing 1, 0.45, and 0.3 u are created and already widely used for a filtration of liquids. Use of PP gives to FM a chemical inertness and stability to aggressive media. By the results of toxicological tests the fine fibre PP filters are recommended for clearing drinking water in household conditions, salines and drugs for injection in organism. The carried out researches have shown that developed FM detains an oil fog (radius of particles 0.14-0.17 u) with efficiency of 99.99% (tests are spent in the L.Ya.Karpov Research Physical Chemistry Institute, Petryanov Laboratory of Filtration, Moscow), and at a filtration of technological air at the Sumy Biofactory and carbon dioxide at the Factory of Champaign Vines "Zolotaya Balka" (Sevastopol) the sterile filtered gaseous media are obtained. The filters combined with a carbon fabrics having, alongside with high selectivity, large adsorption activity are created also. 2. Improvement of drinking water indexes by fine fibre PP filters Together with the international association "Water and Health" the sanitary - chemical and microbiological researches were executed according to efficiency of clearing of drinking water in filter devices "VIN-5" and "Krynichka", in which the filters from PP microfibres, and also filters combined with a carbon fabric are used. The permission of Central Sanitary

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Epidemiologic Service at the Ministry of Public Health of Ukraine on clearing of drinking water by mentioned devices is received. It is shown (Table 28) that PP and combined filters, excepting actually filtering action, essentially improve quality of drinking water: in 3-3.5 times the contents of ammonia, nitrates, zinc, copper, iron, manganese decreases; residual chlorine and led are removed from water completely, in 2-10 times the contents of salts of heavy metals decreases. These are explained by various physico-chemical processes, which take place at precision filtration through fin fibre filters: adsorption, surface phenomena, Brown diffusion, electrostatic interaction. Table 28. Results of laboratory tests of filtering elements in FFF (fine fibre filters) N

Parameters

National Standard for Control Metods

Potable water (initial)

Potable water (after filtering)

Norm on National Standard GOST 2874-82 "Drinking Water"

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Smell, un. Smack, un. Dimness, mg/dm3 Colourness, mg/dm3 Hardness, mg/dm3 N of nitrates, mg/dm3 N of nitrites, mg/dm3 Chlorides, mg/dm3 Sulfates, mg/dm3 Fluorine, mg/dm3 pH, un. Zinc, mg/dm3 Copper, mg/dm3 Cadmium, mg/dm3 Iron, mg/dm3 Residual Cl, mg/dm3 N ammonia, mg/dm3 Dry residue, mg/dm3

GOST 335 1-74 GOST 335 1-74 GOST 335 1-74 GOST 335 1-74 GOST 4151-72 GOST 18826-74 GOST 4 191-82 GOST 4245-72 GOST 4389-72 GOST 4386-81

1 0

<2 <2

20 4.5 1.2 0.3

1 1 0.4 15 4.4 1.0 0.3

22.5 69,0

20.0 60.0

0.4 7.1 0.1

0.4 7.2

GOST 18293-72 GOST 4388-72 SanPIN 4630-82 GOST 40 11 -72 GOST 18 190-72 GOST 4192-82 GOST 18164-72

0.85

0.08 0.001 0.18

0.1 0.2 N/det.

0.05 0.003 0.001 0.09 N/det.

0.1 N/det.

<1.5

<20 <7.0 <45.0 <3.3 <350 <500 <1.5 6.0-9.0 <5.0 <1.0 < 0.001 <0.3 0.3-0.5 <2.0 <1000

3. Influence of PP filters on microflora of drinking water The requirements to water of the central water supply are reglamented by GOST 2874-82 "Drinking Water", according to which the organoleptic properties of water and its chemical composituion are taken into account. The special importance is given to bacteriological parameters of water. The basic parameters describing sanitary - microbiologic character of water are: definition of general microbial seminaty (total number of microbe bodies); definition of bacteria of E.coli type as sanitary-indicative microorganisms; indication of pathogenic microorganisms. General microbial number of water shows the degree of contamination by organic substances. The high meanings of total number unequivocally specify a low sanitary level of water. The essential contents of bacteria of E-coli group characterize a degree of fecal pollution of water and, hence, is the indirect indicator of epidemic danger. The group of pathogenic microorganisms includes, in particular enteropathogenic Escherihia, Salmonella, Shigella, Cholera vibrions etc. The researches on change of microflora in drinking water (before and after the filter) and microflora on a surface of a filtering material were carried out in the Central Sanitary

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Service of Kiev by standard techniques. In Table 29 the characteristics of various microorganisms are submitted. Table 29. The characteristics of pathogenic microorganisms Name Salmonella Shigella Cholera vibrion Escherihia Bacteria ofE.coli type

Deseases caused by microorganisms Salmonellosis, Typhia Shigellosis Cholera

Size of microorganisms, u Till 2 2-4 Length 2-3 Width 0.5 0.5-2 Length 0.6-6 Width 0.3- 1.5

