Cumitech 21: Laboratory Diagnosis of Viral Respiratory Disease

Cumitech IA . Blood Cultures II . June 1982 Cumitech 2 . Laboratory Diagnosis of Urinary Tract Infections . April 1975 Cumitech 3 . Practical Quality Control Procedures for the Clinical Microbiology Laboratory . September 1976 Cumitech 4 . Laboratory Diagnosis of Gonorrhea . October 1976 Cumitech 5 . Practical Anaerobic Bacteriology . April 1977 Cumitech 6 . New Developments in Antimicrobial Agents Susceptibility Testing . September 1977 Cumitech 7 . Laboratory Diagnosis of Lower Respiratory Tract Infections . September 1978 Cumitech 8 . Detection of Microbial Antigens by Counterimmunoelectrophoresis . December 1978 Cumitech 9 . Collection and Processing of Bacteriological Specimens . August 1979 Cumitech 10 . Laboratory Diagnosis of Upper Respiratory Tract Infections . December 1979 Cumitech 11 . Practical Methods of Culture and Identification of Fungi in the Clinical Microbiology Laboratory . August 1980 Cumitech 12 . Laboratory Diagnosis of Bacterial Diarrhea . October 1980 Cumitech 13 . Laboratory Diagnosis of Ocular Infections . May 1981 Cumitech 14 . Laboratory Diagnosis of Central Nervous System Infections . January 1982 Cumitech 15 . Laboratory Diagnosis of Viral Infections . March 1982 Cumitech 16 . Laboratory Diagnosis of the Mycobacterioses . March 1983 Cumitech 17 . Laboratory Diagnosis of Female Genital Tract Infections . August 1983 Cumitech 18 . Laboratory Diagnosis of Hepatitis Viruses . January 1984 Cumitech 19 . Laboratory Diagnosis of Chlamydial and Mycoplasmal Infections . August 1984 Cumitech 20 . Therapeutic Drug Monitoring: Antimicrobial Agents . October 1984

Cumitechs should be cited as follows, e.g.: Greenberg, Laboratory diagnosis of viral respiratory disease. Coordinating Society for Microbiology, Washington, DC.

S. B., and L. R. Krilov. 1986. Cumitech 21, ed., W. L. Drew and S. J. Rubin. American

Editorial Board for ASM Cumitechs: Steven Specter, Chairman; Carl Abramson, W. Lawrence Drew, William J. Martone, John E. McGowan, Jr., Josephine Morello, Roy E. Ritts, Jr., Glenn D. Roberts, James W. Smith, John A. Smith, and Alice S. Weissfeld.

The purpose

of the Cumitech

series

is to provide

of-the-art operating procedures for clinical routine or new methods. The procedures given are not proposed

Copyright

consensus

microbiology as “standard”

0 1986

recommendations

laboratories

by the authors may lack

methods.

Amencan Society 1913 I St., N.W

Washrngton,

which

DC 20006

for Mrcrobrology

the facilities

as to appropriate for fully

stateevaluating

LABORATORY STEPHEN Medicine, LEONARD Center,

DIAGNOSIS OF VIRAL DISEASE

B. GREENBERG, Houston, Texas

of

Department 77030

R. KRILOV, Department New Hyde Park, New York

Medicine,

of Pediatrics,

Microbiology

Schneider

SALLY JO RUBIN, Center, Hartford,

DREW,

Mount

Department Connecticut

Zion

and Immunology,

‘S

Hospital,

Long

Baylor

College

Island

Jewish

California

94120

of

Medical

11042 COORDINATING

W. LAWRENCE

Children

RESPIRATORY

Hospital

of Pathology 06105

EDITORS

and Medical

Center,

and Laboratory

In this Cumitech we focus on viral respiratory infections. The first segmentdeals with specimen collection and transportation; the next gives clinical and virologic data for each of the principal clinical respiratory illness syndromes. Although most of these syndromes can be causedby more than one specific virus, certain viruses have a particularly close association with a definite clinical illness (Table 1). For example, rhinoviruses are the most frequent identifiable cause of the common cold, and parainfluenzaviruses are highly associatedwith croup. For simplicity, we present clinical information on all of the syndromesfirst and then the methodsfor detection or isolation of each virus (Table 2). Sincemany of the technical detailsfor identifying or isolating these agents have been published in Cumitech 15 (1l), the reader is referred to that source for further information. There are several reasonswhy viral studies shouldbe performed in patients with respiratory infections (22). Figure 1 showsthe results of “tracking” the activity of severalrespiratory viruses in the San Francisco Bay area. This type of information is extremely valuable in suggestingprobable diagnosesat specifictimes of the year and in helping to anticipate and limit the spreadof nosocomial infections. Such data reinforce the recognition that viruses other than influenza A also have very circumscribed periods of the year during which they are prevalent (19). Although there are few antiviral drugs, several may be beneficialin respiratory disease.Amantadine hydrochloride, which has definite prophylactic efficacy against influenza A, also has sometherapeutic benefit. Ribavirin, a synthetic nucleoside,hasdemonstratedbenefit in respiratory syncytial virus (RSV) infection, but it must be delivered by aerosol with a cumbersome apparatus(32). Although ribavirin may prove to 1

San Francisco,

Medicine,

St. Francis

Hospital

and Medical

be a relatively broad-spectrum antiviral agent, amantadineis active only against influenza A. Recently there has been an increase in the incidence of pharyngitis due to herpes simplex virus (HSV), especially in young adults infected with either type 1 or type 2 virus (HSV-1 or HSV-2). In many instancesthese patients have been receiving antibiotics for presumedgroup A streptococcalinfections but have not responded to this treatment (26). Although oral acyclovir is not approved for use in this syndrome, it appears to be beneficial and may be approved for use in the near future. Acyclovir is only active againstHSV and varicella-zoster virus (VW). If this trend toward narrow-spectrum antiviral agents continues, viral diagnostic laboratories will be all the more necessary, as will large numbersof technologiststrained in this diagnostic discipline. Even if no therapy is available, the establishment of a definite viral diagnosisis often beneficial in (i) educatingphysiciansand other medical personnel; (ii) eliminating unnecessaryantibacterial agents;and (iii) defining the diseaseprocessand prognosis. SPECIMEN

