43 Cystic Fibrosis Microbiology PETER H. GILLIGAN, DEANNA L. KISKA, AND MARIA D. APPLEMAN COORDINATING EDITOR
MARIA D. APPLEMAN
Cumitech CUMULATIVE TECHNIQUES AND PROCEDURES IN CLINICAL MICROBIOLOGY
Cumitech 1C
Blood Cultures IV
Cumitech 2B
Laboratory Diagnosis of Urinary Tract Infections
Cumitech 3B
Quality Systems in the Clinical Microbiology Laboratory
Cumitech 7B
Lower Respiratory Tract Infections
Cumitech 10A
Laboratory Diagnosis of Upper Respiratory Tract Infections
Cumitech 12A
Laboratory Diagnosis of Bacterial Diarrhea
Cumitech 13A
Laboratory Diagnosis of Ocular Infections
Cumitech 16A
Laboratory Diagnosis of the Mycobacterioses
Cumitech 18A
Laboratory Diagnosis of Hepatitis Viruses
Cumitech 19A
Laboratory Diagnosis of Chlamydia trachomatis Infections
Cumitech 21
Laboratory Diagnosis of Viral Respiratory Disease
Cumitech 23
Infections of the Skin and Subcutaneous Tissues
Cumitech 24
Rapid Detection of Viruses by Immunofluorescence
Cumitech 26
Laboratory Diagnosis of Viral Infections Producing Enteritis
Cumitech 27
Laboratory Diagnosis of Zoonotic Infections: Bacterial Infections Obtained from Companion and Laboratory Animals
Cumitech 28
Laboratory Diagnosis of Zoonotic Infections: Chlamydial, Fungal, Viral, and Parasitic Infections Obtained from Companion and Laboratory Animals
Cumitech 29
Laboratory Safety in Clinical Microbiology
Cumitech 30A
Selection and Use of Laboratory Procedures for Diagnosis of Parasitic Infections of the Gastrointestinal Tract
Cumitech 31
Verification and Validation of Procedures in the Clinical Microbiology Laboratory
Cumitech 32
Laboratory Diagnosis of Zoonotic Infections: Viral, Rickettsial, and Parasitic Infections Obtained from Food Animals and Wildlife
Cumitech 33
Laboratory Safety, Management, and Diagnosis of Biological Agents Associated with Bioterrorism
Cumitech 34
Laboratory Diagnosis of Mycoplasmal Infections
Cumitech 35
Postmortem Microbiology
Cumitech 36
Biosafety Considerations for Large-Scale Production of Microorganisms
Cumitech 37
Laboratory Diagnosis of Bacterial and Fungal Infections Common to Humans, Livestock, and Wildlife
Cumitech 38
Human Cytomegalovirus
Cumitech 39
Competency Assessment in the Clinical Microbiology Laboratory
Cumitech 40
Packing and Shipping of Diagnostic Specimens and Infectious Substances
Cumitech 41
Detection and Prevention of Clinical Microbiology Laboratory-Associated Errors
Cumitech 42
Infections in Hemopoietic Stem Cell Transplant Recipients
Cumitech 43
Cystic Fibrosis Microbiology
Cumitechs should be cited as follows, e.g.: Gilligan, P. H., D. L. Kiska, and M. D. Appleman. 2006. Cumitech 43, Cystic Fibrosis Microbiology. Coordinating ed., M. D. Appleman. ASM Press, Washington, D.C. Editorial Board for ASM Cumitechs: Alice S. Weissfeld, Chair; Maria D. Appleman, Vickie Baselski, B. Kay Buchanan, Mitchell l. Burken, Roberta Carey, Linda Cook, Lynne Garcia, Mark LaRocco, Susan L. Mottice, Michael Saubolle, David L. Sewell, Daniel Shapiro, Susan E. Sharp, James W. Snyder, Allan Truant. Effective as of January 2000, the purpose of the Cumitech series is to provide consensus recommendations regarding the judicious use of clinical microbiology and immunology laboratories and their role in patient care. Each Cumitech is written by a team of clinicians, laboratorians, and other interested stakeholders to provide a broad overview of various aspects of infectious disease testing. These aspects include a discussion of relevant clinical considerations; collection, transport, processing, and interpretive guidelines; the clinical utility of culture-based and non-culture-based methods and emerging technologies; and issues surrounding coding, medical necessity, frequency limits, and reimbursement. The recommendations in Cumitechs do not represent the official views or policies of any third-party payer. Copyright © 2006 ASM Press American Society for Microbiology 1752 N Street NW Washington, DC 20036-2904 All Rights Reserved 10 9 8 7 6 5 4 3 2 1
Cystic Fibrosis Microbiology Peter H. Gilligan Clinical Microbiology-Immunology Laboratory, UNC Hospitals, and Departments of Microbiology-Immunology and Pathology-Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, NC 27514
Deanna L. Kiska Department of Clinical Pathology, SUNY Upstate Medical University, Syracuse, NY 13210
Maria D. Appleman Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033
COORDINATING EDITOR: Maria D. Appleman Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033
Overview of CF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Pseudomonas aeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Clinical Significance . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antimicrobial Resistance and Susceptibility Testing
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Staphylococcus aureus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Clinical Significance . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antimicrobial Resistance and Susceptibility Testing
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Burkholderia cepacia Complex and Related Organisms . . . . . . . . . . . . . . . . . 8 Taxonomy and Clinical Significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Antimicrobial Resistance and Susceptibility Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Burkholderia cepacia Complex-Related Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Burkholderia gladioli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Ralstonia spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Pandoraea spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Inquilinus spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Stenotrophomonas maltophilia and Achromobacter (Alcaligenes) xylosoxidans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Clinical Significance . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antimicrobial Resistance and Susceptibility Testing
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Mycobacterium spp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Clinical Significance . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antimicrobial Resistance and Susceptibility Testing
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Gilligan et al.
CUMITECH 43
Haemophilus influenzae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Clinical Significance . . . . . . . . . . . . . . . . . . . . . . . . Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antimicrobial Resistance and Susceptibility Testing
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Anaerobes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Clinical Significance . . . . . . . . . . . . . . . . . . . . . . . . Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antimicrobial Resistance and Susceptibility Testing
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Enterobacteriaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Clinical Significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Antimicrobial Resistance and Susceptibility Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Streptococcus pneumoniae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Clinical Significance . . . . . . . . . . . . . . . . . . . . . . . . Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antimicrobial Resistance and Susceptibility Testing
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Aspergillus spp. and Other Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Clinical Significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Clinical Significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Laboratory Aspects of Lung Transplantation in CF Lung Disease . . . . . . . . 20 Practical Guide to the Role of the Clinical Microbiology Laboratory in the Management of CF Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Preanalytical Stage (Specimen Selection, Collection, and Transport) . . . . . . . . . . . . . . . . . 22 Analytical Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Postanalytical Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Final Thoughts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
OVERVIEW OF CF
C
ystic fibrosis (CF) is the most common inherited genetic disease in the Western world. It occurs in 1 in 1,900 to 1 in 3,700 live births in people of European ancestry. It is seen much less frequently in Hispanics, Asians, and African Americans (84). In a study of affected families, CF was found to occur at a frequency of 25%, a value associated with an autosomal recessive disorder inherited in a classical Mendelian fashion (10). The early, excess mortality seen in CF is due primarily to chronic lung disease characterized by airway obstruction and chronic infections complicated
by an excessive inflammatory response. In the first part of the 20th century, the genetic nature of CF was not understood. Rather, it was described as a collection of diverse clinical syndromes affecting the pancreas and the alimentary and respiratory tracts. Patients varied in their initial presentations, the ages at which symptoms appeared, the severity of symptoms, and the progression of the disease. Some babies had a blockage of the small intestine called a meconium ileus. Some failed to thrive, could not eat, were underdeveloped, and had muscle weakness. Some neonates initially presented with pancreatitis, persistent diarrhea, or even constipation. Andersen was the first to recognize the syndromic nature of this condition, calling it fibrocystic disease of the pancreas or
CUMITECH 43
mucoviscidosis (9, 140). In 1938, she did a careful analysis of 49 patients, their families, and the causes of death of the patients in relation to their ages at death. She demonstrated that patients dead at 1 week died of intestinal obstruction and that those who died older than 1 week died of respiratory complications due primarily to Staphylococcus aureus lung infection (8). In 1946, with the collaboration of Hodges, Andersen reviewed the fates of the siblings of infants with pancreatic fibrosis and of all the relatives known to their parents. They concluded that CF was an inherited disease (10). In 1989, the genetic defect causing CF was discovered to be on the long arm of chromosome 7 (124, 188, 192). Considerable work has been done since to understand the link between this defect and CF disease. It is now understood that CF is a result of various mutations (more than 1,000 have been observed) in the CF transmembrane conductance regulator (CFTR) gene, the most common being delta phe 508, which is responsible for 70% of the observed mutations. The CFTR protein is a transmembrane regulatory protein present in a variety of cells. It regulates and participates in the transport of electrolytes across epithelial cell membranes and probably across intracellular membranes. A CFTR gene mutation can result in defective cyclic AMP-regulated chloride channel activity that impairs pancreatic, pulmonary, and intestinal functions (243). The CFTR gene can mutate in many ways, including missense and nonsense mutations, frameshifts, splice variations, and in-frame amino acid deletions (243). The effects of the mutations vary. Some result in a normal phenotype, and others result in severe systemic disease. The discovery of the gene responsible for CF and of the mutations responsible for the various disease types allows for genetic testing in people with the characteristic clinical manifestations or family history. One of the manifestations of the abnormal transport of electrolytes in CF patients is an abnormal electrolyte composition in sweat (71). Neonates in families with a history of CF may be screened for the disease by measuring their sweat sodium and chloride concentrations. Unaffected people will have very little sodium and chloride, but those with CF will have sweat chloride concentrations of more than 60 nmol/ liter, two to five times the normal amount (219). The disruption of the exocrine function is the basis of many of the clinical aspects of CF disease. Patients have a basic problem with the way ions and fluids are transported in and out of mucus-producing cells. Mucus secretions should be fluid and thin to lubricate and move through ducts and through various channels in the body. In CF patients, mucus secretions are thick, dry, and sticky so that they clog pas-
Cystic Fibrosis Microbiology
3
sages (46). When microorganisms enter the distal airways in the lungs of CF patients, they are not cleared, and instead of the transient colonization seen in normal lungs, the organisms cause chronic infections, an exaggerated inflammatory response, and progressive bronchiectasis until the airways lose function and the patients suffer respiratory insufficiency. The survival rates of CF patients have increased over the years mainly because of improvements in nutritional regimens and the discovery and development of antibiotics. Before antimicrobials, most CF patients died by the time they were 10 years old, and the causes of death included intestinal obstruction, respiratory failure, pancreatitis, and malnutrition. Today, more than one-third of CF patients are adults (18 years old), and death, when it occurs, is due to respiratory failure from the chronic bacterial lung infections with accompanying airway inflammation (200). A limited variety of organisms have been implicated in chronic CF lung infection. Chronic infections develop very early in the lives of CF patients. At first, CF infants may be intermittently infected primarily by S. aureus, Haemophilus influenzae, and occasionally Pseudomonas aeruginosa. In most individuals, intermittent infection invariably leads to chronic infections (46). Burkholderia cepacia and nontuberculous Mycobacterium spp. appear as important lung pathogens in adolescents and adults (Table 1). As patients reach adolescence, other organisms are isolated from their respiratory secretions, but the role of these organisms in chronic CF lung disease has not yet been delineated. These bacteria of unknown or limited pathogenicity include Stenotrophomonas maltophilia, Achromobacter (Alcaligenes) xylosoxidans, Burkholderia gladioli and other Burkholderia spp., Ralstonia spp., and Pandoraea spp. (151). When molecular methods are used to examine the composition of the microbial flora in respiratory specimens of CF patients, additional unusual, unexpected, and unculturable organisms are detected. Whether these organisms have a role in disease has yet to be determined (177, 190). Despite the expectations following the discovery of the gene responsible for CF disease, a cure has not been developed. However, the survival rate has improved as protocols for patient care and microbiology procedures have improved. The careful detection and identification of the pathogenic organisms with appropriate susceptibility testing can have a huge impact on the fates of CF patients (211). The purpose of this Cumitech is to present the current recommended microbiology procedures for specimens from CF patients. The organisms are discussed in the order of their clinical importance in these patients.