Colibacillosis Intestinal infections

Terms of a survival in water, day 2-93 15-27 4-28 -

Fungi are not sanitary indicative and not reglamented by GOST "Drinking Water". However their presence essentially influences on organoleptic properties and chemical composition of water. Proceeding from the data of Table 29, it was possible to assume that FM with a subtlety of clearing 1; 0.45; 0.3 n will detain effectively microorganisms which are taking place in water, and hence, will change microflora of drinking water. In experiments a potable water is filtered during 8 h of a working day through FM from PP microfibres. The process of a filtration is interrupted for night and on days off. The total duration of work of filters has made 10-15 days. This means after that time the analysis of water on microflora (before and after the filter) and also analysis of microflora on a surface of FM was made. In the selected tests it was determined: total number of microorganisms, number of bacteria ofE.coli type, pathogenic microorganisms and amount of Fungi. The obtained results (Table 30) were compared with parameters of GOST "Drinking Water". The major conclusion following from the analysis of the data in Table 31 consists that on a surface of FM there are no all kinds of microorganisms even after 15 days of operation, i.e. on FM from ultrathin PP fibres any microorganisms do not reproduced. This means that FM does not overgrow by bacteria while exploration. The occurrence of microorganisms in washing waters as well a presence of them in large amount in filtrate after 10 and 15 days of operation are caused by their reproducing and accumulation on surfaces of the filter supplies, in stagnant zones of filter-keeper, in connecting pipelines. These microorganisms represent gram-positive coli and their spores, size of which changes over a wide range: width 0.3-2.2 p., length - 1.7-7 u. Smallest of them pass through pores of FM, accumulate and reproduce on various surfaces, so can be washed off by a current of water. For correspondence the parameters of water on microflora to GOST "Drinking Water", the filtering system has to be subjected periodically by the sterilization procedure. Table 30. Change of microflora in drinking water and on FM after 10-15 days of operation N 1

2

Name of test Drinking water: -before filter -after filter during 1st day -after filter on 10th day -after filter on 15th day Filtering material: -surface before operation -surface after operation

Total number of MO

Amount of MOofEcoli type

Amount of pathogenic MO

Amount of Fungi

2 n/det 500 4000

4 n/det 3 3

n/det n/det n/det n/det

n/det n/det n/det 30

-

n/det n/det

n/det n/det

-

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M. Tsebrenko et al. / Fine Filtering Materials

3

-washing water after 1 0 days -washing water after 15 days

18 250

3 3

n/det n/det

Norm on COST 2874-82 "Drinking Water"

£100

>3

Absence

n/det n/det

-

Table 31. The contents of microorganisms (col./ml) in filtrate

N

Initial water

1 2

0 0

Filtrate After First 15 min portion washing

-

1.7 4.2

After 6 days thermostating First After Initial water portion 15 min washing

60 60

80

40

Total growth

Norm on COST 2874-82 "Drinking Water" >100 >100

This means that FM does not overgrow by bacteria while exploration. The occurrence of microorganisms in washing waters as well a presence of them in large amount in filtrate after 10 and 15 days of operation are caused by their reproducing and accumulation on surfaces of the filter supplies, in stagnant zones of filter-keeper, in connecting pipelines. These microorganisms represent gram-positive coli and their spores, size of which changes over a wide range: width 0.3-2.2 ji, length - 1.7-7 u,. Smallest of them pass through pores of FM, accumulate and reproduce on various surfaces, so can be washed off by a current of water. For correspondence the parameters of water on microflora to GOST "Drinking Water", the filtering system has to be subjected periodically by the sterilization procedure. 4. Giving FM bactericidal property It is known that silver and copper, being are put on various materials, give to last bactericidal property. That is why the microfibres of FM were covered by copper or silver from water dispersion. For comparison we used also PP monofibre with a diameter 0.1-0.2 mm, prepared from the same PP but by traditional technology. The characteristics of the investigated samples are submitted in Table 30. All researches were carried out at the Kiev Company "KVAZAR". It was estimated: the contents of silver or copper in filtrate (potable or deionizing water); the contents of microorganisms in filtrate; overgrowing FM by microorganisms. The content of silver and copper in filtrate was defined by spectral method after preliminary washing FM by deionizing water within 15 min. Was used quartz spectrograph ISP-30. Amount of colonies of microorganisms was calculated with visual microscope on a surface of disk-membrane filter with a pore diameter 0.2 (j. after passing through it of certain volume of filtrate and keeping filter within 2 days at temperature 37 ° in nutritious media of fish-pepton agar. For estimation of overgrow level of FM by microorganisms the disk sample was maintained in potable water during 6 days, washed out fresh portion of water and the contents of microorganisms in the first portion filtrate, in filtrate after preliminary washing within 15 min and on a surface of FM (after thermostating at temperature 37 ° within 5 days in nutritious media) were determined. The results have shown that copper and silver of treated FM are not taken out in filtrate. It testifies to strong fastening these metals on PP microfibres. Samples FM from PP microfibres do not acquire to microorganisms in overgrowing even at increase of amount of colonies in filtered media (see Table 32, 33). Thus, the fact was once again confirmed that on created fine fibre PP materials the bacteria are not reproducing. It means that such FM can be

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M. Tsebrenko et al. /Fine Filtering Materials

recommended for clearing drinking or deionizing water for a long time without sterilizing procedure.