REQUIREMENTS STUDY

FOR VIRAL

In respiratory tract diseaseit is generally only necessaryto submit a specimenfrom the throat or nasopharynx; stool or urine isolates would usually be irrelevant. Nasopharyngealaspirates are superior to swabs,but the latter are considerably more convenient. Throat swabsare probably adequatefor recovering entero- and adenoviruses and HSV, whereas specimensfrom the nasopharynx are definitely superior for recovering RSV and probably better for parainfluenza viruses. Specimensfrom the nasalpassagesare optimal for recovering rhinoviruses, and com-

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AND KRILOV TABLE

CUMITECH

21

1. Respiratory syndromes and associated etiologic agents

Syndrome

Viruses

Upper respiratory tract Common cold . . . . . . . . . . . . . . . . . . . . . . . . . . Rhinoviruses and coronaviruses are most common; others: RSV, parainfluenza viruses, adenoviruses, enteroviruses Pharyngitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adenoviruses, EBV, enteroviruses (coxsackieviruses), HSV, RSV, measles, influenza, parainfluenza viruses Laryngitis .*.................*.......*. Influenza and parainfluenza viruses, rhinoviruses, adenoviruses Croup (laryngotracheobronchitis) . . . . . . . . . Parainfluenza viruses, RSV, influenza viruses, adenoviruses Lower respiratory tract Bronchitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RSV, parainfluenza and influenza viruses; less commonly: adenoviruses, measles virus Bronchiolitis . . . . . . . . . . . . . . . . . . . . . . . . . . . RSV; others: parainfluenza (especially type 3) and influenza viruses, adenoviruses Pertussislike syndrome. . . . . . . . . . . . . . . . . . Adenoviruses (types 1, 2, 3, 5, 12, 19) Influenza . . . . . . . . . . . ..**............... Influenza A and B viruses Pneumonia . . . . . . . . . . . ..‘............... RSV, influenza viruses, adenoviruses, parainfluenza viruses (especially type 3) Pneumonia in the immunocompromised patient . . . . .,......*..................* CMV, VZV, adenoviruses , influenza viruses, RSV, parainfluenza viruses, measles virus, HSV

bined throat and nasal specimensare best for influenza viruses. Procedurefor Obtaining Specimensfor Viral Studies Any material may be usedfor swabs;however, calcium alginate may inactivate HSV and should not be used if this virus is a possible causeof the syndrome under consideration. Obtain specimensas early in the patient’s illnessas possible. Studies with respiratory viruses indicate that the mean duration of viral sheddingmay be 3 to 7 days. For throat cultures, swab the posterior pharynx and tonsillar area. Throat washings obtained by gargling saliva have been suggestedby someworkers but are not clearly superior to throat swabs for virus recovery. For nasopharyngealsamples,obtain a nasopharyngealswabor a nasalwash specimen by using a bulb syringe or a suction apparatus and 3 to 7 ml of buffered saline. A nasal wash specimenis obtained by instilling 2 to 3 ml of mediuminto each nostril and having the patient tilt the head forward to collect the fluid in a paper cup. The contents of the cup are then decantedinto a sterile vial, and a sampleof the fluid is tested for virus.

tically significant differences between modified Stuart, modified Hanks, or Leibovitz-Emory media.Therefore, if short-term transportation is necessary,a Culturette swab (Marion Scientific Corp., Rockford, Ill.) or a similarproduct can be used. It is generally believed that protein (serum, albumin, gelatin) incorporated into a transport mediumenhancesthe survival of viruses in transport and may be especially indicated if more than 12h of transportation is required. Storageof Specimens After a freeze-thaw cycle, significant lossesin infectivity titer occur with lipid-envelopedviruses(HSV, cytomegalovirus [CMV]) but not with agentssuch as adeno- and enteroviruses, which have a coat of protein only. For example, a laboratory strain of HSV held for 1 to 3 days at -20°C and then thawed had reductions in infectious titer of lo* or more. In contrast, when stored for 1 to 3 days at 4°C in Hanks or broth mediumthere was no loss of infectivity in twothirds of the specimens(11). Thus, for shortterm transit or storage, specimens for viral culture should be held at 4°C rather than frozen.

Transportation of Specimens The shorter the interval between collection of Media a specimenandits delivery to the laboratory, the Severaltypes of mediahave been usedfor the greaterthe potential for isolatingan agent. When collection and transportation of viral specimens. feasible, inoculate all specimens other than Most of these have been selectedfor this pur- blood, feces, and tissueinto culture tubes at the pose based either on tradition or on data ob- patient’s bedside.These are then transported to tained by testing the survival of laboratory the laboratory promptly. In general the followstrains of virus. A double-blind prospective ing statementshold, though there may be a few study to compare the recovery of viruses from exceptions. 1. Never leave a specimenat room or the upper respiratory tracts of children, using incubator temperature. three types of transport media, showedno statis-

21

CUMITECH

VIRAL TABLE

3

by”:

Culture

Direct detection

Adenoviruses

+

+/-

+

Coronaviruses

-

-

+

Enteroviruses

+

EBV

-

-

+

CMV

+

+/-

+

HSV

+

+

+

vzv

=t/-

+

+

Serology

Orthomyxoviruses (influenza A, B, and C) Paramyxoviruses (parainfluenza, RSV) Rhinovirus

DISEASE

2. Modes of detection for respiratory viruses Detection

Virus

RESPIRATORY

+

a +, Available methods using commercially obtainable b MAMA, Fluorescent antibody to membrane antigen.

reagents;

2, When it is impossible to deliver a specimenimmediately, it shouldbe refrigerated or packed in shavedice for delivery to the laboratory within 12 h of collection.