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CUMITECH 43
Table 1. Microorganisms in the respiratory tracts of CF patientsa Organisms
Frequency of isolation (%)
Pseudomonas aeruginosa Staphylococcus aureus Burkholderia cepacia complex Stenotrophomonas maltophilia Achromobacter xylosoxidans Burkholderia gladioli Ralstonia spp. Pandoraea spp. Mycobacterium spp. Haemophilus influenzae Streptococcus pneumoniae Enterobacteriaceae Aspergillus fumigatus RSV Influenza virus
59 48 3 10b 9b 1 1 1 13 15 5 5 9 Unknown Unknown
CF population All Predominantly Predominantly Predominantly Predominantly Predominantly Predominantly Predominantly Predominantly Children Children Children All Children All
children and adolescents adolescents and adults adolescents and adults adolescents and adults adolescents and adults adolescents and adults adolescents and adults adolescents and adults
Role in CF lung disease Proven Proven Proven Not provenc Unlikelyd Unlikely Unlikely Possible Proven Likely Unknown Unknown Proven Proven Proven
a
Adapted from reference 151. From reference 34. c From reference 89. d From reference 226. b
PSEUDOMONAS AERUGINOSA Clinical Significance P. aeruginosa is the most important infectious agent involved in chronic lung infection in CF patients (160). In recent years, it has become increasingly apparent that P. aeruginosa can infect these individuals from early infancy onward (239). Overall, P. aeruginosa can be isolated from 60% of all CF patients and from 80% of adults (84). Longitudinal studies suggest that the prognoses in CF are closely linked with the ages at which children become chronically infected with P. aeruginosa (239). Initial lung colonization occurs with nonmucoid, wild-type strains of P. aeruginosa that subsequently give rise to the familiar mucoid variants associated with CF disease. Once the mucoid variant is recovered from the airways, it is nearly impossible to eradicate. Therefore, an important focus in CF care is to prevent the establishment of the mucoid variant in the lung. This is best accomplished by early detection and aggressive antimicrobial therapy (45). Our understanding of the pathogenesis of mucoid P. aeruginosa, like our understanding of the organism itself, is constantly evolving. The seminal event in the development of chronic lung infection with P. aeruginosa is the transformation from the “planktonic,” motile form of the organism (nonmucoid) to the nonmotile form exhibiting the biofilm mode of growth (mucoid) (84). Worlitzsch and colleagues (247) have hypothesized that CF patients have extremely low surface liquid levels, adversely affecting mucociliary clearance. Simultaneously, there is persistent mucus
hypersecretion, resulting in the production of mucus plugs and a thickened mucus layer on the surface epithelium. The interior of this thickened mucus mass is comparatively hypoxic. The planktonic, motile P. aeruginosa penetrates the mucus mass and enters into hypoxic regions, where it grows using anaerobic respiration with nitrate as a terminal electron acceptor (96), so this organism, once thought to be an obligate aerobe, is now defined as an obligate respirer. Under hypoxic stress, the organism converts from a planktonic to a biofilm mode of growth. Anaerobic respiration is directly correlated to the production of alginate and the mucoid phenotype (70, 95, 252). The actual events that result in this conversion from nonmucoid to mucoid growth are not clearly understood. However, mutations in mucA, a gene that down-regulates the synthesis of alginate, is thought to be one mechanism by which this conversion can occur (140, 147, 148). Organisms exhibiting this biofilm mode of growth are refractory to mucociliary clearance and phagocytosis and are more resistant to antimicrobials (96). Lung damage observed in CF patients is primarily due to the inflammatory response to the mucoid P. aeruginosa (173). In particular, high levels of polymorphonuclear cell-derived proteolytic enzymes, especially elastase, can be found in the airways of chronically infected patients (84). The presence of high numbers of polymorphonuclear cells in CF airways is due, at least in part, to suppression or up-regulation of the production of various cytokines in response to the presence of mucoid P. aeruginosa (84). These enzymes may play an important role in the destruction of lung
CUMITECH 43
Cystic Fibrosis Microbiology
5
architecture and accompanying pulmonary failure which is a hallmark of this chronic disease.
Antimicrobial Resistance and Susceptibility Testing
Epidemiology P. aeruginosa is ubiquitous in nature, particularly in aqueous environments in home and medical settings. The organism can be found in sinks, on shower heads, in tap water, on medical equipment (respirators, nebulizers, and humidifiers), and on the surfaces of fresh fruits and vegetables. Epidemiological studies suggest that P. aeruginosa colonization results from exposure to environmental sources rather than person-toperson transmission (29). However, siblings have been shown to carry the same strain, as a result of either cross infection or acquisition from a common source (93). Recent reports of the spread of multidrug-resistant clones among patients in adult and pediatric CF clinics are of concern because patients infected with multidrug-resistant strains may have a worse prognosis than those uninfected (5, 16, 116). Surveillance and molecular typing may be useful tools to monitor cross infection with multidrug-resistant isolates in CF centers.
Since infection with mucoid P. aeruginosa is almost impossible to eradicate, the goal of antimicrobial therapy is to decrease the bacterial load to achieve clinical improvement. After cessation of therapy, the organism load may return to pretreatment levels within weeks to a month, with a subsequent decline in the patient’s clinical condition necessitating another round of antimicrobial therapy. It is easy to see how numerous courses of therapy over many years produce resistant isolates of P. aeruginosa. When P. aeruginosa is first isolated from CF patients, the antimicrobial susceptibility of the organism is similar to that of P. aeruginosa found in nonCF patients. Initial isolates are susceptible (75%) to a wide array of antimicrobials, including piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, imipenem, meropenem, aminoglycosides, and ciprofloxacin (47, 82). Combination therapy, such as that with a -lactam and an aminoglycoside, is typically used to treat pulmonary exacerbations and to delay the development of resistance. But resistance develops readily in P. aeruginosa recovered from CF patients because approximately 30% of CF patients are infected with hypermutator strains (169) with mutations in the DNA repair genes. In P. aeruginosa, the specific gene present is mutS. In strains with this gene, mutation frequencies are much higher, and under repeated rounds of antimicrobial pressure, resistant mutants will be selected (141). It is not uncommon for a CF adult to be infected with P. aeruginosa resistant to all available antimicrobial agents, a scenario that carries a grim prognosis. The susceptibilities of P. aeruginosa isolates from CF patients can be determined by using the reference agar dilution or reference frozen broth microdilution procedure (51). These procedures may require a 24-h incubation period before results are read. Different susceptibility testing strategies have been compared to these reference methods (36, 37). Two commercial automated susceptibility systems, Vitek (bioMérieux, Durham, N.C.) and MicroScan Walkaway (Dade International, Inc., West Sacramento, Calif.) were evaluated and found to have very high rates of very major errors (37), and the use of these automated methods for susceptibility testing of CF P. aeruginosa isolates is not recommended. Both the Etest (AB Biodisk, Solna, Sweden) and Kirby-Bauer disk diffusion susceptibility testing have been shown to correlate with reference MIC testing (36). However, routine susceptibility testing methods with planktonically growing P. aeruginosa isolates are unlikely to be predictive of the activities of antimicrobials against P. aeruginosa growing as a biofilm
Detection P. aeruginosa from CF respiratory specimens is easily detected by culture on a variety of media. Problems arise when mixed morphotypes varying in color, size, and texture are identified. Often these morphotypes are actually members of the same strain that differ in their susceptibility patterns, necessitating tedious isolation procedures to obtain pure subcultures for testing (227). Identification P. aeruginosa organisms grow aerobically and are oxidase-positive, non-glucose-fermenting, gram-negative bacilli. For nonmucoid isolates, identification is based on colonial morphology (no lactose fermentation on MacConkey agar, metallic sheen, and green diffusible pigment), a grapelike odor, and a positive oxidase reaction (129). Identification of mucoid P. aeruginosa is relatively straightforward since very few oxidase-positive, nonfermenting, gram-negative bacilli display this characteristic morphology. The mucoid isolates are usually accurately identified through conventional biochemical testing, mucoid colonial morphology, pigment production, and growth at 42°C (130). However, in CF patients there are atypical P. aeruginosa isolates lacking the characteristic colonial morphology and pigment production, which grow slowly, are auxotrophic, and are comparatively asaccharolytic (84). Their identification may not be possible with commercial identification systems (199, 217) but depends on PCR based on 16S ribosomal sequences (217).
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(159). When susceptibility testing is performed with P. aeruginosa in a biofilm mode, -lactams are less active than when they are tested with P. aeruginosa growing planktonically. Among the -lactams, meropenem appears to be the exception to this observation. Aminoglycosides and ciprofloxacin are equally active against the organism whether it is growing planktonically or as a biofilm (159). These observations further complicate the interpretation of antimicrobial susceptibility results obtained in clinical laboratories that use planktonically growing organisms in susceptibility testing. Because resistance to multiple classes of antimicrobials is so common among isolates from CF patients, the testing of multiple antimicrobials in combination (synergy testing) has been advocated to guide therapy. The U.S. Cystic Fibrosis Foundation funds the synergy testing reference laboratory at Columbia University (website, http://synergy.columbia.edu). This is a Clinical Laboratory Improvement Amendments (CLIA)licensed laboratory. Testing is done by using a microtiter checkerboard technique and determining the fractional inhibitory index (198). Although clinicians use this service frequently, there are no published data demonstrating that the combinations that are effective in vitro correlate with a clinical response. A second approach to synergy testing is to find those cocktails effective against multidrug-resistant strains of P. aeruginosa by testing the bactericidal activity of either two or three drug combinations (1, 45). This multiple-combination bactericidal antibiotic testing (MCBT) is available at the Children’s Hospital of Eastern Ontario, Ottawa, Ontario, Canada. The laboratory is CLIA licensed and can be contacted by e-mail (
[email protected]). A recent randomized, double-blind controlled clinical trial of the efficacy of MCBT versus that of conventional susceptibility testing showed that antimicrobials selected with MCBT results did not improve outcomes compared with drugs selected by conventional testing (3). These data suggest that routine use of MCBT, a very laborintensive method, is not warranted. However, it may be of value in identifying drug combinations that have activity against highly or panresistant organisms. The development of resistance to antipseudomonal drugs has resulted in a search for alternative antimicrobial agents and novel therapeutic approaches. Both aerosolized tobramycin and colistin are extensively used for CF patients. Aerosolized tobramycin has been shown to improve lung function and reduce the P. aeruginosa organism load and the number of pulmonary exacerbations (183). Conventional susceptibility strategies do not predict the efficacy of this aerosolized antimicrobial therapy. Although susceptibility results for tobramycin are
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useful if the organism is susceptible, a report of resistance determined by disk diffusion or the Etest may be meaningless for a patient receiving aerosolized tobramycin because achievable concentrations of 100 to 200 g/ml in airways far exceed the breakpoint based on achievable concentrations in serum. Aerosolized colistin was commonly used before the development of aerosolized tobramycin. In a comparative trial of aerosolized colistin and tobramycin in P. aeruginosa-infected patients, both antibiotics were safe and reduced organism loads but only tobramycin was found to improve pulmonary function (103). Susceptibility testing of colistin in agar-based formats, either disk diffusion or Etests, is not accurate because the large molecule diffuses poorly through the agar matrix. Thus, broth dilution methods are recommended for determining the activity of colistin against P. aeruginosa isolates (104). Azithromycin, a macrolide, is being studied for mucoid P. aeruginosa-infected CF patients (197). There have been three trials involving a relatively small number of patients (n 49) that demonstrated an improvement in pulmonary function after 3 to 6 months (200). Macrolides have been assumed to be inactive against P. aeruginosa, and conventional susceptibility testing has not predicted activity (197). Although its mechanism of action remains unknown, it is thought that azithromycin may be therapeutic because of two activities that improve lung function. First, it has anti-inflammatory activity due to inhibition of neutrophil activity and cytokine production, and second, it inhibits biofilm formation and has relatively low biofilm inhibitory concentrations (concentration at which 50% biofilm inhibition is achieved, 2 g/ml) (159). P. aeruginosa susceptibility testing should include the use of disk diffusion and/or Etests. Multiple morphotypes should be tested in order to detect resistant strains which may not be detected by mixed morphotype testing (157). At the Cystic Fibrosis Center at the University of North Carolina (UNC) Hospitals, Chapel Hill, in vitro susceptibility testing is performed no more frequently than once every 3 months for outpatients. It is also performed upon admission for inpatients and only upon request during the remaining hospital stay. This approach is based on the observation that the P. aeruginosa isolates associated with exacerbation are almost always the same clone as the organism with which the patient is chronically infected. Further studies have demonstrated that the susceptibility patterns of the preexacerbation and exacerbation isolates are fairly constant (2). Synergy studies may be of value for patients who have multidrug-resistant organisms, and the studies
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are available at no charge from the reference laboratory at Columbia University (website, http://synergy .columbia.edu). At UNC Hospitals, synergy testing is done every 6 months on multidrug-resistant isolates.
STAPHYLOCOCCUS AUREUS Clinical Significance S. aureus is one of the bacterial pathogens most frequently recovered from CF airways and is found in approximately 52% of patients (64). In the preantibiotic era, it was the leading etiologic agent of lung infection and a major cause of mortality (9). In contemporary CF populations, it is likely to be the first bacterial pathogen recovered from the airways of infants and young children (84). The percentage of CF patients chronically infected with S. aureus increases throughout childhood and into early adolescence, reaching approximately 60%. The percentage of infected CF patients begins to decline in late adolescence, and approximately 30% of CF adults harbor this organism (84). Although undoubtedly a pulmonary pathogen in the preantibiotic era (9), its current role in CF lung disease is not understood. It was long believed that S. aureus caused changes in the CF airway that enhanced the binding of P. aeruginosa (222). However, little experimental data exist to support this notion. Nevertheless, pulmonary exacerbations associated with S. aureus are treated aggressively with short-term antimicrobial therapy (149). The use of prophylactic antimicrobial therapy to prevent chronic infection has fallen out of favor because of the increased likelihood of the emergence of chronic P. aeruginosa infection in patients receiving the therapy (34, 222). Epidemiology Molecular methods, especially pulsed-field gel electrophoresis, have played important roles in our understanding of the natural history of S. aureus infection in CF patients. Studies using molecular methods to type isolates have shown that CF patients may be chronically infected for as long as 6 years with the same clone of S. aureus (120). In addition, non-CF patients as well as CF patients can transmit methicillin-resistant S. aureus (MRSA) to CF patients in hospital settings (88). Specific clones of S. aureus isolated from multiple CF patients have been described, although most CF patients harbor a unique clone (120). The molecular epidemiology of S. aureus in CF patients is very similar to that of P. aeruginosa: unique clones infect individual patients over the long term, cross infection among CF patients can occur, and multidrug-resistant organisms can be transmitted between patients.