Table 32. The characteristics of the investigated samples

N 1 2 3 4 5 6 7

Character of pretreatment without processing without processing powder of silver covering suspension of silver covering powder of copper covering without processing without processing

Type of sample FM from PP microfibres PP monofibre with diameter 0.1-0.2MM FM from PP microfibres FM from PP microfibres FM from PP microfibres FM from polyamide (PA) microfibres Film from PA

Table 33. The data on overgrowing samples of FM by microorganisms Contents of colonies on a surface of FM Control sample FM after keeping for 6 days

number of a sample

1 0 0

2 -

4 0 0

3 0 0

Total overgrowing

6 0 0

5 0 0

7 10 Total overgrowing

5. Reception of FM from mixes with polyoxymethylene From the earlier executed researches it is known that polyoxymethylene (POM) - the polymer of formaldehyde - is capable to eliminate in moisture media very insignificant quantity of formaldehyde being antiseptic. As a result a product from POM have bactericidal properties. Therefore a research of influence of the POM additives on properties of FM from PP microfibres represented scientific and practical interest. The additives of POM in amount 5, 10, 20, 30% were introduced at a stage of polymers mixing. From three-components mix it was obtained FM, in which POM was as microfibres (alongside with PP microfibres), and this provided the obtaining developed surface. Table 34. Influence of the POM additives on microstructure of extrudates from mixes PP/PA of ratio 40/60

1.6 2.3 1.5 0.8 0.9

6.3 6.2 6.7 5.3 5.4

«

38 38 24 44 31

0.1 0.5 0.2 0.1 0.5

8.6 1.3 0.5 1.5 0.4

£

s «

41.0 16.2 11.5 16.0 1.9

Outer shell Numeral %

8.1 5.4 5.3 4.2 5.4

s?

Numeral %

5.2 5.0 5.2 5.3 5.5

Films

Numeral %

9.5 50 40 18 21

£ «

Average diameter, u

40 53 70 42 57

1

Particles

Numeral %

6.3 6.2 6.1 6.3 6.7

Short fibres Average diameter,

Numeral %

0 5 10 20 30

Average diameter,

Content of POM, %

Long fibres

5.1 1.4 1.3 4.4 6.0

£

i 47 31 47 70 75

The results of microscopic researches have shown that the additive of 5-10% POM essentially improves the formation of PP fibres in a matrix of co-PA (Table 34) and allows to increase the content of PP in a mix up to 40%. For comparison a monofibre from initial POM was formed by traditional technology. Amount of eliminated formaldehyde at heating FM and monofibre was estimated by the IR-spectroscopy. The results have eliminates the same

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M. Tsebrenko et al. /Fine Filtering Materials

quantity of formaldehyde as from monofibre of 100% POM. It is explained that POM as microfibres has the very developed surface. Last promotes the processes of destruction with elimination of formaldehyde. This opens a new way of giving to filtering materials from PP microfibres of bactericidal properties. It is very important that quite small amount of POM can be introduced into a mix of polymers. References [1] [2]

Tsebrenko, M.V. (1983) Fibrillation of the mixtures of crystallizable, amorphous and poorly crystalline polymers, Intern. J. Polymeric Mater., V. 10, pp. 83-119. Tsebrenko, M.V.(1991) Ultrathin Synthetic Fibres (Russ), Khimiya, Moscow.

Preparedness Against Bioterrorism and Re-Emerging Infectious Diseases J. Kocik et al. (Eds.) IOS Press, 2004

239

Antibacterial adsorption-filtering materials for individual protection means of organism 3.COMPOSITE MATERIALS BASED ON POLYURETHANES AND ACTIVE CARBON Yu. SAVELYEV, L. ROBOTA, O. SAVELYEVA, N. KARTEL*, V. STRELKO* Institute of Macromolecular Chemistry ofNAS of Ukraine, Kiev, Ukraine, e-mail: [email protected] *Institute for Sorption and Problems of Endoecology ofNAS of Ukraine, Kiev, Ukraine, e-mail: [email protected]

1. Introduction Polymer composite materials containing an active carbon have attracted a lot of attention because of their protective, filtering/adsorptive properties and pollution free status. Varying the carrier polymer material, the fraction and nature of adsorbent, and the geometry it is possible to produce protective composite adsorptive materials of different application. Block, elastic, and immobilized composite adsorptive materials could be effectively used as single piece personnel protection as well mat-shaped active components of air- and water-cleaning systems. The main factors defined the properties of composite polymers are the adhesion of the polymers to the solid surfaces as well as the polymer high elasticity and deformation resistance during the composite formation. Both chemical and physical bounds may be formed in the polymer-filler interface. The formation of particular number of quite steady bounds irrespective of their nature is supposed to be the main factor based on the theory of adhesion [1]. So the problem consists not in the nature of the strong bounds, but in it quantity necessary for optimal properties. Improvement of the strength properties can be reached as the result of changing the conditions of the internal stress relaxation in the boundary layer. The use of the two component binding with different adsorption to the filler surface or of the blockcopolymers with selective interaction of the blocks with different nature with the filler surface due to their different affinity with the surface may be perspective from this point of view. First of all it is typical for the polyurethanes (PU) based on aromatic and aliphatic hydrazine derivatives and polyethers and polyesters. There are a lot of the polar groups in PU, such as NH-, -C-O-C-, -C(O)OC-, available to create the bounds with the active centers of carbon surface which have a high degree of valence unsaturation. Putting of the ionic groups into the hard block of PU stipulates for appearance of the centers capable of forming the ion-molecular hydrogen bonds and electrostatic bonds (ion-ion, ion-dipole, etc) [2]. The introduction of the carbon component at the stage of polyurethane formation can lead to the interruption of the