-, not routinely

Comments

Culture and IF are preferred methods of diagnosis. Significance of isolate must be interpreted in relationship to serotype and clinical findings. Diagnosis not routinely available, Significance of isolate must be interpreted in relationship to type isolated and clinical findings. Nonspecific heterophile antibodies (e.g., Monospot) are most readily available, but not reliable in children ~4 years old. Serology for virus-specific antigens is also available. Culture is most readily available. Rapid diagnostic methods reported include IF, molecular hybridization, and electron microscopy. Culture and IF are both preferred to serology. Significance of isolate must be interpreted in relationship to clinical findings. Direct detection by nonspecific (e.g., Tzanck preparation, electron microscopy) and specific (e.g., IF) techniques is often superior to culture in speed and sensitivity. FAMAb is the most sensitive serological method; enzyme immunoassay and anticomplement IF are also sufficiently sensitive for most uses. For RSV, direct detection (IF, ELISA) approaches the sensitivity and specificity of viral culture. For influenza and parainfluenza viruses, direct antigen detection is not as available or as sensitive as viral isolation. Culture is the only routinely available method for rhinovirus detection. available or not consistently

reliable.

Service (Fed. Reg., vol. 45, no. 141, 21 July 1980)and in the Department of Transportation and Interstate Quarantine regulations (49 CFR, Section 173.386.388,and 42 CFR, Section 72.25, Etiologic Agents). Copies of these regulations Transport Regulations can be obtained from the Biohazards Control Specific requirementsfor shippingspecimens Officer, Centers for DiseaseControl, Atlanta, have been publishedby the U.S. Public Health Ga. In essence, regulations require that the

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CUMITECH

21

15 10 5 15 10 5 15 10 5 15 IO 5 0

FIG.

specimen be wrapped in sufficient absorbent material to absorb the entire contents of the specimen in case of leakage or breakage. The wrapped specimen must be enclosed in a durable watertight container, which in turn is enclosed in another outer shipping container. The watertight container must be secured with shock-absorbent material or tape so that it does not become loose as the dry ice sublimates or shaved ice melts. UPPER RESPIRATORY

INFECTIONS

Cold

The common cold is an acute, self-limited viral infection of the epithelial surfaces of the upper airway that is characterized by nasal dischargeand stuffiness,sneezing,rhinitis, and throat irritation. In infants, clinical manifestations can be more severe than in older individualssincenasalobstruction in this agegroup can significantly interfere with feeding and sleeping. Fever, irritability, and restlessnessare also more frequent in infants. In an adult, temperature elevation of more than 1°C is unusual. With rhinoviruses, sneezing, nasal discharge, and obstruction usually begin together and increasein severity over 2 to 3 days. Similar patterns of upper respiratory tract symptomshave beendescribedfor coronavirus colds (38). Most colds last 1 week, but in about 25% of patients they last up to 2 weeks. There are few physical findingson examination. The nasalpassagesmay be obstructed by mucus, and there may be pharyngeal erythema and exudate. There are no characteristic differences in clinical findings amongthe viruses that cause the commoncold syndrome. Although hay fever

and vasomotor rhinitis give similar symptoms, their recurrent and chronic nature are soon recognizedby the patient as not being typical of a commoncold infection. The incubation period of the common cold is usually 2 to 3 days. Children tend to have more coldsper year than adults, with the peak beingin the early school years when the annual average number is three to eight per child (13). The respiratory viruses causing colds are found in the picornavirus, coronavirus, and myxovirus families (10). Rhinoviruses account for approximately 25% of all colds in adults (28, 30, 35). Coronavirusesare difficult to study but are thought to be important as a causeof 10 to 20% of all colds. RSV and parainfluenzaviruses are responsiblefor colds especially in infants and young children, but alsoin adults. Enterovirusesand adenoviruseshave alsobeen implicated in upper respiratory tract illnesses,but their role in causingcoldlike symptomsalone is poorly defined. Standard virus isolation procedures will only detect the etiologic virus 60% of the time. Due to the self-limited and generally benign course of most colds, specific diagnostic studies are infrequently performed. If indicated, this is best done by isolation of live virus or detection of virus antigen in nasalsecretionsobtained by the nasalwash technique or by nasopharyngeal swabs. Nasal washingsare less convenient but have a higher yield. These specimensare optimal for recovering rhinoviruses and RSV, while influenza and parainfluenza viruses are recovered best from combinedthroat and nasalspecimens.Serologic tests are available for a number of these agents, especiallv coronaviruses, but