Cystic Fibrosis Microbiology
7
Detection Two issues are important to consider when selecting isolation medium to recover S. aureus from respiratory secretions. First, the complexity of CF microflora and the presence of P. aeruginosa require that a selective medium be used to ensure adequate detection of S. aureus. The second issue is the frequent presence of auxotrophic strains of S. aureus which develop aberrant colony morphologies (small, nonhemolytic, nonpigmented colonies with a fried-egg appearance) on standard isolation medium such as sheep blood agar or chocolate blood agar (86, 121). The term “smallcolony variants” (SCV) refers to these mutants (125) that may enhance the persistence of S. aureus in CF airways. Trimethoprim-sulfamethoxazole therapy can serve as a selective pressure for SCV expression, with mutants typically showing auxotrophy for thymidine (86, 121). Auxotrophy for hemin and menadione may also occur but less frequently (121). For many years, mannitol salt agar (MSA) has been the selective and differential medium for the recovery of S. aureus from respiratory specimens from CF patients. It is highly inhibitory for gram-negative rods including P. aeruginosa and B. cepacia, is reliably differential due to the ability of S. aureus to ferment mannitol, and gives typical colony morphology for thymidine auxotrophs (86). Recently, a new medium, CHROMagar Staph aureus agar (CSA), has been developed by BD Diagnostics, Sparks, Md. Like MSA, it is selective (using colistin, nalixidic acid, and an unspecified antifungal agent) and differential (using proprietary chromogenic reagents) and supports the growth of auxotrophs. The initial evaluation of CSA versus MSA for the isolation of S. aureus from CF throat and sputum specimens showed that CSA was excellent, with slightly improved detection (sensitivity, 100%) compared to MSA (89.4%) (81). Flayhart and colleagues proposed that S. aureus isolates on CSA giving the appropriate chromogenic reaction can be rapidly identified and reported because over 95% of isolates they tested gave appropriate reactions (81). It is likely that similar results would be obtained with MSA but that evaluation has not been recently done. The basic recommendation is that either MSA or CSA should be used to isolate S. aureus from respiratory tract specimens from CF patients. Although CSA is more expensive than MSA, it provides identification results more rapidly. Overgrowth by P. aeruginosa may occur on CSA, affecting the recovery of S. aureus from a small subpopulation of CF patients. Identification The identification of S. aureus is easily accomplished by either the traditional tube coagulase test or the
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wide variety of slide latex agglutination tests that are available. There are no reports in the literature of CF-specific S. aureus isolates that are not accurately identified by either approach. Antimicrobial Resistance and Susceptibility Testing The 2004 U.S. CF patient registry report showed that 15% of CF patients harbor MRSA (64). It is important to identify patients with MRSA because these organisms can be transmitted between hospitalized patients and cause serious infections that are more difficult to treat than those caused by methicillinsusceptible isolates. Nonauxotrophic S. aureus CF strains can be identified using CLSI-recommended techniques (51). Effective approaches for the detection of MRSA strains from CF patients include cefoxitin disk screening, the use of oxacillin screening plates, latex agglutination for the detection of PBP2a, or molecular detection of the mecA gene. Testing auxotrophic or SCV S. aureus isolates may be slightly more complex and require non-culture-based techniques, latex agglutination for PBP2 and mecA PCR, to detect methicillin resistance accurately (128). The frequency with which susceptibility testing of CF S. aureus strains should be performed has not been systematically studied.
BURKHOLDERIA CEPACIA COMPLEX AND RELATED ORGANISMS Taxonomy and Clinical Significance B. cepacia was first recognized as an important opportunistic pulmonary pathogen in CF patients in the mid-1980s. Originally categorized as a Pseudomonas sp., it was transferred to the genus Burkholderia (248) with B. cepacia as the type species. At
Table 2.
Burkholderia cepacia complex: taxonomy and epidemiology
Genomovar
a
that time, clinical research groups at three different North American CF centers recognized that this organism could cause a progressive, invasive, and fatal bacteremic disease now known as the cepacia syndrome. The syndrome, characterized by high fevers, rapid decline in pulmonary function, and bacteremia, is an unusual occurrence in the CF patient population (111, 224, 225). The mortality of cepacia syndrome is significant, with 20% of newly infected patients dying within 6 months (224). The other 80% of patients have a disease course very similar to that seen in patients infected with P. aeruginosa, with a gradual, continuous decline in lung function eventually resulting in death due to cardiopulmonary failure (224). These data are somewhat confounded by the fact that some of the B. cepacia-infected patients were also chronically infected with P. aeruginosa. Subsequent studies have confirmed this observation of the double infection (115). As pathogenicity and epidemiology studies of B. cepacia were accomplished using modern classification methods including 16S rRNA sequence analysis, fatty acid and whole-cell protein analyses, and DNADNA hybridization methods (57, 232), it became increasingly clear that B. cepacia was a complex of at least nine different species (Table 2). Among the species of the B. cepacia complex, Burkholderia multivorans (genomovar II) and Burkholderia cenocepacia (genomovar III) are recovered most frequently from CF patients (58, 97, 136). Two studies, one with CF transplant patients and the other with CF adults, have demonstrated that B. cenocepacia is the more pathogenic of the two (12, 115). Although both B. multivorans and B. cenocepacia can cause the cepacia syndrome, most cases are caused by B. cenocepacia (143). The pathogenicity of B. cenocepacia is attributed to a unique “cable pilin,” a biofilm mode
Species designation
Frequencya,b (%)
Pathogenicity in CF patientsc
I II
B. cepacia B. multivorans
5 20–40
Unknown Pathogenic
III
B. cenocepacia
50–80
Very pathogenic
IV V VI VII VIII IX
B. B. B. B. B. B.
stabilis vietnamiensis dolosa ambifaria anthnia pyrrocina
1 5 5 1 Case report Case report
Unknown Unknown Unknown Unknown Unknown Unknown
Frequency with which the genomovar is recovered from CF patients infected with the B. cepacia complex. Data from references 97, 137, and 147. Data from references 119 and 147. d Data from references 97 and 147. b c
Clonal spreadd Not documented Infrequent; most strains obtained environmentally Frequent; spread of three distinct clones well documented Not documented Not documented Documented; unusual Not documented Not documented Not documented
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of growth, siderophore production, a particularly toxic lipopolysaccharide, and the ability to invade and survive intracellularly (143). Epidemiology Organisms belonging to the B. cepacia complex are environmental organisms found in soil and on plants (54, 143, 180). B. cepacia (genomovar I) is a plant pathogen causing soft rot in onions (143). Several species within the complex are of interest as plant growth promotion or bioremediation agents (54). The CF community considers the potential infectious disease risk to CF patients to be greater than the potential benefit from the agricultural and environmental uses of these agents (54), especially since it has been observed that CF patients can be colonized by both B. cenocepacia and B. multivorans from the environment. Of the nine different species within the B. cepacia complex, B. multivorans and B. cenocepacia, the two species causing most infections in CF patients, have been the most studied in epidemiology investigations. Person-to-person spread has been well documented with B. cenocepacia and has been observed with B. multivorans (44, 97, 108, 223). B. cenocepacia has been demonstrated to be genetically highly heterogeneous. One specific B. cenocepacia strain designated Toronto or ET 12 is widely disseminated among CF populations in both North America and Western Europe (223), and careful epidemiologic studies have documented its person-to-person spread (90). Subsequent studies showed that this organism produces an unusual adhesin, called cable pilin, which may have a significant role in dissemination (223). Later studies have found that this adhesin appears to be unique to the ET 12 clone and to closely related genotypes (12, 108, 136). The cable pilin-positive strains may spread not only among CF patients but also from CF to non-CF patients (108). Further genetic studies revealed that there are three widely disseminated clones of B. cenocepacia: the original Toronto or ET 12, the PHDC strain first seen in the middle Atlantic states in the United States, and a third Midwest strain found in the U.S. central region (44, 143). Patients infected with B. cepacia have significantly reduced long-term survival rates compared with those not infected with that organism. Since the organism can spread between patients, prevention of organism transmission is of paramount importance to patients’ outcomes. Prevention or infection control protocols have social and therapeutic impacts on patients. Since the B. cepacia complex is found in approximately 3% of U.S. CF patients and approximately 15% of Canadian CF patients, the control protocols affect many patients. The organism typically is first seen in adolescents and young adults.
Cystic Fibrosis Microbiology
9
Segregation of B. cepacia-infected CF patients from uninfected CF patients has proven highly successful (200). This separation can be difficult to enforce with adolescents, for whom being part of the group is particularly important. It is also recognized that cohorting of B. cepacia complex-infected patients may not prevent the spread of different clones among these patients since it has been documented that one clone may be replaced with a different clone of greater virulence (26). In addition, B. cepacia complex-infected patients may be excluded from official Cystic Fibrosis Foundation social functions and educational conferences. Finally, in most transplant centers, B. cepacia-infected CF patients are not considered to be candidates for lung transplantation (151). Detection P. aeruginosa, with its fast rate of growth and large, mucoid colonies, can obscure the colonies of the B. cepacia complex when MacConkey and eosinmethylene blue (EMB) agar media are used to isolate nonfermenting, gram-negative rods from CF respiratory secretions (211). After the recognition of the importance of B. cepacia in CF lung disease, selective isolation media were quickly developed. These media, Pseudomonas cepacia agar (85) and oxidationfermentation polymyxin bacitracin lactose (OFPBL) agar (237), recovered B. cepacia complex organisms more efficiently than MacConkey agar. Although most P. aeruginosa isolates were suppressed on these new media, other organisms could break through, especially other non-glucose-fermenting organisms such as B. gladioli, Ralstonia species, and S. maltophilia, as well as yeasts and molds (100). Subsequently, a third medium called B. cepacia selective agar (BCSA) was developed (100). This medium is superior to both P. cepacia agar and OFPBL agar, being more sensitive (greater number of isolates recovered) and more specific (fewer non-B. cepacia complex organisms recovered). BCSA is recommended as the medium of choice for the recovery of the B. cepacia complex from clinical specimens. Identification Accurate identification of clinical isolates belonging to the B. cepacia complex is critical to patient care and outcome. Several studies have shown that commercial systems misidentify the B. cepacia complex and misidentify other species such as B. gladioli and Ralstonia pickettii as B. cepacia. Even the most accurate systems identify only 85% of B. cepacia complex isolates accurately (31, 130, 150, 210). Seven hundred seventy isolates tentatively identified as belonging to the B. cepacia complex were referred to the Cystic Fibrosis Foundation-sponsored B. cepacia Research Laboratory and Repository, Ann Arbor,
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Mich., for confirmatory identification using PCR assays (150). With the molecular method, 88 (11%) were found not to be B. cepacia complex organisms. Those most frequently misidentified as B. cepacia complex organisms were B. gladioli (n 28), Stenotrophohomonas sp. (n 12), Pseudomonas sp. (n 11), Alcaligenes sp. (n 7), and Ralstonia sp. (n 2). Twenty-six remained unidentified at the species level. Systems that gave misidentifications included Vitek GNI Plus, MicroScan GNP, API 20NE, Remel N/F, and conventional biochemicals (150, 210). In addition, 20 isolates which were submitted as “species other than B. cepacia complex” were subsequently identified as B. cepacia (150, 210). This observation is troubling because of the potential for transmission of the organism from individuals considered to be “B. cepacia free” to susceptible individuals, an infection control crisis due to a laboratory misidentification. Any organism identified phenotypically as either a B. cepacia complex organism or a closely related organism, including B. gladioli and Ralstonia spp., should have its identity confirmed by molecular means. In addition, any isolate from a CF patient identified by a commercial identification system as a “nonfermenting gram-negative rod” or “unidentified” should be referred for molecular identification (210). The B. cepacia Research Laboratory and Repository at the University of Michigan, Ann Arbor, has developed several PCR assays using a variety of sequence targets to identify the different species of the B. cepacia complex as well as closely related organisms. PCR targets include recA and 16S rRNA sequences for the different species within the B. cepacia complex (180); 23S rRNA sequences for B. gladioli and S. maltophilia (240, 241); 16S rRNA sequences for Ralstonia mannitolilytica, R. pickettii, and A. xylosoxidans (55, 138); and the gyrB gene for Pandoraea spp. (55). Identification of B. cepacia complex isolates by molecular methods should be considered definitive for the purposes of patient management. Antimicrobial Resistance and Susceptibility Testing B. cepacia complex organisms recovered from CF patients are typically resistant (90%) to aminoglycosides, fluoroquinolones, colistin (polymyxin E), imipenem, and cephalosporins. If the patient’s isolate has not yet been challenged with antimicrobials, it may be susceptible to piperacillin-tazobactam, trimethoprim-sulfamethoxazole, ceftazidime, and minocycline. Resistance will be variable for meropenem (1, 156, 201). Emergence of resistance, following several courses of antimicrobial therapy with agents to which the organism was initially susceptible, is the norm. It is not unusual for these organisms to be resistant to all