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reactive chains on the well-extended surface and as the result to diminution of dimension network density. At the same time adsorption of the prepolymer molecules at the solid surface may facilitate formation of the three dimensional structure of the polymer matrix due to the orientation of the molecules and leads to the increase of specific surface and pore volume of the polymer matrix. The creation of the polyurethane based composite sorption materials in the form of block and foams let to produce of the goods of required geometrical forms, resistance to the mechanical stresses, elasticity, and impossibility of pollution of the filtered objects with carbon dust. 2. Experimental 2.1. Materials The KAU-type active carbon with particle size of 0.5-1 mm and dust-like powder, pore volume on benzene Ws = 0.60 cm3/g and specific surface area Ssp = 1140 m2/g was used. Synthesized polyurethanes based on aliphatic diol (PU1) [3], unsymmetrical dimethyl hydrazine (PU2), hydrazinium-hydrazide (PU3) were used as polymer matrix [4], as well as commercial available polymers polyvynilalcohol (PVA), polyvynilchloride (PVC). Two types of composite sorption materials (CSM) were developed. 2.2. Composite preparation In the first type, named by "hard" CSM, the active carbon was suspended in the polymer solution of different concentrations followed by hardening the composite in the vacuum oven at temperature 40-70 °C for complete solvent removal. The general structure of the polymer: H-[CE-DIC-PE-DIC]n-EG, where PE - polyether, DIC - diisocyanate, CE - Chain extender, EG - end group, or H - atom, The features of the starting reagents are submitted in Table 35. Table 35. The features of the starting reagents Active carbon KA U Granularity, mm 0,5-1

Bulk density, Volume of sorption g/cm3 pores (Ws), cm3/g 0.412 0.60 Polymeric solutions

Structure of the elementary chain Name l.PVC -(CH2-CHCl-)n 2. PVA -(CH2-CHOH-)n * 3. PU-1 * 4. PU-2 * 5. PU-3 -[R1CONH-R2-NHCO-R3-OCONH-R2-NHCO-]x-

Specific surface area (SSD), m2/g 1140

Solution DMFA Water DMFA DMFA DMFA

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The second type of composite sorption materials, named by "elastic" CSM, was prepared by introduction of the active carbon into the polyurethane system during the foaming of the latter. The general structure of the polymer in this type of CSM is the follows:

where PE - polyether, polyester, DIC - diisocyanate, CE - chain extender, CLG - cross-linking group.

Synthesis of elastic CSM: Reaction of a chain's growth

CARBON MATERIAL

Reaction of gas-educing

ELASTIC KSM

Formation of the bonds with cross-links

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Chain extenders (initiators)

Obtained foam polyurethane's are characterized by rather different physical data (Table 36). Table 36. Some features of foam polyurethane's NoNo

Samples

Density (p), g/cm3

Water adsorption (cm3/g)

I.

PP-l

0.043

0.419

2.

PP-2

0.070

0.152

3.

PP-4

0.041

0.238

4.

PP-6

0.038

0.290

5.

PP-8

0.031

0.257

6.

PP-l I

0.056

0.585

7.

PP-12

0.043

0.346

8.

PP(D)-13

0.058

0.828

9.

PP(D)-14

0.041

0.495

Y. Savelyev et al. / Composite Materials

243

10.

PP(D)-15

0.051

0.406

11.

PP(D)-16

0.038

0.614

12.

PP(D)-17

0.049

0.518

13.

PP(D)-18

0.027

0.209

3. Measurements Pore structure parameters were investigated using the determination of the pores volume on benzene Ws, cm3/g (exhauster method), determination the values of the specific surface area on argon Ssp, m2/g, mercury porosimetry investigation on the high pressure (Pore Sizer M9200, "Cultronics France"). Level of contamination of the physiological solution (Mdust/McsM) xl00%, where MdUSt - mass of the dust content in the physiological solution after contacting with CSM, MCSM - mass of the initial CSM Polymer investigations were carried out using Differential Scanning Calorimeter (DSC) Small Angles X-ray Scattering (SAXS) Boundary phase nature was analyzed using IR-spectroscopy.

4. Results and discussion 4.1. The "Hard" type CSM Decrease of adsorption properties of the initial carbon materials after composite formation was estimated by change of absolute value of pores volume on benzene. The dependencies of the pores volume values on benzene Ws on the polymer concentration and the values and specific surface area SeEtof the sorbents are shown on the Fig. 43 and Table 37.

Figure 43. Dependence of the pores volume on benzene of the CSM (a) and of the active carbon (b) vs the polymer content in the CSM (C,%).

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Table 37. Data of the specific surface area for sorbents Sorbents KAU (initial) KAU-PVA

KAU-PVC

KAU-PU1

KAU-PU2

KAU-PU3

Polymer content in CSM, %

SSP, m2/g

S SP L!>M /s S p KAU xl00%

.