CUMITECH

21

are generally less useful because of the need to collect acute- and convalescent-phase specimens over a 2- to 3-week period. In addition, infants may not always mount titer rises with acute infection; conversely, in certain situations seroconversions may not indicate acute infection. Significant antibody titers in young infants may also reflect passive acquisition of maternal immunoglobulins. These issues will be addressed in more detail as the particular agents are reviewed. Pharyngitis Acute pharyngitis is an inflammatory disease of the mucous membranes and structures of the throat, frequently involving the nasopharynx, uvula, and soft palate as well. The syndrome is characterized by soreness or irritation of the throat, but severe pain or difficulty in swallowing are not characteristic. Other frequently observed signs and symptoms are fever, headache, nausea, vomiting, abdominal pain, and cervical adenitis. Pharyngitis can also be a prominent part of the infectious mononucleosis syndrome. The diagnosis of pharyngitis requires objective evidence of inflammation (i.e., erythema, exudate, ulceration, or a combination of these). Overall, this syndrome is most common in cold-weather months, and the particular etiology correlates with the prevalence of the involved agent. For example, rhinoviruses may cause pharyngitis in the spring and fall. When there are associated nasal symptoms, the etiologic agent is almost always a virus. Pharyngitis without nasal involvement is often due to group A streptococci, but viruses cause a majority of even these cases, Adenoviruses are the most common cause of a primary pharyngitis, especially in children under 6 years of age (41). Types l-7, 7a, 9, 14, and 15 are the most commonly isolated. Adenoviruses cause a follicular pharyngitis, and exudative lesions are common as well. In older children and adults, conjunctivitis, if present, strongly suggests a diagnosis of adenovirus infection. Fever, malaise, headache, myalgias, chills, and dizziness can be present as well. In influenza, pharyngitis is often a major complaint, but other symptoms such as myalgias, headache, cough, and coryza are frequently present as part of the syndrome. Exudative tonsillitis or pharyngitis also occurs in approximately half of the cases of infectious mononucleosis due to Epstein-Barr virus (EBV) (18). These findings are generally associated with other features of this syndrome: malaise, fatigue, fever, cervical adenopathy, headache, and splenomegaly. Pharyngitis with ulcerative or vesicular lesions, or both, over the soft palate and uvula as well as the pharynx can be seen with enteroviral (e.g., type A coxsackie-

VIRAL

RESPIRATORY

DISEASE

5

virus) and primary HSV infections. This type of enteroviral disease is manifest as a febrile illness with marked sore throat and dysphagia, hence its pseudonym “herpangina.” HSV disease can be associated with fever and tender cervical adenopathy. An associated gingivostomatitis, if present, can be helpful in diagnosing this form of HSV disease (29). Patients with measles can have pharyngitis as well as Koplik spots on examination of the oral cavity. Although a scratchy throat or mild pharyngeal erythema may be seen with other respiratory viruses (rhinoviruses, RSV, parainfluenza viruses), it is not as prominent a finding as the rhinitis. With RSV and parainfluenza viruses, components of lower respiratory disease may be present as well. In most cases of pharyngitis, the main goal of diagnosis is to separate group A streptococcal disease from other causes because of the differences in treatment and potential complications. The patient’s history and physical findings, epidemiologic factors (e.g., season, known on-going viral outbreak in the community), and other associated findings as noted above may suggest a particular etiology and may dictate a throat culture for either group A streptococci or a virus. For the latter, a nasopharyngeal wash or swab is useful for culture, especially if nasal symptoms are prominent. Throat washings or swabs may be preferable for viral culture if there is prominent pharyngitis. Laryngitis Acute laryngitis is usually characterized by hoarseness. Although it may be common in children and associated with airway obstruction, it is rare to find this complication in adults. Laryngitis often is found in association with the common cold or influenza syndrome. Hoarseness may be present in 20% of cases of common respiratory illness. It frequently occurs with midwinter illnesses and correlates with the presence of cough and sore throat. Although many of the major respiratory viruses have been reported to cause hoarseness, the most common causes include parainfluenza and influenza viruses, rhinoviruses, and adenoviruses. On examination, the patient with acute laryngitis may have a reddened and edematous mucosa with superficial ulcerations. If there is acute epiglottitis, the epiglottis is often red and greatly swollen. Most patients recover from laryngitis within 10 days. For the most common causes, a nasopharyngeal wash or throat swab is probably the specimen of choice. Croup Croup (laryngotracheobronchitis) is an agerelated syndrome of varying degrees of inspiratory stridor, a barking cough, and hoarseness

6

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due to obstruction in the region of the larynx and subglottic trachea. It occurs in children 3 months to 3 years of age, with a peak incidence in year 2 of life (3 1). It generally begins as an upper respiratory tract infection with rhinorrhea, a sore throat, and mild cough. Over 1 to 2 days there is development of hoarseness, inspiratory stridor with supraclavicular and suprasternal retractions, and a worsening cough which has been likened to the barking of a dog. The course of the disease fluctuates, with exacerbations frequently occurring at night. It generally resolves over 3 to 7 days. Severe cases are associated with significant hypoxia, and, rarely, intubation or a tracheostomy is necessary to bypass the obstruction. Some evidence of lower respiratory disease (e.g., rales, wheezing) is frequently noted as well. Roentgenograms of the neck reveal subglottic narrowing of the trachea and a normal epiglottis. In occasional cases and especially in older children, acute laryngitis with hoarseness and evidence of upper respiratory infection occurs without the croupy cough. Parainfluenza viruses, especially type 1, are the most important causes of croup, with RSV second in frequency. Influenza virus and adenoviruses may also cause this syndrome. LOWER RESPIRATORY

INFECTIONS

Bronchitis/Tracheobronchitis Bronchitis is a clinical syndrome characterized by fever, cough, and rhonchi (12). Frequently there is also pain beneath the sternum. The cough is described as dry, harsh, and brassy, which reflects the acute inflammation of the larger airways including the trachea and large and medium-sized bronchi. Upper respiratory infection manifest by rhinitis, pharyngitis, or both is frequent initially. Over 1 to 2 days this progresses to the marked tracheobronchial symptoms which characterize acute bronchitis. These symptoms last 4 to 6 days and are followed by a l- to 2-week recovery period during which cough and low-grade fever may persist. In bronchitis associated with influenza, systemic symptoms of fever, chills, and myalgias are prominent. Overall, RSV is the most common cause of acute bronchitis, especially in very young children. Parainfluenza viruses, especially type 3, are second in frequency. Other viral etiologies in children include adenovirus type 7, influenza A, and measles. In measles, which is now much rarer in the United States due to large-scale vaccination, bronchitis is part of the clinical syndrome which includes rash, mucous membrane involvement, conjunctivitis, and fever. Influenza A can cause a very severe bronchitis, especially in children with first-time exposure.