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antimicrobial agents when individual agents are tested (1). Like mucoid P. aeruginosa, B. cepacia is almost impossible to eradicate with antimicrobial therapy. Recently, the Clinical and Laboratory Standards Institute (CLSI) (51) published breakpoints for disk diffusion as well as MICs of ceftazidime, meropenem, minocycline, and trimethoprim-sulfamethoxazole. Isolates of the B. cepacia complex should be tested and reported with an interpretation only for these antibiotics. If other antibiotics are tested by an MIC method such as the E test, results should be reported without interpretation. Synergy testing is used to examine combinations of antimicrobials that may have activity against B. cepacia. Three approaches have been reported including the use of double E tests, checkerboard synergy testing, and MCBT (1, 144, 201). Often double drug combinations fail to demonstrate efficacy, and triple drug combinations are required to show beneficial in vitro activity. It should also be noted that in vitro antagonism may occur with a frequency similar to that of synergistic combinations (1, 144). Synergy testing, regardless of the approach taken, is expensive, and a randomized, double-blind control trial did not demonstrate an outcome benefit when MCBT was compared to standard susceptibility testing (3, 45). Burkholderia cepacia Complex-Related Organisms The emergence of B. cepacia complex organisms as important pathogens in CF patients has stimulated a more thorough search for other “new” pathogens in CF patients, with the recognition that a variety of organisms, either not previously recovered from humans or infrequently recovered from them, may be detected in the respiratory secretions of CF patients. The catalyst of this observation was the highly selective B. cepacia isolation media that allowed the recovery of new organisms (85, 100, 237). In addition, because of improvements in CF therapy, especially that with antipseudomonal antimicrobials, CF patients are living longer, and their respiratory tracts provide an ideal environment for the growth of these organisms. The pathogenic role of the newly recognized and infrequently recovered organisms (see Table 3) has not been as carefully studied as that of the B. cepacia complex. Burkholderia gladioli B. gladioli was the first of these new organisms to be recognized in the lungs of patients with CF. It was initially recovered on OFPBL medium and mimicked the appearance of B. cepacia (48). The organism pro-
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Cystic Fibrosis Microbiology
duced a diffusible yellow pigment and appeared to use lactose, a key feature of this selective and differential medium for the recognition of B. cepacia. The API 20NE (bioMérieux) system effectively identifies the B. cepacia complex, but it does not have B. gladioli in its database. In studies using this identification system, B. gladioli was frequently misidentified as B. cepacia (130). B. cepacia complex isolates have also been misidentified as B. gladioli. In a small outbreak involving four CF patients, isolates were identified as B. gladioli by phenotypic means (244) and were subsequently identified by molecular analysis as the ET 12 strain of B. cenocepacia (52). Because of problems with the accurate identification of B. gladioli, its possible role in CF lung disease is currently unclear (19, 117). The application of molecular methods using PCR targets in regions of the 23S rRNA has been successful in accurately identifying B. gladioli (241) and, in the future, will allow accurate identification and improved understanding of its epidemiology and pathogenicity. Methodical susceptibility studies of large numbers of isolates of B. gladioli have not been performed, and antibiotic efficacy breakpoints particular to them have not been developed. Based on P. aeruginosa breakpoints, B. gladioli isolates tend to be more susceptible to antibiotics than B. cepacia, particularly to aminoglycosides. Ralstonia spp. Several different species of the genus Ralstonia have been recovered from respiratory specimens from CF patients (56) (Table 3). These organisms can grow on agar selective for B. cepacia (100). Like B. gladioli,
Table 3. Selected non-glucose-fermenting rods other than those of the Burkholderia cepacia complex recovered on B. cepacia selective agars Growtha on: Organism Burkholderia gladioli Ralstonia pickettii Ralstonia mannitolilytica Ralstonia gilardi Ralstonia respiraculi Ralstonia insidiosa Inquilinus limosus Pandoraea pnomenusa Pandoraea apista Pandoraea sputorum Pandoraea pulmonicola
PC agarb
OFPBLc
BCSAd
ND ND
ND ND ND ND ND ND ND ND ND
ND ND
, most strains grow on this medium; , most strains fail to grow on this medium; ND, no data. Pseudomonas cepacia agar (85). c Reference 241. d Reference 100. a
b
11
with the use of phenotypic identification schemes these organisms can be misidentified as B. cepacia, and B. cepacia can be misidentified as Ralstonia spp. (130, 210). A number of PCR assays targeting the different species have been developed to differentiate them from the B. cepacia complex. Currently there are no data to indicate their pathogenicity (55). Specific data on the antimicrobial resistance of Ralstonia spp. recovered from CF patients are not available. R. pickettii is typically resistant in vitro ( 50% susceptible) to colistin, ceftazidime, ticarcillin-clavulanate, meropenem, amikacin, tobramycin, and gentamicin. Organisms are usually susceptible (70% susceptible) to ceftriaxone, cefepime, piperacillin, piperacillin-tazobactam, imipenem, fluorquinolones, and trimethoprim-sulfamethoxazole. Clues that the isolate may be a Ralstonia species are meropenem resistance and imipenem susceptiblity since the reverse are typically characteristics of the B. cepacia complex (196). Pandoraea spp. Pandoraea is a recently described genus originally recovered on BCSA from respiratory secretions from CF patients (53, 65). These organisms can be difficult to distinguish from both the B. cepacia complex and Ralstonia spp. by conventional means but can be easily distinguished from them by fatty acid analysis (53). Pandoraea spp. are asaccharolytic, and this characteristic can be a clue to their identity. Unfortunately, selected isolates of the B. cepacia complex and Ralstonia may exhibit similar phenotypes so molecular methods using gyrB are the most accurate means of differentiating Pandoraea from Ralstonia and the B. cepacia complex (55). Clinically, there are indications that Pandoraea may be more virulent than either Ralstonia spp. or B. gladioli. Pandoraea apista was shown to cause bacteremia in a CF patient (114) and caused a small outbreak in six CF patients in which patient-to-patient spread was likely (118). But because the patients were also infected previously or concurrently with recognized CF pathogens, primarily P. aeruginosa, it is difficult to determine what role this organism may have had in the lung disease of these CF patients. Pandoraea spp. tend to be multidrug resistant, with elevated MICs of -lactams, aztreonam, and aminoglycosides for these organisms, but varied in their susceptibility to the fluoroquinolones (65, 114). Isolates may be intermediately susceptible to doxycycline, imipenem, or ceftriaxone. Because such a small number of isolates have been tested (65, 114), susceptibility should be determined on a case-by-case basis by using an MIC method. Etests may be useful in testing these organisms but have not been validated for this purpose.
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Inquilinus spp. Inquilinus limosus is yet another non-glucosefermenting, gram-negative rod that has been recovered from the respiratory tracts of a small number of CF patients. It can grow on BCSA but may fail to grow on MacConkey agar (238). Identification to the species level has been based on 16S rRNA sequence analysis (55, 238). There are insufficient data to comment on the clinical relevance of this organism in CF lung disease. The organism demonstrates in vitro resistance to trimethoprim-sulfamethoxazole, aminoglycosides, penicillins, cephalosporins, colistin, and polymyxins. Ceftazidime, imipenem, and ciprofloxacin have shown in vitro activity (238). Because there are no CLSI standards, MIC methods must be used to determine in vitro activity.
STENOTROPHOMONAS MALTOPHILIA AND ACHROMOBACTER (ALCALIGENES) XYLOSOXIDANS Clinical Significance S. maltophilia and A. xylosoxidans are two nonglucose-fermenting, environmental, gram-negative rods that are being recovered with increasing frequency from CF patients. In a study of 995 CF patients of 6 years of age, S. maltophilia was isolated in 9% and A. xylosoxidans was isolated in 7% of cases (35). In the 2004 U.S. CF patient registry, 12% of CF patients were found to harbor S. maltophilia (64). CF patients with S. maltophilia tend to be in mid- to late adolescence or young adulthood (64, 68, 89, 145). Unlike that with P. aeruginosa, chronic infection with S. maltophilia is unusual, occurring in approximately 10% of cases (84), and most CF patients are only transiently infected or colonized (145). Oral quinolone antibiotic use, more than two courses of intravenous antibiotics, isolation of Aspergillus fumigatus, and oral steroid use have been shown to be associated with an increased risk of S. maltophilia acquisition (89, 91, 145, 201). CF patients with S. maltophilia frequently are coinfected with P. aeruginosa. There is growing evidence that S. maltophilia does not play a significant role in the deterioration of lung function, which is the hallmark of CF lung disease (89, 145). Given the frequently transient nature of S. maltophilia infection, aggressive treatment in CF patients may not be warranted. There is less published on the clinical role of A. xylosoxidans than on that of S. maltophilia, but based on what is available, the two bacteria appear to have similar roles. With both, patients are usually in adolescence when the organisms are acquired, tend to be coinfected with P. aeruginosa, are only transiently colonized or infected, and do not have an
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increased rate of pulmonary deterioration associated with either organism (226). Patients receiving antimicrobials were more likely to acquire S. maltophilia than A. xylosoxidans when compared to controls. Because of the lack of data, it is not possible to be absolutely certain that A. xylosoxidans does not play an important role in CF lung disease. Epidemiology S. maltophilia is readily recoverable from CF patients’ home and hospital environments. Sink drains and faucets appear to be the most common sources (68). Interestingly, CF patients with S. maltophilia are usually colonized or infected with a genotype different from that found in the home environment (68). Although most patients have a unique genotype, sharing of S. maltophilia genotypes has been observed, especially among siblings (68, 132, 231). Those individuals who are persistently colonized with S. maltophilia often have sequential genotypes, with no more than half of the patients being consistently infected or colonized with a single genotype (132, 231). The same level of epidemiologic investigations has not been done with A. xylosoxidans. However, the data that are available are similar to those for S. maltophilia. Most A. xylosoxidans patients have a unique genotype, and shared genotypes are most frequent among sibling pairs or other epidemiologically linked individuals. Unlike those with S. maltophila, most patients with persistent infection or colonization with A. xylosoxidans have a single genotype (132). Detection Both S. maltophilia and A. xylosoxidans can be readily recovered on MacConkey and EMB agars. Both organisms also grow on all three B. cepacia selective agars (BCSA and OFPBL and P. cepacia agars), making it important to differentiate them from the B. cepacia complex (100). A selective medium for the recovery of S. maltophilia containing vancomycin, imipenem, and amphotericin B (VIA agar) has been used in a research setting but has not found widespread clinical use (68). Identification Both S. maltophilia and A. xylosoxidans have distinct phenotypic identification patterns (87, 207) that should allow their differentiation from the B. cepacia complex. However, B. cepacia reference laboratories report receiving both of these organisms misidentified as B. cepacia (100, 150). Commercial identification systems generally can differentiate S. maltophilia from the B. cepacia complex (130). However, misidentification of S. maltophilia as the B. cepacia complex by API 20NE, the Vitek GNI panel, and MicroScan GNP has been reported (210). There
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is no evidence that B. cepacia complex organisms are misidentified as S. maltophilia (31, 130, 150, 210). A key phenotypic characteristic of S. maltophilia is the ability to degrade DNA, i.e., a positive DNase test. It is recommended that tubes with negative tests be incubated a full 72 h to avoid false-negative readings (87). There is sufficient overlap with other biochemical tests that DNase-negative strains of S. maltophilia (which are rare) will need to be differentiated from the B. cepacia complex by molecular means. Because S. maltophilia can be misidentified as B. cepacia by phenotypic means, PCR assays using a 23S rRNA sequence were developed for S. maltophilia. The evaluation of this PCR assay demonstrated that it could accurately differentiate S. maltophilia from a variety of non-glucose-fermenters from CF patients, including members of the B. cepacia complex, B. gladioli, R. pickettii, and A. xylosoxidans (240). This PCR assay is currently used by the B. cepacia Reference Laboratory and Repository at the University of Michigan (J. LiPuma, personal communication). Reference laboratories also report the misidentification of A. xylosoxidans as B. cepacia (100, 210). B. cepacia may also be misidentified as A. xylosoxidans, although less frequently (202, 210). MicroScan GNP, Rapid Neg ID, API 20NE, and RapID NF Plus were the commercial systems that misidentified A. xylosoxidans as B. cepacia (210). Differentiating A. xylosoxidans from the B. cepacia complex by phenotypic means can be challenging. A. xylosoxidans typically oxidizes xylose, but not lactose, maltose, or sucrose. B. cepacia complex strains also frequently oxidize xylose, but most strains are reported to be positive for lactose, maltose, and sucrose. However, there is sufficient variability in the sugar oxidation patterns among the members of the complex that differentiation of selected strains of this complex from A. xylosoxidans can be difficult. A. xylosoxidans is also lysine decarboxylase negative, and most strains of the B. cepacia complex are positive, but since certain B. cepacia strains may be lysine decarboxylase negative, identification is further confused (87, 207). A PCR assay based on the 16S ribosomal sequence of A. xylosoxidans (138) can distinguish A. xylosoxidans from other non-glucose-fermenters, including the B. cepacia complex, R. picketii, B. gladioli, and S. maltophilia. The assay will not distinguish between species of Achromobacter (138), but because there is no evidence that any other species plays a role in CF lung disease, this may not be important. Antimicrobial Resistance and Susceptibility Testing Both S. maltophilia and A. xylosoxidans are multidrug-resistant organisms (202, 205). Recently, the CLSI (51) published susceptibility testing standards
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for S. maltophilia, giving breakpoints to use with both MIC and disk diffusion methods for minocycline, levofloxacin, and trimethoprim-sulfamethoxazole. No testing standards are currently available for A. xylosoxidans. Susceptibility data based on broth microdilution MIC methods using P. aeruginosa breakpoints have been reported for both organisms as well as results of checkerboard synergy testing (40, 201, 202, 205). S. maltophilia is typically resistant to the carbapenems due to the presence of a chromosomally encoded zinc metallocarbapenemase (139). In addition, it is also resistant to aminoglycosides and most -lactams. Ceftazidime may show activity, but development of resistance over time is common. Occasional strains may be susceptible to -lactam– -lactamase inhibitor combinations such as piperacillin-tazobactam and ticarcillin-clavulanic acid, but typically 75% are resistant. Levofloxacin appears to be more active in vitro than ciprofloxacin, but resistance to the fluoroquinolones is common. Minocycline and trimethoprim-sulfamethoxazole are the most active agents against S. maltophilia (40, 205). However, trimethoprim-sulfamethoxazole resistance is more common in isolates from CF patients than in non-CF isolates (196, 205). Synergy was observed for fewer than half of the isolates of S. maltophilia for the combination of either ciprofloxacin or trimethoprim-sulfamethoxazole with either piperacillin-tazobactam or ticarcillin-clavulanic acid and the combination of azithromycin and trimethoprimsulfamethoxazole (201, 205). Susceptibility studies with A. xylosoxidans recovered from CF patients showed that this organism is resistant to aminoglycosides, fluoroquinolones, and trimethoprim-sulfamethoxazole. Inhibitory activity was reported with imipenem, meropenem, ceftazidime, ticarcillin-clavulanic acid, piperacillin, piperacillin-tazobactam, and minocycline for only 40 to 60% of the isolates tested (202). In vitro synergy was seen with 10% of isolates, so synergy testing for combination therapy is not likely to be helpful in the management of patients infected with A. xylosoxidans (201, 202).