1140 738 813 977 1010 1054 1068 702 815 1063 437 977 1110 795 1080 1092 1100

100

15 8 8 13 5 4 5 2 1 7 3 0.5 6.5 0.5 0.45 0.2

65 71 86 88.5 92 94 62 71 93 38 86 97 70 95.5 96 96

R, nm Figure 44. Data of the mercury porosimetry investigations for the CSM and KAU volume pore distribution on equivalent radii

The ratio of the pore volumes of CSM and initial active carbon was calculated accordingly equation:

where: Vsexp and Vs'n are the pores volume on benzene of the CSM and of initial carbon, respectively. But it should be taken into account that some quantity of the polymer is presented in CSM, so the resulting ratio will be:

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where: n - is the share of the polymer in the composition. It was determined that there was some difference between Nj and N2. The decrease of adsorption properties of initial carbon in the CSM is higher in compositions based on PVA and PVC (20-40%) than in those based on PU1, PU2 and PU3 (2-10%). The obtained composites are considered as a polymer matrix filled with carbon. Active carbon inserted into the polymer with block of different polarity and flexibility (PU1, PU2, PU3) is mainly concentrated in the soft blocks microregions, which form an essential part of polymer. The association of the hard blocks and formation of the domains promotes the movement of the carbon into interdomain space. This leads to redistribution of the intermolecular bonds at the expense of the partial screening the polar groups of the macrochain by active carbon. The weaker the intermolecular bonds in the soft blocks the deeper the hard blocks segregation. It should be noted that self-association of the hard blocks according the DSC and SAXS data decreases in the row: PU1 > PU3 > PU2. So the PU1-KAU composite adsorbent was determined as most effective due to the less screening of the pore structure. For comparison the homopolymers PVA, PVC forms the homogeneous dispergated system "active carbon-polymer". As the carbon filler and the polymer matrix have different thermal expansion coefficients there exist a possibility of destruction of the adhesive layer at the interface boundary due to shrinkage of the polymer matrix during the hardening of the composite. That is why the high elasticity and resistance against the deformation forces are necessary conditions for polymer matrix. Whereas the PVA and PVC form hard films (relative elongation of 10-50%, tensile strength of 46-54 MPa), it is expedience to use plasticizers. For comparison the relative elongation and tensile strength for polyurethane matrixes are the follows: PU1 and PU2: 350-600%, 12-25 MPa, PU3: 200%, 7 MPa. There are a lot of polar groups in polyurethane macrochain, such as -NH, -CO, -COC-, -COO-, available to interact with the surface of active carbon because of high valency unsaturation of the latter. According to IR-spectroscopy data of "urethane-active carbon" model system the interaction between nelectrons of active carbon and urethane carbonyl group may be supposed. It is likely the transformation of carbonyl bond of urethane group: >C=O; that is confirmed by slump of C=O bond intensity (1660 cm-1) and appearance of a new bond (1485 cm-1). The hydrophobic properties of CSM are conditioned by hydrophylicity of its polymer matrix. The values of the water absorbability for the initial polymers are: PVA - 138%, PVC - 0.006%, PU1 - 3.1%, PU2 - 3.71%, PU3 - 20.2%. Level of contamination for physiological solution (0.9% NaCl) after contacting with the initial carbon material and CSM has been determined. 0.5 g of material was put in a glass flask, 20 ml of a physiological solution and heparin (2500 iu) as surfactant were added, then the flask was shaken during 15 min and the contamination level was then studied using the electronic scanning microscope. Analysis of the microphotos confirms that the surface of the initial material is grainy and determines the weakly attached particles, which can be separated from the surface of carbon grain during the perfusion of the liquid. The CSMs have smoother surface and non-of weakly attached particles. The sorption ability of the initial carbon material and CSM was estimated using the methylene blue (319 a.u.m.) relates to a class of low molecular substances (100-500 a.u.m.), and vitamin B12 (1355 a.u.m.) presented a class of "medium" (500-5000 a.u.m.) molecules. The influence of the polymer immobilization of the active carbon on sorption-kinetic characteristics of sorbents was estimated by sorption-kinetic investigation in static conditions [5]. Comparing the coefficients of initial activity for studied materials, we calculated that immobilization by polymers leads to negligible decreasing of the sorption ability of both the

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"medium" and low molecular substances. However for the CSM with 5-7% polymer content these coefficients were decreased in 1.2 - 1.5 times. The analysis of results obtained for hard CSM showed the practically identical tendency of the pores frame parameters Ws and Ssp to diminution with the increase of the polymer content in CSM. Up to 10% of polymer content the changes in pores frame parameters have a linear dependence. The losses of sorption pores volume and specific surface area were estimated as polymer content in composite. At more than 10% of polymer content in CSM the significant decreasing of pores volume values is observed. According to conducted investigation the composite components are not inert to each other. So the character of pores volume value changes may be described by rectilinear low (compare with [6]):

where x - is the mass part of polymer in the mixture. The almost rectilinear site of Ws dependence in composite on polymer content may be expressed as follows:

In other words, the presence of polymer in CSM inactivates a carbon component by filling some part of pores. To reveal what exactly pores will be filled by polymer, a method of mercury porosimetry was used. It allows receiving a differential distribution of the pores volume on radiuses from 3 to 106 nm. The mercury porograms of the initial carbon and based composites with polymer content of 0.5 and 2 % are presented in Fig. 44 Quantitative data of obtained results are given in Table 38. Table 38. Composition and properties of CSM Example 1 2 3 4 5 6 7 8 9 10