CUMITECH

21

(Influenza B probably causes less severe bronchial disease than influenza A.) In adults, influenza viruses are probably the most common cause of bronchitis/tracheobronchitis; however, parainfluenza virus, RSV, and adenoviruses are also important. Tracheobronchitis and bronchitis are most commonly reported in adults during the winter months and epidemic periods when influenza is present in the community. Nonviral causes of bronchitis include Mycoplasma pneumoniae, Bordetella pertussis, and Haemophilus influenzae. Although the etiologic agents overlap in croup and acute bronchitis, the larynx and subglottic trachea are not involved in bronchitis. Diagnosisis madeby viral detection in nasopharyngeal specimens. Bronchiolitis Bronchiolitis is an acute viral lower respiratory tract infection unique to children lessthan 2 years of age, with the peak occurrence from 2 to 8 months (62). The diseaseis characterized by expiratory wheezing, air-trapping tachypnea, retractions, nasalflaring, grunting, irritability, and dehydration (due to poor oral intake). Pneumonia, otitis media, mild conjunctivitis, and diarrhea are frequently associatedfindings. Radiographic examination of the chest demonstrates hyperinflation with depresseddiaphragmsand hyperlucency of the parenchyma. Prominent vascular markings radiating from the hila and areasof atelectasiscan also be seen, RSV is the major causeof bronchiolitis, especially during epidemic outbreaks and in cases requiring hospitalization, where it can account for more than 80% of cases.Parainfluenzaviruses, especially type 3, are the secondmost commonly isolatedvirusesin this disease.Other less frequently isolated agents in bronchiolitis include adenoviruses, rhinoviruses, influenza virusesA and B, and enteroviruses (32). Respiratory specimens,preferably obtained by the nasalwash technique, are best for isolating the offending agent. Serologic determinations are not generally very helpful since, in the agegroup most affected, passively acquired maternal antibody to many of the potential pathogensis still present. PertussislikeSyndrome Pertussisor whooping cough is a prolonged respiratory illness characterized by recurrent paroxysmsof coughing, posttussivefatigue, and vomiting. Classically, although not always seen, the paroxysm is associatedwith cyanosis and endswith an inspiratory whoop. This phase of the illness can last 1 to 2 weeks, and recovery ranges from 4 to 10 weeks from the onset of illness. Bordetella pertussis is the prototypic cause of this syndrome, but it has also been

CUMITECH

VIRAL

21

reported in patients from whom only an adenovirus has been isolated. Types 1,2,3,5, 12, and 19 are the adenoviruses that have been implicated in this syndrome (14,37). Even in the absence of isolation of 23.pertussis, care should be used in attributing this syndrome to an adenovirus, for a number of reasons. Isolation of B. pertussis requires collection of suitable specimens,their rapid processing,and the useof freshly prepared selective media. If this procedure is not routinely performed, its sensitivity may be markedly decreased.In addition, as mentioned, adenovirusesare capableof latent or recrudescentinfection, making interpretation of a positive finding potentially diacult. Influenza Influenza can cause a characteristic illness with fever, chills, sore throat, and systemic complaints. There may be predominance of common cold or pharyngeal symptoms, but cough is very common, The incubation period for influenza is 2 to 5 days, and illnessesusually occur in epidemicpattern throughout a community during the winter months. The clinical finding of pneumoniawith influenza can be a manifestation of the virus infection itself or be due to secondary bacterial infection caused primarily by Streptococcus pneumoniae, but also by Staphybcoccus aureus, H. influenzae, and a vtiety of other unrelated bacteria. If the influenza virus causesthe pneumoniadirectly, there is a high mortality rate. Influenza virus can be isolated from nasopharyngeal wash or throat swab.When an epidemicof influenza is present in a community, it is usually easy to make a clinical diagnosisof this infection. Pneumonia

RESPIRATORY

DISEASE

7

agents; older children get lesssevere diseaseas a rule. Influenza viruses A and B are not as prevalent asRSV and parainfluenzaviruses, but during epidemics,children of all agesmay develop lower respiratory tract infection due to these viruses. Adenoviruses are often isolatedin children with pneumonia,but their commonasymptomatic carriage makes determination of the etiologic significancedifficult at times. Because of this, the actual frequency of adenoviral pneumonia is uncertain; nevertheless, severe pneumonia, especiallywith types 3,7, and 21, is well documented(9). Severe pneumonia is also the most serious complication of measles.Although pneumoniais often due to secondary bacterial infection, primary measlespneumonia may exist as well. Enteroviruses, rhinoviruses, and coronaviruses causesomecasesof pneumonia,but thesecases are generally milder than those with the above agents, and the incidence of such diseaseis uncertain. VZV, EBV, CMV, and HSV can causean interstitial pneumonitisin congenitally or neonatally infected infants as well. Viral pneumoniain infants and young children generally presentsafter a l- or 2-day prodrome of coryza, low-gradefever, and decreasedappetite (8). Its onset is marked by increasedcongestion and fever, vomiting, .coughing, and increased fretfulness. Apneic spells have been observed in infants with viral pneumoniadue to RSV, parainfluenzavirus, and influenza viruses. Respiratory specimenssubmitted for viral diagnosisare generally from the upper respiratory tract. Since the viruses associatedwith pneumonia (other than adenoviruses)are rarely carried asymptomatically, such isolatesare still significant and at least imply an associationwith the lower respiratory disease.Nasal washes,nasopharyngeal swabs, and throat swabs are the specimensmost frequently obtained for viral detection. If the patient requires intubation, tracheal aspirates may be obtained, although nasopharyngealwashes are still the preferred specimens.In addition, if proceduresto obtain lower respiratory tract specimens(e.g., bronchoscopy, lung biopsy) are clinically indicated, someof the material obtained shouldbe submitted for viral diagnosisas well. As with most of the entities discussed,serologic diagnosisis in generallessusefulthan viral isolation or antigen detection.