MYCOBACTERIUM SPP. Clinical Significance Mycobacterium tuberculosis is rarely detected in CF patients and is not considered an important pathogen in CF lung disease. In prospective studies of infection or colonization with nontuberculous mycobacteria (NTM), these organisms were recovered from 10 to 13% of CF patients (171, 209). In a national study in the United States, the Mycobacterium avium complex (MAC) was the predominant organism recovered
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from 72% of the NTM-positive patients, and in a study conducted in Paris, Mycobacterium abscessus was recovered from 52%. In both studies, Mycobacterium kansasii and Mycobacterium gordonae were infrequently recovered, with M. gordonae considered either a contaminant or a saprophyte (92). It should be noted that the French population was primarily pediatric and that the average age of U.S. patients was 26 years. Only 37% of CF patients in the U.S. study met the American Thoracic Society’s microbiologic definition of NTM disease, which is three positive sputum cultures with no positive acid-fast bacillus (AFB) smears, two positive cultures with one positive AFB smear, or a positive bronchoscopically obtained culture or AFB smears (7). In the French study, two-thirds of the M. abscessus-infected or -colonized patients met the bacteriologic definition of NTM disease (209). In the U.S. study, patients with M. abscessus were more likely to meet the American Thoracic Society’s case definition of disease. These data as well as some published case reports (60, 63, 206) suggest that patients with consistently positive M. abscessus cultures have clinical disease due to this organism. Pathologic findings in these patients may include granulomatous inflammation with areas of focal necrosis and tuberculoid granulomas with Langerhans histiocytes (63, 229). In addition, patients who were persistently culture positive for NTM, mainly M. abscessus, showed evidence on high-resolution computed tomography scans of lung disease progression consistent with NTM disease (63, 171). The high-resolution computed tomography scans showed cystic lesions or cavities, segmental consolidation, and nodules in peripheral regions of the lung (171). Interpretation of these findings is difficult because they are also observed in CF patients from whom NTM are not recovered. However, the presence of cultures persistently positive for NTM, particularly M. abscessus, indicates that the progression in lung disease may be due to these organisms. Such patients require therapy appropriate for the species of NTM with which they are infected. Epidemiology NTM are environmental organisms found in soil and in water. Molecular epidemiology studies using pulsed-field gel electrophoresis indicate that patients are typically infected with individual clones of either M. abscessus or the MAC. This indicates that personto-person spread or common source acquisition of these organisms does not occur with any degree of frequency (171, 209). Detection As that of many other organisms in CF patients, culture detection of NTM is complicated by the pres-
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ence of mucoid P. aeruginosa. Even rapidly growing NTM may take several days to grow, by which time cultures are overgrown with P. aeruginosa. N-Acetyl cysteine-NaOH decontamination of respiratory specimens prior to culture is widely used to reduce contaminating flora. Unfortunately, it is relatively ineffective in reducing contamination due to P. aeruginosa. Contamination rates due to P. aeruginosa of between 25 and 50% have been reported (18, 133, 242). Sequential treatment of CF respiratory specimens with 0.25% N-acetyl cysteine–1% NaOH followed by 5% oxalic acid treatment reduces contamination rates to approximately 5% (171, 242). It has been reported that these sequential treatments of CF specimens result in reduced recovery of NTM, especially with specimens containing small numbers of organisms (18). Improved recovery can be obtained by using only 5% oxalic acid treatment for those specimens that show bacterial overgrowth. There are two issues with this approach. First, it is much more labor-intensive and expensive to reprocess 25 to 50% of specimens. Second, since only specimens with low numbers of organisms will be compromised by the sequential approach, it is likely that these cultures represent colonization rather than infection. Cultures for NTM should be done on solid and liquid media (7). A comparison of the BACTEC 460TB and BACTEC MGIT 960 showed better recovery of NTM with the BACTEC 460TB system (133). BACTEC 460TB was also superior to the MB/BACT ALERT 3D system for the detection of NTM. In particular, MB/BACT ALERT was inferior in the detection of Mycobacterium chelonae (176). Identification Molecular methods are preferred to phenotypic ones for the identification of NTM. The MAC, M. kansasii, and M. gordonae are efficiently identified using nucleic acid hybridization methods that detect species-specific RNA sequences in 16S rRNA (GenProbe, San Diego, Calif.) (234). Phenotypic means (biochemical testing, high-pressure liquid chromatography analysis of mycolic acid, or the combination of both) can be used to differentiate the rapidly growing mycobacteria, although these methods may have difficulty distinguishing M. abscessus from M. chelonae. In addition, biochemical tests may require extended incubation times, making phenotypic identification cumbersome (249). Sequencing of the hsp65 gene or the 16S-23S intergenic gene region can accurately differentiate M. abscessus from M. chelonae (131, 187). Alternatively, PCR amplification of the hsp65 gene followed by restriction enzyme analysis or melt curve analysis using the LightCycler can accurately differentiate these two organisms (208, 249). Based on sequence analysis of
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the 16S-23S intergenic gene region, M. abscessus isolates can be divided into two different genotypes, types I and II. Type 1 strains are more common in CF patients (131).
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HAEMOPHILUS INFLUENZAE
biotic-resistant strains (191). Haemophilus parainfluenzae is occasionally isolated from CF patients but appears to play a limited role in the pathogenesis of lung damage (107, 178, 185), although one study reported H. parainfluenzae as the most frequent Haemophilus species isolated from these patients (74). In the majority of studies, Haemophilus spp. are isolated most frequently from young CF patients, in whom the point prevalence is 30 to 40% in children under 10 years of age (73, 178, 185, 195). In contrast, the point prevalence is only 10% in patients over 10 years of age (73). This low prevalence may reflect the frequent presence in this age group of mucoid P. aeruginosa, which obscures the growth of Haemophilus. Use of selective media for Haemophilus with an adult CF population demonstrated a prevalence of 34%, similar to the prevalence seen in children (27). Despite its frequent isolation, the contribution of Haemophilus to CF lung pathology has not been definitively resolved. H. influenzae had no effect on cell detachment in an in vitro epithelial cell model, which suggests that this organism does not directly cause tissue damage (233). In patients greater than 6 years of age, isolation of H. influenzae was not associated with impaired pulmonary status as measured by forced expiratory volume in 1 s (167). A pathogenic role may be suggested since Haemophilus is isolated significantly more often during acute CF pulmonary exacerbations than at other times. An increased isolation rate was also noted for asymptomatic patients just prior to the development of an acute exacerbation (185). In addition, it has been postulated that early infection with organisms such as Haemophilus or S. aureus may damage the respiratory epithelium, leading to increased attachment of, and subsequent colonization by, P. aeruginosa (30, 140). Although no placebo-controlled trials have been conducted to establish whether treatment of Haemophilus decreases the frequency of exacerbations or subsequent colonization with P. aeruginosa, antibiotic therapy has been recommended for CF patients harboring Haemophilus regardless of their clinical status (178, 185). The organism responds well to antimicrobial therapy, with concomitant resolution of symptoms and eradication of the organism in at least 70% of cases (113, 178, 185).
Clinical Significance Nonencapsulated strains of H. influenzae are frequently isolated from the lower respiratory tracts of CF patients. But unlike P. aeruginosa, H. influenzae does not chronically colonize CF airways, although occasional long-term persistence (6 months) can occur (38, 105, 152, 178). Persistence appears to be associated with the presence of hypermutable, anti-
Detection H. influenzae can be isolated from throat swabs, sputa, and bronchoscopically obtained specimens. Throat swabs have been compared with bronchoalveolar lavage specimens for detecting the presence of H. influenzae in the lower airways (182, 195). The sensitivity has ranged widely, with higher sensitivity seen for younger patients and with the use of selective
Antimicrobial Resistance and Susceptibility Testing The mycobacteria most frequently recovered from CF patients, the MAC and M. abscessus, are not susceptible to agents typically used to treat M. tuberculosis infections (235). CF patients are heavily pretreated with antibiotics prior to considering therapy for NTM infection, and their NTM strains may be resistant. In addition, these organisms can be very difficult to eradicate with antimicrobials, requiring months of therapy (80). Because therapy is long and drug efficacy is not predicted by species identification (246), susceptibility testing should be done on all clinically significant isolates from CF patients who will be treated. Testing of NTM is typically done by using a broth-based method, either macro- or microdilution (246). MAC infections are typically treated with macrolides in combination with a second drug (80). The MAC is usually susceptible to macrolides (clarithromycin or azithromycin) in vitro if the organism has not had prior exposure to the drug (164, 246). The use of azithromycin in the treatment of mucoid P. aeruginosa may increase exposure of the MAC to the macrolides, increasing the likelihood of initial resistance and the need for susceptibility testing (164). Macrolides are also used to treat M. abscessus infections, again in combination with other antimicrobials to which this organism may be susceptible. Macrolide resistance is more common in M. abscessus than among the other rapidly growing mycobacteria (33, 249, 250). M. abscessus isolates are frequently susceptible to amikacin and may be susceptible to linezolid, cefoxitin, or imipenem (235, 250). Since resistance can develop during antimicrobial therapy, when patients show no clinical improvement or show clinical deterioration and still have a positive culture result, susceptibility testing should be repeated. If the initial isolate was not tested, both the initial and recent isolates should be tested (80, 246).