Polymer content in CSM, % mass. 5 2 1 7 5 3 0.5 6.5 0.5 0.2

Ws, cm3/g

Ssp, m2/g

Dustiness, %

0.59 0.58 0.55 0.53 0.53 0.55 0.65 0.60 0.64 0.66

702 815 1063 437 830 977 1088 795 1080 1100

absence 0.1 0.15 absence 0.1 0.1 0.2 0.1 0.05 0.05

As is obvious, the changes in porous structure of carbon material took place in area of macro- and mesopores: their volumes and specific surfaces were decreased in 1.5-2 times at the low polymer content (about 2%) in composite. The Ws and Ssp indexes practically were not changed in this region. Tendency of augmentation of medial radius of macropores and decreasing of medial radius mesopores was also observed. It may be conclude, that increase of polymer content in CSM leads to a progressing degradation of transport system of active carbon with further retardation of kinetic processes of adsorption.

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4.2. The "Elastic" type CSM Table 39. Composition and properties of the elastic CSM Example 1 2 3 4 5 6 7 8 9 10

Contents of components in the CSM (% mass.) carbon/polymer 23/77 33/67 41/59 47/53 50/50 55/45 60/40 23/77 33/67 55/45

Ws, cm3/g

S(sp.),m2/g

Dustiness,%

0.55 0.57 0.58 0.62 0.64 0.66 0.48 0.57 0.60 0.66

1086 1082 1065 1065 1080 1100 815 1006 1080 1100

absent absent absent absent absent absent absent absent absent absent

The analysis of obtained data shows, that parameters of porous frame (Ws and S sp ) of elastic CSM depend on the ratio of carbon and polymer in composite (%). At the carbon content more than 50%, CSM lose elasticity because of "extinguishing" the foam by carbon component. The optimum ratios of polymer-active carbon (dust) are established to be from 3050% to 70-50%. The Ws and Ssp values for CSM at these ratios a little differ from the same indexes of initial carbon and have practically identical tendency to change. Mercurial porograms CSM-1, CSM-2 (with the 33% content of a carbon material) and initial KAU demonstrate that the porous carbonic frame did not undergo essential changes. Estimating the effect of the filler on structure and properties of linked polyurethane's one should take into account the influence of the solid surface on chemical bonds network formation. Sorption effectiveness of the obtained materials seems to be achieved due to the formation of the network structure. It will allow finding a wide application for these materials in future. 4.3. Giving bactericidal activity to PU matrixes The compounds having biological activity interact with receptor by the certain part (biophore), mainly owing to formation of rather weak non-valent bonds. Among these interactions the main role Van-der-Vaals forces are acting. Alongside with its the large role play hydrogen bonds. Particularly, it is necessary to note the forces of the electrostatic interaction. The heteroatoms (N, O, S, P), heterocycles, groups -C=C-, aromatic structures as a whole can enter into composition of biophore. And if for monomeric compounds above mentioned is the determining factor in ensuring to it of the biological activity, for polymers it is necessary to take into account one more factor. The polymeric matrix, on the one hand, can promote availability of biophores, and with another - to block its. The structural modification of polyurethane's allows to adjust them supramolecular organization, latter allows to adjust both availability, and place of biophore's localization. Incorporation of the (macro)heterocyclic fragments into polyurethane macromolecule is one of the way to obtain of polymers with biological activity. (Macro)heterocyclic compounds are perspective agents from this point of view to formation of relatively weak non-valent bonds, which are answered the definite active centers of these compounds.

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Linear polyurethane's of the general structure: H-[- CE - 1C - PE - 1C -]-EG were synthesized, where:

1C: aromatic or aliphatic diisocyanates; PE : oligoethers or oligoesters; Polyurethane foams were synthesized based on compounds I and II, oligoether and oligoester, aromatic diisocyanate and activators of the urethane formation process. • Linear polymers based on sulfonyl hydrazide of the crown ethers (I, HI) possessed of high fungicidal and bacteriostatic activity. • Introduction of quinoxaline (II) and thiazol (IV) derivatives into polyurethane macrochain intensify of polymer bactericidal activity. • Polyurethane foam based on compounds I, II are possessed of high biocompatibility and bactericidal activity. Bactericidal action of the polymers was studied as regards to Staphylococcus aureus, Pseudomonas aeruginisa, Bac. Subtilis, E. coli and some others. The polymers are possessed by bactericidal activity of prolonged action (at least 4 years) as regards to the microorganisms under study. Histotoxicity of the polymers were investigated by the tissue culture method. Histotoxic indexes of the studied polymers were in the range of 0.76-0.84, thus toxic action of the polymers on the tissue culture cells is absent. Haemolitic action of the polymers was studied. The polymers had been undergoing of the haemolysis on 0.9%, e.g. these polymers does not contain the haemolitic active compounds. Fungicidal activity of the polymers were investigated as regards to some kinds micromicetes as test-organisms: Aspergillus terreus Thom, Chaetomium varioti Bainier, Penicillium funiculosum Thom, Penicillium chrysogenum Thom, Penicillium cyclopium Westling, Trichoderma viride Pers. ex Fr.