Pneumonia,defined as an infiltrative disease of the lungs, is most frequently viral in infants andchildren, with an estimatedrisk ashigh as24 casesper 1,000children under 1 year of ageand decreasingthereafter (25). Viral pneumonia in adultsis unusualand is associatedpredominantly with either influenza or adenovirus infection. Pneumoniais associatedprimarily with signs and symptoms of respiratory tract infection, including cough and shortnessof breath. Chest X ray will show evidence of an infiltrate in the lungs, and usually there are physical diagnostic findings compatible with infection. In younger patients, the most frequently diagnosedagents and those causingthe most severe diseaseare RESPIRATORY INFECTIONS IN RSV, influenza viruses A and B, and the parainIMMUNOCOMPROMISED PATIENTS fluenza viruses. Of the parainfluenza viruses, type 3 is most commonly associatedwith pneuHerpesviruses monia. RSV and parainfluenza viruses cause pneumoniamainly in infants and young children Immu.nocompromisedpatientsare subjectto a undergoing their primary infection with the number of potentially very severe viral respira-

8

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tory infections. Many of these are due to herpesviruses (CMV, HSV, VZV). Since these agents are capable of maintaining latent infection, clinical infection may result from either primary infection or reactivation of latent virus. In patients with congenital or acquired defects in cellmediated immunity (e.g., severe combined immunodeficiency syndrome, DiGeorge’s syndrome, Wiskott-Aldrich syndrome, newborn malignancy, organ transplantation, AIDS [acquired immune deficiency syndrome], severe malnutrition), this family of viruses poses a major threat. CMV. CMV infection is frequent and occasionally severe in the at-risk individuals described above. CMV infection is the most common illness and the leading cause of death in renal transplant recipients. It usually presents 1 to 3 months after the transplantation procedure as a syndrome of fever, nonproductive cough, dyspnea, hypoxemia, leukopenia, and arthralgia (55). It is also associatedwith severe and frequently fatal interstitial pneumonitis in bone marrow transplant recipients, with a meantime to onsetof 6 to 8 weeksafter transplantation (46, 47). Other patients at risk for this type of pneumonitis include individuals with cardiac, liver, or kidney transplants, AIDS, or cancers (especially hematologicmalignancies)and premature newborns 2 to 4 weeks after multiple blood transfusions; possible sourcesof virus include blood products, the engrafted organ or bone marrow, sexual transmission, and reactivation of the host’s latent virus. CMV can be isolatedfrom urine, throat washings,buffy coat, lung biopsy, and autopsy specimens.In all cases,results needto be interpreted with caution since asymptomatic sheddingcan occur in these patients. The presenceof CMV inclusionsin tissueincreasesthe likelihood that the virus is causingthe disease.When CMV is isolated from lung it is necessary to rule out other bacterial, fungal, chlamydial, or protozoan agents(e.g., Pneumocystis carinii) which may be primarily responsiblefor the illness(1). HSV. HSV-1 and HSV-2 can cause disseminated diseasein immunocompromisedpatients, involving the lungs, liver, adrenal glands, and brain, aswell asseverelocal disease.Newborns are the most severely affected, but disseminated HSV diseasehas also been reported in the severely malnourished, in the iatrogenically immunosuppressed,in patients with WiskottAldrich syndrome, and in individuals with severe burns or eczema(7). Patients with hematologic malignanciesand those who have received bone marrow transplants are at great risk of developing severe HSV infections, but visceral disseminationwith pulmonary involvement is uncommon. Diagnosismay be delayed because

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the typical cutaneous lesionsmay be absent or maskedby the underlying diseaseprocess(e.g., severeeczemaor burn lesions)(3). HSV-2 is the more common causeof neonatal disease,while the other patients described develop disease more commonly with HSV-1. HSV can be identified by demonstrating the characteristic cytological changesof multinucleated giant cells and “ballooning” cytoplasm on cells scrapedfrom the baseof lesions,fixed, and stained with Wright or Giemsa stains (the Tzanck test). This test cannot distinguish HSV from VZV and is only 50% as sensitive as viral isolation (17). VZV. VZV is the causative agent of varicella (chicken pox) and herpeszoster (shingles).Varicella is the primary infection, whereas zoster representsrecrudescenceof latent virus. A syndrome of life-threatening varicella with visceral involvement, especially pneumonitis, with high mortality rate has been reported in infected children with leukemia or lymphoma, aplastic anemia, or bone marrow transplants and those treated with prednisoneor cytotoxic drugs (48, 59). Others at great risk include patients with congenital T-cell deficiencies such as DiGeorge’s syndrome, severe combined immunodeficiency, cartilage-hair hypoplasia, and Nezelof s syndrome.Disseminatedvaricella hasbeen reported in children on high-dosecorticosteroid therapy for conditions without primary T-cell defects (e.g., nephrotic syndrome, rheumatic fever, severe asthma)(59). Herpes zoster occurs in patients who have had varicella previously. Patients with Hodgkin’s diseaseor bone marrow or renal transplants are more likely to develop extensive or disseminatedskin lesions (beyond the initial dermatome), but visceral dissemination and mortality are very rare (63). The virology laboratory can offer important information in this setting. Specific diagnosisis mandatory in high-risk patients or their contacts to guide the use of antiviral therapy or the appropriate prophylactic use of varicella-zoster immune globulin. As a varicella vaccine becomesmorewidely available, the laboratory will be able to offer information on patients who are seronegative and are thus potential candidates for vaccination, as well as on those who seroconvert once vaccinated. The vesicular skin lesions need to be differentiated from those caused by certain enteroviruses, bacterial agents,hypersensitivity reactions, or HSV. Biopsiesof visceral lesionssuchasvaricella pneumonia and encephalitis may also be used for diagnosis.Second, determiningthe immunestatus in high-risk individuals exposed to VZV infections can help guide the administration of varicella-zoster immuneglobulin.