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agar for Haemophilus. With their high negative predictive values, throat swabs appear to be most useful in ruling out the presence of H. influenzae in the lower respiratory tract (15, 195). Direct detection of H. influenzae in sputum and lung tissue has been attempted by using PCR amplification and by using immunoperoxidase-conjugated monoclonal antibody staining. With these methods, the presence of H. influenzae could be demonstrated in sputa and lung tissues of CF patients despite negative cultures (153, 154). The clinical significance of these findings is unknown. The low isolation rate of H. influenzae in previous studies and from patients of 10 years of age appears to be the direct result of inhibition and overgrowth by P. aeruginosa (186, 189). Results from several studies suggest that the use of selective media for culture of CF respiratory specimens increases the isolation rate of H. influenzae (24, 83, 155, 189). This has been shown to be true for non-CF patients as well (42, 109). The most common selective medium formulation includes bacitracin (300 g/ml) in a chocolate agar or a supplemented blood agar base (74, 83, 189). Bacitracin has a suppressive effect on most gram-positive oropharyngeal organisms but has little effect on Enterobacteriaceae, yeasts, and Pseudomonas spp. (109). Cultures are typically incubated anaerobically at 35 to 37°C for 24 to 48 h to suppress the growth of P. aeruginosa, although complete suppression is not always achieved and may not result in increased isolation of Haemophilus (151). The necessity of selective media and anaerobic incubation is controversial. In one of the original studies with selective media, the authors stressed that it is necessary to correlate both clinical and bacteriological data (109). However, in many of the studies which show selective media to be beneficial, culture results were not interpreted in light of the clinical status of the patient. Also, in patients already colonized with P. aeruginosa and/or S. aureus, the clinical relevance of isolating small numbers of Haemophilus is questionable. Therapy directed against the former organisms would also be active against Haemophilus, which is susceptible to most of the antimicrobials used for treating infections in CF patients (73, 126). Identification Isolation of Haemophilus requires further identification and -lactamase testing. Identification of H. influenzae is based upon typical colonial morphology, a characteristic “mousy” odor, Gram stain morphology, and X (hemin) and V (NAD) factor requirements (127). X factor dependence can be determined by growth factor-based or porphyrin-based methods. There are also commercial biochemical identification systems (Haemophilus identification test kit by
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Remel, Haemophilus identification agar quad by Remel, API NH by bioMérieux, and Haemophilus identification card by bioMérieux) available. Results of a comparison of the above methods have shown that porphyrin-based assays are the most reliable means of identifying H. influenzae (162). Antimicrobial Resistance and Susceptibility Testing Since 30 to 40% of Haemophilus strains in the United States are resistant to ampicillin by -lactamase production (32, 76), all respiratory isolates of Haemophilus should be tested for -lactamase with a simple method such as the chromogenic cephalosporin-based test, e.g., nitrocefin disks. Typically, -lactamase-negative isolates are susceptible to ampicillin and other -lactam agents and -lactamase-positive isolates are susceptible to -lactam-inhibitor combinations. There are exceptions as outlined below. Of concern are the -lactamase-negative ampicillin-resistant (BLNAR) strains that have been isolated at low frequencies in the United States (0.9% of all isolates) (76) but at higher rates in other parts of the world. Surveillance studies performed in Spain and Japan reported isolation rates of 9.3 and 3.0%, respectively (146, 168). These strains are resistant (MIC for these strains, 4 g/ml) or intermediately resistant (MIC for these strains, 2 g/ml) to ampicillin, and MICs of amoxicillin-clavulanic acid and expanded- and broad-spectrum cephalosporins for these strains are increased (73, 168). CLSI recommendations (51) are that these strains should be considered resistant to -lactam-inhibitor combinations and to expanded-spectrum cephalosporins. BLNAR strains are susceptible to parenteral and oral broadspectrum cephalosporins (73, 168). There is currently no standardized testing procedure or universal definition for BLNAR strains (21). At this time, CLSI broth or agar dilution methods with Haemophilus test medium are preferred for testing of ampicillin against Haemophilus (21). Although all -lactamase-positive strains of Haemophilus have been considered susceptible to -lactam-inhibitor combinations and cephalosporins, there have been reports of strains resistant to amoxicillin-clavulanic acid (73, 75, 102, 146, 168). These strains are rare (0.1 to 1.1%) and remain susceptible to broad-spectrum cephalosporins and quinolones. Additional susceptibility testing to detect the resistance mechanisms described above is not recommended for routine use at this time since the frequency of these resistance mechanisms is low and the clinical implication is poorly understood. In addition, these strains still remain susceptible to antibiotics frequently used to treat CF patients, such as
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cephalosporins and quinolones. More comprehensive susceptibility testing may be considered in cases where a patient demonstrates predominant growth of Haemophilus in repeated cultures despite appropriate therapy.
ANAEROBES Clinical Significance Anaerobic organisms do not appear to play an important role in pulmonary infections in CF patients. Aspiration of oral secretions, leading to anaerobic pneumonia and pulmonary abscess, is rarely seen in this population (134). Anaerobic cultures of CF lung tissue obtained by thoracotomy have revealed few patients with anaerobic colonization of the lower respiratory tract (228). In addition, patients found to have an anaerobic organism(s) by culture were also colonized with P. aeruginosa, which suggested a minor, if any, role for anaerobes in this setting. In a study by Jewes and Spencer (112), anaerobes were isolated by semiquantitative culture of sputa from approximately 24% of CF patients. Anaerobes were isolated just as frequently from patients with and without pulmonary exacerbations, which suggested a colonizing, rather than a pathogenic, role for these organisms. Occasionally, putrid sputa from CF patients have been noted. In this scenario and in cases of suspected aspiration, diagnostic measures and therapy for anaerobes should be considered. Detection Bronchoscopically obtained specimens (tissue and bronchial brush specimens) are the only appropriate respiratory samples for anaerobic culture. Tissue samples should be kept moist and transported to the laboratory immediately. Alternatively, depending on the size of the tissue, it may be placed in an appropriate anaerobic transport system. Bronchial brush specimens may be placed in a sterile vial containing 1 ml of prereduced tryptic soy or chopped meat broth. There is some evidence that bronchoalveolar lavage samples may be useful in the diagnosis of anaerobic lung infections (99). If the laboratory chooses to accept bronchoalveolar lavage fluid for this purpose, the specimen should be transported immediately in a plugged, air-free syringe and processed within 10 min of receipt (99). Even though bronchoscopic specimens are less likely to be contaminated with oral anaerobic flora than are sputum specimens, they are by no means sterile. Therefore, quantitative culture is recommended to distinguish organisms in significant numbers which correlate with the presence of pneumonia (22). The minimal culture setup for anaerobes should include a nonselective anaerobic blood agar, a selec-
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tive anaerobic blood agar with kanamycin and vancomycin, and an anaerobic phenylethyl alcohol agar plate that will suppress the growth of facultative gram-negative rods and swarming Clostridium spp. Culture plates can be examined after 48 h of incubation. For negative cultures, a total incubation period of 7 days is acceptable. If the presence of Actinomyces spp. is suspected, the culture plates should be held for 14 days. A more detailed description of anaerobic bacteriology is found in the WadsworthKLT Anaerobic Bacteriology Manual (119). Identification Many anaerobic isolates can be presumptively identified with a few simple tests (20, 119). A costeffective approach using colonial and cell morphology and bile susceptibility and spot indole testing has been published (165). Definitive identifications can be accomplished with commercially available kits, prereduced anaerobically sterilized biochemical tests, and metabolic end-product analysis with gas-liquid chromatography. Presumptive identifications are appropriate in the setting of anaerobic pneumonia since multiple species may be isolated. Antimicrobial Resistance and Susceptibility Testing There are a number of effective antibiotics for the treatment of anaerobic infections, such as -lactams, metronidazole, carbapenems, and -lactam-inhibitor combinations (6), but resistance to virtually all of them has been detected (11, 158, 184, 203, 215). Methods to determine MICs for anaerobic isolates include agar dilution, broth microdilution, and the E test (119, 193). The specifics of anaerobic susceptibility testing are described in the NCCLS document M11-A6 (166) and the Wadsworth-KLT Anaerobic Bacteriology Manual (119).
ENTEROBACTERIACEAE Clinical Significance Enterobacteriaceae are not chronic colonizers of CF airways and appear to play only a minor role in CF lung infection (35, 105). These organisms are found in approximately 1 to 4% of CF patients and are associated with 10% of acute respiratory exacerbations (23, 106). They are isolated more commonly in children 0 to 5 years of age, before chronic colonization with P. aeruginosa is established (105). Detection All Enterobacteriaceae grow well on MacConkey agar, so special selective media are not required, although the presence of these organisms may be
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obscured by mucoid P. aeruginosa. If they are detected in cultures of bronchoscopic specimens or as predominant organisms in sputum cultures, all isolates should receive complete identification and susceptibility testing. Identification Escherichia coli is the most common member of the Enterobacteriaceae isolated from CF patients, followed by Proteus spp., Klebsiella spp., and Citrobacter spp. (23, 105, 106). Enterobacter spp., Providencia spp., Morganella spp., and others are seen less frequently. In one study, highly mucoid strains of E. coli were reported to be present in the sputa of 12% of CF patients (142). E. coli and Proteus spp. can be identified on the basis of morphology and spot testing (165). Identification of other members of the Enterobacteriaceae is typically done using a commercial identification system. Antimicrobial Resistance and Susceptibility Testing Susceptibility testing should be done routinely for all clinically significant Enterobacteriaceae from CF patients. The susceptibility patterns may be unpredictable because of the multiple courses of antibiotics given to these patients. Testing should include a method for detecting extended-spectrum -lactamase (ESBL) production in isolates of E. coli and Klebsiella spp. A recent survey indicates that approximately 5% of the CF isolates harbor ESBLs (175). Two cases of lung colonization with ESBL-producing Enterobacteriaceae strains in CF patients have been reported previously (39).
STREPTOCOCCUS PNEUMONIAE Clinical Significance A recent study has shown a Streptococcus pneumoniae incidence of 5.5% in CF patients (67). Like that of Haemophilus, the isolation rate is highest in children 10 years of age (67, 105). Infection or colonization is generally transient, but the organism has been associated with exacerbations (23, 67, 106). Most patients are infected with unique clones, but in a Spanish CF population, a widely dispersed multidrug-resistant Spanish clone of S. pneumoniae 23F was recovered from multiple CF patients (67). Recommendations for administration of Prevnar, the 7valent conjugate pneumococcal vaccine, are applicable to infants with CF. The vaccine has reduced the number of invasive pneumococcal infections in infants and toddlers overall, although there are no specific data relative to CF patients.
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Detection S. pneumoniae grows well on commonly used media including sheep blood agar, chocolate agar, horse blood agar, and sheep blood agar containing colistin and nalidixic acid. Although S. pneumoniae may be obscured by the overgrowth of P. aeruginosa on nonselective media, the routine use of sheep blood agar containing colistin and nalidixic acid as a selective medium for S. pneumoniae has been shown to have little clinical value (74). Identification Identification of S. pneumoniae is based upon Gram stain morphology, colonial morphology and hemolysis, and an additional confirmatory test, e.g., a bile solubility, optochin susceptibility, latex agglutination, or DNA probe test. The DNA probe test is considered a “gold standard” but can be expensive for routine identification, considering the number of organisms that would have to be tested on a daily basis (69). There are reports of bile-insoluble or optochin-resistant isolates of S. pneumoniae, but these are typically infrequent occurrences. For the most part, the bile solubility and optochin susceptibility tests are accurate tests for identifying S. pneumoniae. There are some reports of bile solubility testing’s being less reliable than optochin susceptibility testing, particularly if the sodium deoxycholate is applied directly to colonies on an agar plate (drop test) or if the test is applied to isolates that lack the characteristic morphology of S. pneumoniae (41, 161). However, one study reported excellent sensitivity and specificity for the drop test, comparable to those of optochin susceptibility testing. Common sense would dictate that a second confirmatory test should be done if a bile-insoluble or optochin-resistant organism with the colonial and Gram stain morphologies of S. pneumoniae is encountered. The latex agglutination tests for S. pneumoniae antigen have variable specificities since many strains of nonpneumococcal alpha-hemolytic streptococcal species share capsular antigens similar to those of S. pneumoniae (123). When applied to colonies with typical S. pneumoniae morphology, the sensitivity and specificity of the latex test are high (41, 123). Antimicrobial Resistance and Susceptibility Testing All respiratory isolates of S. pneumoniae should be tested for penicillin resistance. This can be done using a 1-g oxacillin disk or a penicillin MIC method, such as the Etest. Isolates resistant by the oxacillin disk method should be reported as presumptively resistant to penicillin, with MIC confirmation to follow. Oxacillin-sensitive isolates are con-
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sidered susceptible to those -lactam agents approved for treatment of pneumococcal infections. Approximately 39% of S. pneumoniae strains in the United States possess some level of resistance to penicillin, with about 26% displaying high-level resistance (MIC for these strains, 1 g/ml) (76). In a recently described S. pneumoniae-positive CF population in Spain, the rate of pencillin resistance was even higher, with 73% of strains showing some level of resistance and MICs for 46% being 2 g/ml. These strains were frequently multidrug resistant as well (67). Since these strains are often resistant to other oral antimicrobial agents such as erythromycin and trimethoprim-sulfamethoxazole, these agents should be tested routinely on all penicillin-resistant respiratory isolates and for patients with -lactam allergy.