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5. Conclusion The new composite sorbents of two types, hard and elastic, were developed. It was shown that decreasing of adsorption properties of the initial carbon material is less than 7% at the polymer content in hard composite of 1-5%. The optimum ratio of polymer-active carbon (dust) in elastic composite is 30-50% to 70-50%. The main factor determined the high effectiveness of polyurethane application is the microphase separation in polymer and formation of hard and soft block regions. The association of hard blocks causes the motion of carbon to the interdomain area (region of the soft block) with subsequent redistribution of intermolecular bonds at the expense of screening the polymer polar groups by the active carbon and prevention of their interaction. The interaction between Ti-electrons of active carbon and urethane carbonyl group may be occurred. The primary factor of differing the sorption is the difference of the chain extender structures. Sorption efficiency of the elastic composite materials was achieved due to the formation of the network structure. For blockcomposite sorption materials the changes in porous frame of a carbon material took place in area of macro- and mesopores: their volumes and specific surfaces were decreased in 1.5-2 times at the small contents (about 2%) of polymer in a composite, where such indexes as Ws and Ssp practically did not change. Parameters of porous structure for elastic composites are not differed essentially from initial carbon at optimal "active carbon-polymer" ratio and have the same trend to change. The carried out researches have shown that introduction of various biological active compounds (biophores) in the PU matrixes during their synthesis open effective way to prepare both polymers and composites on their base with active carbons possessing definite bactericidal properties against pathogenic microorganisms and fungi.

References [1] [2] [3] [4]

[5] [6]

Lipatov, Yu.S. (1991) Physico-chemical bases of filling of polymers, Khimiya, Moscow. Savelyev, Yu.V. (2000) Polyurethane-urea ionomers based on macroheterocyclic polymers, Polymer & Polymer Composites, vol. 8, Nl, pp. 27-30. Savelyev, Yu.V., Veselov, V.Ya., and Grekov, A.P. (1991) Linear polymers based on hydrazine, in Polymer Films and Coverings", Leningrad, pp. 108-109. Savelyev, Yu.V., Veselov, V.Ya., and Grekov, A.P. (2000) Investigations of the reactions of 1,1dimethyl hydrazine with alkylhaloids and epoxy compounds, Ukr. Chem. J. (Russ), vol. 66, N12, pp. 110-114. Marushko, S.Z., Kartel, N.T., and Savelyev, Yu.V. (1996) Composite sorbents "Active CarbonePolymer" and their characteristics", in CARBON'96: Intern. Conf on Carbon (Newcastle), vol. 2, pp. 463-464. Fedorov, N.F., Ivakhnyuk, G.K., and Babkin, O.E. (1995) Modern problems of the theory of adsorption, in VIIIntern.Conf. "Theoretic Adsorption Aspects", Moscow, PAIMS, vol. 1, pp. 88-96.

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List of Tables and Figures Table 1. Table 2. Table 3. Table 4. TableS. Table 6. Table7. Table 8. Table 9. Table 10. Table Table Table Table Table Table Table

11. 12. 13. 14. 15. 16. 17.

Table 18. Table 19. Table 20. Table 21. Table 22. Table 23. Table 24. Table 25. Table 26. Table 27. Table 28. Table 29. Table 30. Table 31. Table 32. Table 33.

Priorities for CDC's global infectious disease strategy, 2001-2002 Critical biological agents for public health preparedness List of infectious agents, adopted by Bulgarian Army Medical Service as possible biological agents Basic terms definitions Infectious diseases morbidity in the country ORM Threat Matrix Infected Personnel by Country Affected U.S. Conferees by German State Stages of Smallpox Infection (U.S. Army Office of the Surgeon General) Comparison between a conventional HAZMAT (HMI) incident and one involving a Weapon of Mass Destruction (WMD) Human Susceptibility to Brucella spp Examples of DoD Electronic Medical Surveillance Systems Components of JBAIDS Select Block I and II Agents Mass units associated with various bacterial spores and treatments Operational characteristics of three in vitro hybridoma culture methods Comparison of the three different cell culture methods examined in this study Inactivation of coliphages in water supply in dependence on time duration of GDP effect Inactivation of coliphages in drinking water depending on duration of GDP affect Dynamics of inactivation of the polio virus in drinking water under GDP influence Inactivation of coliphages in waste water in dependence on duration of GDP influence Dynamics of inactivation of the polio virus in the filtrate of native waste water under GDP influence Decontamination of water contaminated with S.aureus and Micobacterias under GDP influence Decontamination of water containing of polio virus under GDP influence Comparative sorption-bactericidal efficiency of carbon metal-containing adsorbents The structural - sorption characteristics of carbon materials Sorption ability of carbon materials to Cholera vibrion Results of laboratory tests of filtering elements in FFF (fine fibre filters) The characteristics of pathogenic microorganisms Change of microflora in drinking water and on FM after 10-15 days of operation The contents of microorganisms (col/ml) in filtrate The characteristics of the investigated samples The data on overgrowing samples of FM by microorganisms

19 27 48 48 49 95 115 116 118 148 163 181 187 187 193 200 200 219 219 220 220 221 221 221 229 230 230 234 235 235 236 237 237

252

Table 34. Table 35. Table 36. Table 37. Table 38. Table 39. Figure 1. Figure 2. Figure 3.

Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. Figure 11 a. Figure lib. Figure lie. Figure lid. Figure 12. Figure 13. Figure 14. Figure 15 Figure 16. Figure 17. Figure 18. Figure 19. Figure 20. Figure 21. Figure 22. Figure 23. Figure 24. Figure 25. Figure 26. Figure 27. Figure 28. Figure 29. Figure 30. Figure 31. Figure 32.