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EBV. Identification of EBV infection in the clinical laboratory is dependent upon serological tests since isolation or direct detection of the virus is not routinely available. Heterophile antibodies of the immunoglobulin M (IgM) class to animal erythrocytes are present in the majority of patients with an acute EBV infection. Rapid slide tests (e.g., Monospot) measuring horse erythrocyte agglutination are very sensitive, although they may remain positive for up to a year after EBV infection. Rare false-positives have been seen with lymphomas or hepatitis. Heterophile antibody responses are much less commonly observed in children less than 2 years of age with an infectious mononucleosis syndrome including tonsillar pharyngitis and respiratory symptoms (40). Measurement of EBV-specific antibodies may be helpful in establishing the diagnosis in this setting. Kits to measure IgG antibodies to the viral capsid antigen and to measure the nuclear antigen of EBV serum by immunofluorescence (IF) are commercially available. Viral capsid antigen antibody responses occur early in the course of the illness but may remain elevated for life, limiting the utility of this antigen in diagnosing an acute infection. Nuclear antigen antibody rises later in the acute illness and usually enables one to date the timing of the infection. For a more detailed discussion of EBV serology, see Cumitech 15 (11). Adenoviruses Gf the nonherpes DNA viruses, adenoviruses have been reported to cause severe disease with extensive pulmonary and hepatic involvement in immunocompromised patients. The lung involvement can progress to an interstitial pneumonitis, pleural effusion, necrotizing bronchiolitis, or all of these. These findings have been described in renal and bone marrow transplant recipients and newborns as well as in patients with other congenital or acquired immunodeficiencies (54, 64). Adenoviruses in these instances can be isolated from upper or lower respiratory tract specimens by the methods described previously. Adenoviruses have also been obtained from urine and, rarely, blood cultures. Urine and upper respiratory tract isolates must be interpreted in light of the clinical setting since latent or persistent shedding may exist. In addition to earlier cautions regarding adenovirus serology, these patients may be unable to mount an antibody response. Orthomyxo- and Paramyxoviruses Orthomyxo- and paramyxovirus (influenza A, RSV, parainfluenza viruses) diseases appear to be of no higher incidence in immunocompromised patients than in the general population, but in these patients these viruses persist in

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respiratory secretions for increased lengths of time. In addition, instances of dissemination of the viral infection beyond the respiratory tract have been reported (63). Diagnostic methods are described for these agents in earlier sections. Measles is a paramyxovirus that can cause overwhelming disease in patients with protein calorie malnutrition, malignancy, or other defects in cell-mediated immunity. The syndrome is one of fever, necrotizing giant-cell pneumonitis, encephalitis, hemorrhagic dermatitis, renal insufficiency, and shock (7). Due to successful immunization practices, measles is rare in the United States at present. Nasopharyngeal and conjunctival secretions, urine, blood (leukocytes), and biopsy material can all be used for virus isolation or the detection of viral antigen. IF staining of specimens is presently the diagnostic method of choice, with good antisera available commercially. Although cell lines will grow measles virus, isolation is rarely attempted due to the rarity of isolates, the time required, and the technical complexity of the procedures (49). Serological studies are also possible, although antibody responses may not be seen in immunodeficient patients. Hemagglutination inhibition (HI), complement fixation (CF), and neutralizing antibodies appear with the development of the macular rash and reach peak titers in about 10 days. IgM antibodies appear with IgG antibodies but decline to undetectable levels in 30 to 90 days, An enzyme-linked immunosorbent assay (ELISA) for measles antibody is available with at least a 20-fold higher sensitivity than HI or CF tests. All reagents are commercially available except measles antigen, which can be obtained by infection of susceptible cell cultures. Specific IgG and IgM can be identified separately with this technique. The Cl? test offers the advantage of being commercially available and more convenient to use in many laboratories (49) . LABORATORY

DETECTION

OF VIRUSES

Detection of Adenoviruses Direct detection of adenoviruses in clinical specimens by IF is possible using commercially available antibodies to the hexon (the common antigen) of adenoviruses. IF tends to be negative in specimens from patients carrying the virus incidentally (44). Thus, although less sensitive than viral isolation in cell culture, IF may be more helpful in determining the etiologic significance of an adenovirus isolate. A nucleic acid sandwich hybridization technique for detection of adenovirus DNA has recently been described (57) but is not routinely available as yet.

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For viral isolation, primary human embryonic kidney cells are optimal but are expensive and may not be routinely obtainable. Heteroploid cell lines such as HeLa or HEp-2 cells may be used as well and are commercially available. IF staining or ELISA procedures using antihexon antibody are used to confirm cell culture isolates as adenoviruses (36). Neutralization with typespecific antibodies is the method of choice for type-specific identification of an isolate if indicated. Antisera for adenovirus types 1 through 7 are commercially available, and reference antisera for other types may be obtained from the American Type Culture Collection, Rockville, Md. The methodology for these techniques is described in detail in the Manual of Clinical Microbiology, 4th edition (15). Serological responseto adenovirusescan be measuredwith HI and neutralization CF tests. The CF test detectsa group-specificantigen,anda fourfold or greaterrise in titer indicatesa current infection. Unfortunately, children under 10 years of age often do not mount a CF responseto adenovirusinfection. Microneutralization and HI methods can be used to test for type-specific antibodies;the former is more sensitiveand specific (15). The laboratory finding of an adenovirus mustbe interpretedcautiously sincetheseviruses are capable of latency and recrudescencein asymptomaticpatients.Asymptomatic viral sheddingcan persistfor up to 18monthstier an acute adenoviralinfection. Still, recovery of an adenovirus from a throat or conjunctival specimenin the appropriateclinical settingis suggestiveof causality, especiallyif no other potential pathogensare isolated. A fourfold rise in antibody titer to an adenovirusin addition can be helpful in linking it to an etiologic role, although not all infections result in serologicresponsein infants and young children (11).