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colonized (17, 50). In addition, not all patients atopic for Aspergillus and colonized with this organism will develop ABPA and some nonatopic CF individuals will (220). Obviously, other factors involved in the development of ABPA have not been determined. Scedosporium apiospermum has been recovered with some frequency from CF patients. In one study, this organism was recovered from 8.6% of CF patients (49). This filamentous fungus can often be found in conjunction with Aspergillus. Both share common antigens, some of which are likely to be allergens in susceptible CF individuals (49). One study suggests that Scedosporium apiospermum can cause a syndrome similar to ABPA and will respond to the same treatment (oral corticosteroids, voriconazole, or itraconazole) used to treat ABPA (49). Epidemiology
ASPERGILLUS SPP. AND OTHER FUNGI Clinical Significance The most common fungi recovered from CF patients are Aspergillus fumigatus and Candida albicans (17, 49). Although the vast majority of laboratories will culture sputum samples and report the presence of both (211), their clinical significance is quite different. C. albicans is typically viewed as a harmless saprophyte in the respiratory tracts of CF patients (84). A. fumigatus is the primary fungus associated with allergic bronchopulmonary disease (220). Other Aspergillus spp. may be recovered from CF patients but are seen infrequently. Allergic bronchopulmonary aspergillosis (ABPA) occurs in 1 to 15% of CF patients, with the Cystic Fibrosis Foundation registry reporting the prevalence at about 2% (222). The large variation in ABPA prevalence is not surprising given the lack of consensus on a case definition since clinical (presence of wheezing) and radiographic (bronchiectasis and pulmonary infiltrates) findings are similar to those seen in the natural progression of chronic bacterial lung infection in this population. The pathogenesis of ABPA is due to allergic reactions to Aspergillus growing in the CF airways. The characteristic thickened airway mucus is believed to trap the inhaled spores, with resulting hyphal growth of the organism at the epithelial cell surface. Aspergillus produces a protease which may damage the lung epithelium and trigger the production of proinflammatory cytokines and a chronic allergic inflammatory response (220). ABPA is more common in individuals who are atopic for Aspergillus, suggesting that a preexisting allergy to Aspergillus is important in disease pathogenesis. The pathogenesis of ABPA is not well defined, so it is not understood why only a small percentage of CF patients develop ABPA when approximately 50% of CF patients are
Both Aspergillus and Scedosporium are soil organisms transmitted to susceptible people by inhalation of the organisms’ spores and hyphal fragments (49, 220). Molecular epidemiology studies suggest that person-to-person spread of fungi does not have clinical relevance (50, 66). Patients may be colonized with a variety of genotypes of Aspergillus, with one genotype often predominating (50). Detection Routine culture for fungi is not recommended for CF patients, although the organisms may grow on bacterial isolation media (151). When fungal isolation is attempted, it is recommended that antibacterial agents with antipseudomonal activity be incorporated into the medium (17). The diagnosis of ABPA is based on clinical and immunologic findings, not on culture results. A recent consensus document on ABPA in CF patients has listed the following criteria (220): 1. Clinical signs include acute or subacute deterioration which is not due to another etiology. Relevant clinical signs include wheezing, cough, increases in sputum production, change in pulmonary function, and decreased exercise tolerance or exerciseinduced asthma. 2. Relevant laboratory findings include a serum immunoglobulin E (IgE) concentration of 500 IU/ml and the presence of IgE antibodies to A. fumigatus and/or IgG antibodies to A. fumigatus. 3. The presence of an immediate reaction to a skin test with Aspergillus can be used in lieu of IgEspecific antibodies to A. fumigatus. 4. Radiographic changes show infiltrates, mucus plugging, or bronchiectasis (demonstrated by chest computed tomography) which has not improved with antibacterials and chest physiotherapy.
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VIRUSES Clinical Significance Approximately 6 to 30% of acute pulmonary exacerbations in CF patients are associated with serological or cultural evidence of a viral infection (172, 174, 179). As in non-CF patients, the incidence of viral infections is highest in children. CF patients do not appear to be more prone to developing respiratory viral infections than the normal population, but the subsequent effects on pulmonary function are more severe (101, 172, 214, 236). Infants (2 years old) with CF are at significant risk for development of lower respiratory tract infection, for hospitalization, and for deterioration in lung function as a result of a respiratory viral infection (101). This is particularly true for infections with influenza A virus or respiratory syncytial virus (RSV), where significant morbidity is seen (4, 61, 179). Because RSV infection tends to occur early in life, this virus is postulated to play a role in the initiation and early progression of lung damage in CF patients (4). There is some suggestion of synergistic pulmonary deterioration in patients with chronic P. aeruginosa colonization and concurrent viral infection (174, 221). There is also an association of early acquisition of P. aeruginosa in infants hospitalized for respiratory viral infections (14). One study reported prevalent adenovirus infections in CF patients and suggested an association with lung function deterioration in patients who demonstrated seroconversion (194). Severe pulmonary exacerbations have been observed in CF patients with primary Epstein-Barr virus (EBV) infection and chronic P. aeruginosa colonization (245). Conversely, infection with rhinovirus, which is relatively common in CF patients, has not been associated with a decline in clinical status (214). With the above-mentioned exceptions, the role of respiratory viruses in CF pulmonary disease is poorly understood. Detection A number of studies have reported difficulty in isolating viruses from CF respiratory samples even when serological results indicate an acute viral infection (77, 101, 179, 181, 214, 236). In some instances, this can be attributed to improper or prolonged specimen transport and storage conditions, but these do not explain all of the unsuccessful isolation attempts. The problem may be related to the thick, mucoid respiratory secretions of CF patients or to an unidentified inhibitory factor(s) in these secretions. Whatever the case may be, rapid, nonculture techniques are needed to adequately detect viruses in these patients. In this regard, a number of assays are commercially available or in development, including nucleic acid amplication, antigen detection, and direct immuno-
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fluorescence assays. Rapid immunofluorescence and antigen detection tests are commercially available for RSV and influenza virus, the two viruses of main concern for CF patients. These tests are performed only on a seasonal basis in temperate regions, usually from November through April. Commercial immunofluorescence tests are also available for adenoviruses (216). Nasopharyngeal washes or aspirates are the preferred specimens for respiratory virus culture and antigen testing. Nasopharyngeal and throat swabs are often submitted for viral testing but are considered suboptimal specimens because of the insufficient numbers of mucosal cells that are collected by swabbing. In some laboratories, washes have not been as successful as nasal and throat swabs for the immunofluorescent-antigen assays because mucus, squamous cells, and blood cells may obscure the reactions (213). Others have found the washes to be very successfully used. Bronchoalveolar lavage and transbronchial biopsy samples are useful specimens for diagnosing lower respiratory tract infections. Specimens should be collected within 24 to 48 h of symptom onset for best diagnostic yield. Specimens should be placed in an appropriate viral transport medium and transported immediately on wet ice to the laboratory (213). Appropriate cell lines should be inoculated to ensure the isolation of the viral pathogens most common in CF patients, i.e., influenza A and B viruses, parainfluenza viruses 1 through 3, RSV, and adenovirus (174). Useful cell lines include primary rhesus or cynomologous monkey kidney, continuous rhesus monkey kidney, HEp-2, MRC-5, mink lung, Madin-Darby canine kidney, and A549 cells. Most positive cultures are detected within 1 week, but negative cultures should be held for 2 weeks. Isolation of many of the respiratory viruses has been adapted to the shell vial method which can detect virus 24 to 48 h postinoculation. Identification Identification of virus isolates typically relies on the cytopathic effect, hemadsorption studies, and immunofluorescence staining (216, 251). Some laboratories also use molecular techniques. For a detailed discussion of virus identification, see reference 216.
LABORATORY ASPECTS OF LUNG TRANSPLANTATION IN CF LUNG DISEASE Over the past decade, lung transplantation has become a life-saving intervention for CF patients with cardiopulmonary failure. It is estimated that approximately 4% of CF patients received lung transplantation between 1991 and 2001 (137). Complications of lung transplantation are the second leading cause of
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death in CF patients after bronchiolitis obliterans syndrome. In the first year posttransplantation, infection is the leading cause of death. Organisms frequently associated with posttransplant infections include cytomegalovirus (CMV), Aspergillus, B. cepacia, and M. abscessus (12, 25, 72, 78, 98, 122, 206). Studies have shown that CF patients already infected with B. cepacia before their transplant will have much shorter survival times than CF patients infected with other organisms (12, 43, 137). Because the same clone infecting patients during the pretransplant period is found to infect them posttransplant (218), infection with the B. cepacia complex has become a contraindication for lung transplantation at the majority of North American transplant centers (135). Death of B. cepacia-positive recipients in the immediate posttransplant period is typically due to B. cepacia sepsis and wound infection. Data suggest that outcome is worse with B. cenocepacia than with the other species in the B. cepacia complex, but the numbers are still quite small (12). However, these data emphasize the need for determining the specific species of the B. cepacia complex with which a transplant recipient is infected in order to understand the comparative virulence levels of these organisms posttransplant. CMV is a cause of serious morbidity and mortality in CF patients who have undergone lung transplantation (13, 25, 78, 253). CMV prophylaxis and monitoring of patients for the presence of CMV are important parts of posttransplant management (135, 253). CMV disease typically appears between 1 and 3 months posttransplant. The most common clinical presentation is a febrile mononucleosis or leukopenia, but some patients may have more extensive organ involvement including interstitial pneumonitis, hepatitis, and/or gastrointestinal disease (62). Often CMV disease cannot be distinguished clinically from organ rejection. Because these two conditions are treated very differently, rapid, sensitive, and specific tests are needed to diagnose CMV disease. Transplant patients can chronically shed CMV in urine and saliva; therefore, cultures of these specimens are nonspecific in terms of diagnosing or predicting symptomatic CMV disease. Alternatively, blood cultures (using buffy coats) are a more specific indicator of disease. Although CMV isolation may be useful for antiviral susceptibility testing, many laboratories are using non-culture-based detection assays for CMV. Direct detection of CMV in blood by antigenemia, hybrid capture, and PCR amplification methods has increased the sensitivity, positive predictive value, and turnaround time for diagnosing CMV disease in immunosuppressed patients (204). CMV antigenemia and quantitative CMV PCR are the main methods used to monitor lung transplant recipients (135). These tests are able to generate quan-
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titative results in terms of the number of infected cells per slide or the amount of DNA (picograms or copies per milliliter) in blood. In many instances, these tests are positive before the onset of symptoms, predicting which patients will develop CMV disease. They can be used to institute early antiviral therapy and to monitor therapy. Quantitative thresholds for determining patients at high risk for symptomatic infection vary between assays and among different transplant populations. In general, a high viral load correlates with the development of symptomatic disease. In some cases, a low viral load that rapidly increases also carries with it a significant risk of disease. Most transplant centers monitor viral loads weekly, with retesting of low-positive patients within 2 to 4 days to detect rapidly rising levels. The antigenemia assay detects the viral protein pp65 expressed in blood leukocytes during the early phase of CMV replication, and the number of pp65positive cells has been shown to correlate with disease. Specimens for this assay must be processed within 8 h of collection, and any delay will decrease the number of detectable pp65-positive cells in the blood specimen. The test is not standardized, so results from different laboratories may be difficult to compare. In addition, only cell-associated virus is detected and free viruses in the blood will not be detected (230). Quantitative amplification assays differ with respect to the nucleic acid targets detected and the methods used for target amplification. CMV can be amplified from both cellular and acellular fractions of blood, although the level of CMV DNA is higher in leukocytes than in plasma. The results of these assays are typically available the same day of specimen receipt. The reader is referred to several excellent reviews on the current methods available for quantitation of CMV (28, 94, 212). Posttransplant lymphoproliferative disease due to EBV is a major cause of morbidity and mortality in pediatric CF lung transplant recipients. It occurred in 21% of patients with 45% mortality in one series (59). Monitoring for EBV by quantitative PCR is performed in approximately one-third of CF lung transplant programs. In addition, prophylaxis with oral acyclovir and ganciclovir may be used in recipients who are EBV seronegative and receive organs from EBV-positive donors (135). Aspergillus fumigatus is frequently found in the airways of CF patients both pre- and posttransplant. It is the most common cause of post-lung transplant fungal infection. It can cause invasive pulmonary infections complicated by pulmonary hemorrhage, tracheobronchial aspergillosis near the site of anastomosis, and wound dehiscence. Since infections can be associated with fatal outcomes (98), posttransplant prophylactic antifungal therapy with either
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aerosolized amphotericin or itraconazole and fluconazole is frequently used (135). Galactomannan monitoring for detection of invasive aspergillosis in CF patients post-lung transplant appears to have little utility. The sensitivity of the assay is low (30%) in lung transplant recipients with invasive aspergillosis and is negative in those with tracheobronchial aspergillosis. In addition, false-positive results were frequently reported from CF patients during the first 2 weeks posttransplant (110). M. abscessus is a pathogen that can be refractory to treatment and cause fatal outcomes in CF transplant recipients. Infections typically occur in the early posttransplant period in patients who have been infected pretransplant (98, 125, 206). Although a retrospective review of mycobacterial infections in transplant recipients showed that nontuberculous infections were rare (125), 2% of CF patients are colonized or infected with M. abscessus (170). Given the poor prognosis, the prevalence of M. abscessus in CF patients, and the prolonged therapy required, it may be prudent to screen for the organism and treat patients during the pretransplant period.
PRACTICAL GUIDE TO THE ROLE OF THE CLINICAL MICROBIOLOGY LABORATORY IN THE MANAGEMENT OF CF PATIENTS The life expectancy of CF patients has shown steady improvement, in large part due to improved management of their chronic infections (151). Part of this improvement can be attributed to improved recogni-
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tion of pathogens and susceptibility testing of these organisms. In this section, practical issues will be discussed for each stage of laboratory testing of respiratory specimens obtained from CF patients. Preanalytical Stage (Specimen Selection, Collection, and Transport) Expectorated sputum specimens are the specimens of choice for attempting to determine the microbial flora associated with CF lung disease. These specimens are easily obtained and correlate reasonably well with bronchoscopically obtained specimens (84, 228). However, CF patients younger than 8 years of age often cannot produce sputum. In those patients, there are two options. One is a throat swab obtained by placing the swab in the posterior pharynx and having the child gag or cough on the swab. The other is a bronchoscopically obtained specimen, typically a bronchoalveolar lavage specimen. Bronchoalveolar lavage specimens are considered to be the gold standard for CF bacteriology (195). Respiratory specimens for virus detection should be sent to the laboratory on wet ice and kept at 4°C until processed (213). The frequency with which bacterial, fungal, and mycobacterial cultures should be obtained from CF patients has not been defined. It is clear that for many infectious agents, chronic infection is the rule. Because of the expense involved in processing specimens, it is incumbent on the microbiologist or pathologist to work closely with their CF clinician colleagues to determine the best approach at their institution. Certainly a minimum of an annual cul-
FIGURE 1. Culture of CF sputum specimen from which P. aeruginosa is detected on MacConkey agar (left) and B. cepacia is detected on BCSA (right). Reprinted from reference 151.