Influence of the POM additives on microstructure of extrudates from mixes PP/PA of ratio 40/60 The features of the starting reagents Some features of foam polyurethane's Data of the specific surface area for sorbents Composition and properties of CSM Composition and properties of the elastic CSM

237 240 242 244 246 247

Microarray Technology 14 Epidemiology and Laboratory Capacity (ELC) program, fiscal years 1995 through 2000 20 Selected examples of how ELC program resources were used by states and large public health agencies to improve infectious disease surveillance and outbreak response 21 Estimated impact of PulseNet if the 1993 multistate food-borne outbreak of Escherichia coli O157:H7 had occurred in 1998 22 Conceptual diagram of the Laboratory Response Network 29 Algorithm developed for the rapid rule-out of Bacillus anthracis by clinical laboratories. 30 Environmental and clinical specimens processed by LRN laboratories 34 The Risk Management Paradigm 101 Conference Attendees by City in Germany 116 Smallpox Presentation and Progression over Time (SBCCOM) 118 1 st Generation Caseload 119 2nd Generation Caseload 120 3rd Generation Caseload 120 4th Generation Caseload 121 Infected Caseload - First 60 days for Baden-Wiirttemberg 121 Infected Caseload - First 60 days for Germany 122 Infected Caseload - First 60 days for Area of Responsibility 122 Effectiveness of Ring Vaccine (Baden- Wiirttemberg) 123 Effectiveness of Ring Vaccine (Germany) 124 Effectiveness of Ring Vaccine (Area of Responsibility) 124 Total Patient Caseload (Baden-Wiirttemberg) 125 Total Patient Caseload (Germany) 125 Total Patient Caseload (Area of Responsibility) 126 Projected Fatalities 126 Treatment Network (Baden-Wiirttemberg) 127 Treatment Network (Germany) 128 Treatment Network (Area of Responsibility) 128 Medical Personnel Requirements (Baden-Wiirttemberg) 129 Medical Personnel Requirements (Germany) 129 Medical Personnel Requirements (Area of Responsibility) 130 Key elements of WMD preparedness 153 Elements of a successful WMD Training Program. The arrows on the slide show the WMD prerequisite training classes 154 Relationship between Crisis Management and Consequence Management 155 Steps in an effective planning process 156 Effective elements of a WMD response - Plan, Train, Exercise, and Revise 157

253

Figure 33. FACS analysis showing specific binding of fluorescently-labeled mAbs to B. anthracis spores Figure 34. Schematic of antibody displaying filamentous phage particle Figure 35. ELISA detection of (A) MS2 virions and (B) recombinant MS2 coat protein using purified anti-MS2 Fab antibody Figure 36. Rep-PCR fingerprint of different Bacilli Figure 37. Method of protein identification using the Mascot search engine Figure 38. Super-imposed average gels non-induced versus induced Figure.39. Pilot plasmachemical plant Figure 40. The influence of ratio Solid: Liquid on the adsorption of the trypsine by carbon fabric Figure 41. Scheme of ultrathin fibres formation and SEM microphotos of obtained product Figure 42. Microfibrillar structure of ultrathin polypropylene fibres Figure 43. Dependence of the pores volume on benzene of the CSM and of the active carbon vs the polymer content in the CSM Figure 44. Data of the mercury porosimetry investigations for the CSM and KAU volume pore distribution on equivalent radii

192 196 197 208 210 213 224 231 232 233 243 244

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Author Index AganB. Ahrens, J. T. Anderson, P.E. Asokliene, L. Bartoszcze, M. Bellenkes, A. H. Berencsi, G. Bray, D. Chomiczewski, K. Cosivi, O. Csohan, A. DelVecchio, V.G. Donlon, M.A. Elzer, P.H. Faludi, G. Finke, E. Fock, R. Grigoriev, A. Hanson, E. Kapustin, A Kartel, N. Kellogg, R. Khan, A.S. Kim, M. H. Kriz,B. Kutateladze, M. Kyncl, J. Lukens, D.C. Mayboroda, M. Meyer, R.F. Miller, M.J. Morse, S.A. Negut, M.

12 114 195 66 170 89 60 26 73 9 60 207 191 161 60 58 58 226 12 76 226,232,239 26 195 195 44 54 44 195 232 26 26 17,26 137

Netesov, S. Nichelson, D. Niedrig, M. Niemeyer, D. Niemeyer, D.M. Niemeyer, Debra M. O'Connell, K.P. Oyston, P.C.F. Park, J.T. Pavlin, J.A. Penkov, E. Perry, S.R. Pivovarov, A.A. Plochev, K. Price, R. Quinn-Doggett, K. Rezanova, V. Robota, L. Roweley, R. Savelyev, Yu. Savelyeva, O. Shvets, D. Stopa, P. J. Strelko, V. Thompson, R.G. Tibbetts, C. Tsebrenko, I. Tsebrenko, M. Twardowski, T. Valdes, J.J. Vigus, R.A. Woodall, J.

105 26 37,58 12 179 185 195 164 195 3 47 26 215 47 105 147 232 239 12 239 239 226 147 226,239 195 12 232 232 132 195 147 105,111

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