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ture for these agents should be the norm, and more frequent cultures should be done as the patient’s clinical condition dictates. Regardless of the specimen type obtained, good microbiology practices are important. The specimen must be properly labeled and efficiently transported. With the trend toward consolidation of microbiology services, specimens are often sent significant distances under various environmental conditions. If plating cannot be performed at the site of specimen collection, specimens should be transported at 4°C. P. aeruginosa survives at this temperature for 24 h, and most of the other pathogens of CF patients will survive at this temperature for a minimum of 1 to 2 h. Analytical Stage Sputum specimens for bacterial culture are often screened to determine if they are heavily contaminated with upper respiratory flora from saliva, as evidenced by the presence of high numbers of squamous epithelial cells. Although such contamination is a common occurrence in CF patients (41% of cases in one study), all these specimens typically contain purulent material that grows gram-negative bacilli such as P. aeruginosa. Laboratorians should also consider that patients may travel significant distances (100 miles) for an outpatient visit at a CF clinic. These patients may have already begun their journey home by the time the specimen is evaluated, making collection of a second, better specimen impractical. For all these reasons, it is recommended that Gram stain examinations for specimen quality should not be the basis of rejection of sputum specimens from CF patients (163). Bacteriology Culture Processing Although there are a limited number of organisms that need to be detected in CF patients, the high prevalence of mucoid P. aeruginosa, which may obscure colonies of other organisms, makes it necessary to use a variety of selective media to isolate specific organisms. CF specimens should be plated onto media that will support the specific isolation of S. aureus, H. influenzae, P. aeruginosa, and the B. cepacia complex (Fig. 1). Recommendations for the types of media to use to accomplish this isolation are given in Table 4. Plates should be held for 3 to 5 days, depending upon the specific organism. Bronchoalveolar lavage specimens should be cultured quantitatively on both selective and nonselective media by using dilutions that will allow the detection of between 103 and 107 CFU/ml (195). The decision of which organisms will be identified and undergo antimicrobial susceptibility testing will vary from institution to institution (211). Most laboratories will identify and report any
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amount of P. aeruginosa, S. aureus, B. cepacia complex, H. influenzae, and S. maltophilia organisms from sputum specimens. S. pneumoniae and Enterobacteriaceae should probably be identified and have susceptibility testing done only if they are predominant. Although the importance of molecular methods for the identification of non-glucose-fermenting rods has been emphasized, only 5% of non-glucose fermenters could not be adequately identified using conventional methods (79). This underscores the importance of “traditional” laboratory practices in the performance of these cultures. CF samples are among the most labor-intensive specimens submitted to the clinical microbiology laboratory. As such, the laboratorian and clinician should agree on a cost-effective approach to the workup of these specimens. Since CF patients are chronically infected and have the same bacteria isolated repeatedly, susceptibility testing is not necessary for each culture. At the Cystic Fibrosis Center of the UNC Hospitals, non-glucose fermenters are identified and have susceptibility testing performed no more frequently than every 3 months unless the patient is admitted. At the time of admission, the patient isolate will be identified and susceptibility testing will be performed. Repeat cultures during the hospital course are not encouraged unless there is a major clinical complication. Patients with MRSA have susceptibility testing done annually once infection is established (151). The techniques to determine susceptibilities of common bacterial agents in CF patients are listed in Table 4. Culture results from throat swab specimens may have a different significance from those from sputum specimens. Throat swabs are reasonably specific to use for the detection of the presence of CF pathogens in the lower airways. However, these specimens lack sensitivity (50%) for P. aeruginosa in young children so the absence of these organisms from throat swabs does not mean that they are absent from the lower airways (195). The bacterial load in broncoalveolar lavage specimens which is significant and necessitates identification and susceptibility testing has not been well defined. For some organisms, such as P. aeruginosa, the B. cepacia complex, and S. aureus, any number of organisms in a bronchoalveolar lavage specimen is probably significant. With quantitative cultures, it is unclear whether to use 103, 104, or 105 CFU/ml as the breakpoint for identification and susceptibility testing of different isolates in bronchoalveolar lavage specimens. The lower the breakpoint for the workup, the higher the sensitivity and the lower the specificity. The reverse is true for the high breakpoint: lower sensitivity, higher specificity. Since studies show that median counts for S. aureus, H. influenzae, and P.
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Table 4. Practical guide to isolation, identification, and susceptibility testing of organisms frequently recovered from CF respiratory secretions Identification method(s)
Isolation mediuma
Incubation conditions
Horse blood or chocoate agar with or without 300 g of bacitracin MSA or CSA
35–37°C, 72 h, 5–10% CO2
Conventional
-Lactamase
35–37°C, 72 h, ambient air
Conventional
Pseudomonas aeruginosa
MacConkey or EMB
35–37°C, 72 h, ambient air
Conventional
Burkholderia cepacia
BCSA
Molecular
Stenotrophomonas maltophilia Achromobacter xylosoxidans Burkholderia gladioli, Ralstonia spp., Pandoraea spp., Inquilinus spp. Streptococcus pneumoniae
MacConkey or EMB
35–37°C, 96–120 h, ambient air 35–37°C, 72 h, ambient air 35–37°C, 72 h, ambient air 35–37°C, 96–120 h, ambient air
Oxacillin screening plate, cefoxitin disk screen, latex agglutination for PBP2a Disk diffusion, Etest, no automated susceptibility systems Disk diffusion, Etest, broth microdilution MIC Disk diffusion, Etest, broth microdilution MIC Broth microdilution MIC
Organism(s) Haemophilus influenza
Staphylococcus aureus
MacConkey or EMB BCSA, MacConkey, EMB
Conventional Conventional, molecular Molecular
35–37°C, 3 days, 5% CO2
Conventional
Enterobacteriaceae
Colistin-nalixidic acid agar, horse blood or chocolate agar MacConkey or EMB
35–37°C, 72 h, ambient air
Conventional
Nontuberculous Mycobacterium spp.
7H9 broth, LöwensteinJensen
37°C, 6 wks (broth), 8 wks (slant), ambient air
Aspergillus spp.
Fungal media containing gentamicina
30°C, 3 wks, ambient air
Conventional for rapid growers; sequence analysis, DNA probe for MAI Conventional
Susceptibility test(s)
Broth microdilution MIC
Oxacillin screen, Etest, or broth microdilution MIC Disk diffusion, automated susceptibility systems, broth microdilution MIC Broth microdilution MIC for rapid growers
Not applicable
a
Aspergillus frequently grows on primary bacterial isolation media including BCSA, MSA, and horse blood agar.
aeruginosa are all at least 104 CFU/ml (195), this number may be a reasonable breakpoint to ensure adequate sensitivity and specificity. Synergy testing of antibiotics for P. aeruginosa and the B. cepacia complex has been discussed above. Most laboratories report, identify, and perform susceptibility testing for the other bacteria, such as S. pneumoniae and enterics, only if they are predominant. It is unknown how frequently laboratories should identify and perform susceptibility testing on Ralstonia spp., B. gladioli, Pandoraea spp., Inquilinus, and A. xylosoxidans. Mycobacteriology Culture Processing All respiratory specimens from CF patients that are cultured for mycobacteria should have direct smears done, not to detect specimen quality but to examine for organisms. Positive smears indicate high organism burdens. In patients with M. abscessus, a positive smear may suggest active infection rather than colo-
nization (7). CF respiratory specimens cultured for mycobacteria need extensive decontamination to prevent P. aeruginosa overgrowth. Although M. tuberculosis is rare in CF patients (151), NTM can be recovered from 13% of CF patients (170). Standard approaches to identification and susceptibility testing are recommended. Fungal Culture Processing The major fungal pathogen in CF patients is Aspergillus. It can grow on several of the selective bacterial media (151). Aspergillus can chronically infect or colonize CF patients. When Aspergillus is first detected in a bacterial culture, it should be identified to the species level and reported. After the initial isolation, the frequency of identification and reporting should be based on clinician expectations and needs. When fungal cultures are specifically requested for CF patients, selective fungal media are required because of the potential for bacterial over-
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growth, especially with P. aeruginosa. Selective fungal media containing gentamicin, amikacin, or ciprofloxacin should be used because of their activity against strains of P. aeruginosa and because media containing these antimicrobials will enhance the recovery of molds from CF respiratory specimens (17). Although Candida species are frequently recovered from CF respiratory specimens (17), there is no evidence that these organisms play a role in chronic CF lung disease (84). Therefore, Candida should not be reported for respiratory cultures from CF patients. Viral Specimen Processing There are few data on how best to process specimens from CF patients to detect viruses directly by antigen testing, direct fluorescent-antibody testing, or culture. Molecular detection of respiratory viruses holds great promise for CF patients. However, studies are needed to determine the best means for recovery of viral nucleic acids from the complex milieu of CF respiratory secretions. Postanalytical Stage There are two major issues, taxonomy and reporting of synergy studies, which the microbiology laboratory must address when they report results for CF patients. As more and more genera of bacteria are found in CF respiratory secretions, it is incumbent upon the microbiologist to be prepared to explain the significance of the new taxa. Currently, little is known about many of the new taxa (Table 1). When organisms are reclassified to a new genus, it is important to continue to report the old genus in parentheses with the new genus name, e.g., Stenotrophomonas (Xanthomonas) maltophilia, for some period (at least 1 year) so that clinicians can become familiar with the new taxonomy. Reports of synergy testing results can be challenging, especially for MCBT when double and triple combinations are used and 298 drug combinations are tested (45). Microbiologists and clinicians must configure these reports to make the information understandable and accurate, which can prove to be a major task. For testing performed at the synergy testing reference laboratory at Columbia University (website, http://synergy.columbia.edu), clinicians should be encouraged to obtain a login code so that they can review results for their patients directly online.
FINAL THOUGHTS Over the past 25 years, the microbiology of lung disease in CF patients has become increasingly complex. At the same time, the life expectancy of CF patients has increased markedly but certainly not as much as the CF patients and their families have hoped. Clini-
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cal microbiologists are key members of the health care team that has fought resolutely on behalf of these patients. We have made important contributions to the patients’ management by recognizing emerging pathogens, developing new diagnostic approaches for detecting pathogens, studying the pathogens’ epidemiology, recognizing emerging drug resistance problems, and developing novel susceptibility test methods to determine antimicrobial combinations effective against multidrug-resistant organisms. As clinical microbiologists, we must continue to dedicate ourselves to improving our understanding of the microorganisms that are responsible for so much suffering among individuals with CF. REFERENCES 1. Aaron, S. D., W. Ferris, D. A. Henry, D. P. Henry, D. P. Speert, and N. E. MacDonald. 2000. Multiple combination bactericidal antibiotic testing for patients with cystic fibrosis infected with Burkholderia cepacia. Am. J. Respir. Crit. Care Med. 161:1206–1212. 2. Aaron, S. D., K. Ramotar, W. Ferris, K. Vandemheen, R. Saginur, E. Tullis, D. Hassee, D. Kottachchi, M. St. Dennis, and F. Chan. 2004. Adult cystic fibrosis exacerbation and new strains of Pseudomonas aeruginosa. Am. J. Respir. Crit. Care Med. 169:811–815. 3. Aaron, S. D., K. L. Vandermheen, W. Ferris, D. Fergusson, E. Tullis, D. Haase, Y. Berhiaume, N. Brown, P. Wilcox, V. Yozghatlian, P. Bye, S. Bell, F. Chan, B. Rose, A. Jeanneret, A. Stephenson, M. Noseworthy, A. Freitag, N. Paterson, S. Doucette, C. Harbour, M. Ruel, and N. MacDonald. 2005. Combination antibiotic susceptibility testing to treat exacerbation of cystic fibrosis associated with multiresistant bacteria: a ramdomised, double-blind, controlled clinical trial. Lancet 366:463–471. 4. Abman, S. H., J. W. Ogle, N. Butler-Simon, C. M. Rumack, and F. J. Accurso. 1988. Role of respiratory syncytial virus in early hospitalizations for respiratory distress of young infants with cystic fibrosis. J. Pediat. 113:826–830. 5. Al-Aloul, M., J. Crawley, C. Winstanley, C. A. Hart, and M. J. Walshaw. 2004. Increased morbidity associated with chronic infection by epidemic Pseudomonas aeruginosa strain in CF patients. Thorax 59:334–336. 6. Alridge, K. E., D. Ashcraft, K. Cambre, C. L. Pierson, S. G. Jenkins, and J. E. Rosenblatt. 2001. Multicenter survey of the changing in vitro antimicrobial susceptibilities of clinical isolates of Bacteroides fragilis group, Prevotella, Fusobacterium, Porphyromonas, and Peptostreptococcus species. Antimicrob Agents Chemother. 45:1238–1243. 7. American Thoracic Society. 1997. Diagnosis and treatment of disease caused by nontuberculous mycobacteria. Am. J. Respir. Crit. Care Med. 156:S1–S25. 8. Andersen, D. H. 1938. Cystic fibrosis of the pancreas and its relation to celiac disease: a clinical and pathologic study. Am. J. Dis. Child. 56:344–399.
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