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latter only for OC-43) have been described as well. None of these is routinely performed by diagnosticlaboratories.

Detectionof Picornaviruses Rhinoviruses.Detection of rhinovirus antigens directly by IF or immunoperoxidase staining techniques in clinical specimensis not feasible due to the large number of serotypesthat would need to be tested. Serologic diagnosisof rhinovirus infection is also impractical due to the large number of serotypes and the lack of a simple, reliable method for detecting antirhinovirus antibody. A singlemicroneutralization technique for quantitative measurementof rhinovirus antibody responsesis well described, but it has been used mostly in large-scaleserologicalsurveys or epidemiologicalstudies. Virus isolation in cell culture is the diagnostic method of choice for rhinoviruses. Procedures for viral isolation are described in detail in the Manual of Clinical Microbiology, 4th edition (50). Human diploid cells (e.g., WI-38) are the most commonly used and available for rhinovirus isolation. Primary cultures of humanembryonic kidney or lung have also been used (34). Rhinovirusesgrow optimally at 33°Cin a roller drum apparatus. Typical rhinovirus cytopathic effect (CPE) begins as a focal rounding and swellingof the cell layer and eventually spreads to involve the entire cell sheet. If suchan isolate is acid labile (lo@fold reduction of virus titer at pH 3), it can be presumptively identified as a rhinovirus. Definitive identification requires neutralization testing with type-specific rhinovirus immune sera. Such sera are not routinely available, and such typing is generally not warranted on an individual clinical isolate. Enteroviruses. The enteroviruses include the echo-, coxsackie-, and polioviruses as well as Detectionof Coronavirus enteroviruses 68 through 71 and hepatitis A Coronaviruses,as noted above, are probably (enterovirus 72). There are at least 69 distinct frequently responsiblefor cold syndromes(60). serotypes within the family. As a result, adePart of the uncertainty over their exact role quate antisera for routine methods of direct relates to the difficulties in detecting these immunologic detection of these agents are not agents.No practical method of direct examina- available. Serologic testing is similarly limited. tion for coronavirus antigen exists, although the In epidemicsor situations where a limited numagent is structurally recognizable on electron ber of serotypes are suspect (e.g., hand-footmicroscopic examination of clinical specimens and-mouth diseaseor myocarditis), a microneu(51). In addition, coronavirusesare very difficult tralization procedure for detecting antibody has to culture and are not isolated by routine meth- been described,but it is not routinely available ods. They require organ cultures of ciliated (45). In most instances, then, viral isolation is epithelial tissues, and their isolation is per- the best method. The cell lines used in most formed primarily as a research method only. laboratoriescan be readily usedto culture group Serological diagnosis of coronavirus infection B coxsackievirus, echovirus, and poliovirus. requires demonstrationof a fourfold or greater Primary monkey kidney and human kidney or increaseby CF or neutralization tests. At pres- cell lines, e.g., WI-38, are best for growing these ent, antisera are only available for two sero- agents.Inoculation of sucklingmice is the methtypes, 229Eand (X-43. ELISA and HI tests(the od of choice for isolating group A coxsackievir-

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uses (49, although few laboratories routinely infect these animals because of the great expense involved. Presumptive diagnosis of an enteroviral isolate can usually be made based on the characteristic CPE in conjunction with the clinical history and time of the year the specimen was obtained. A history of recent oral polio vaccination should be determined in young infants and children since these attenuated strains will grow in cell culture. Specific identification of the serotypes is accomplished using intersecting pools of hyperimmune sera. This procedure is not routinely indicated or available in most clinical laboratories. Still, isolation of an enterovirus in throat or feces specimens does not prove an etiologic association, since prolonged carriage after an infection can occur and the finding should be interpreted in light of the clinical findings. Although demonstration of a fourfold or greater rise in serotype-specific antibody titer may be helpful in documenting an etiologic association, serologic testing is usually not necessary. Detection of Herpesviruses CMV. Monoclonal antibodies to CMV can be used for IF tests on lung tissue. This offers the advantage of rapidity, but this technique is generally less sensitive than viral isolation and the reagents are just becoming available in most clinical laboratories (56, 58). Other direct detection methods such as electron microscopy and molecular hybridization techniques have been described for the detection of CMV in urine, throat swabs, and peripheral leukocytes, but their relative sensitivity has not been established and they are not routinely available in most diagnostic laboratories (2, 27, 56). Human fibroblast cells (e.g., WI-38, 350Q) best support the growth of CMV. The appearance and extent of CPE depend on the titer of virus, but in general, CPE usually appears within 10 to 14 days (56). The typical CPE consists of enlarged, rounded, refractile cells in appropriate host cells and is highly suggestive of CMV. The presence of intranuclear inclusions by IF or hematoxylin and eosin staining can be used as additional confirmation. Rapid culture detection has been achieved by staining and by treatment of cell cultures with monoclonal antibodies after just 1 night of incubation (24). A variety of serological tests are available for measuring anti-CMV IgG. The CF test is the most frequently used, and all reagents are commercially available, A variety of other serological tests have been described including anticomplement IF, indirect IF antibody, immune adherence hemagglutination, enzyme immunoassay, and indirect hemagglutination. The reagents needed for most of these are available

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commercially, and several are marketed as complete kits (e.g., indirect IF antibody, enzyme immunoassay, and immune adherence hemagglutination). Comparison studies have shown that all of these are sensitive and specific for measuring CMV antibodies (56). In addition, several methods for measuring anti-CMV IgM antibodies are available. These measurements need to be controlled for the presence of rheumatoid factor (an anti-IgG IgM antibody produced in some rheumatologic, vasculitic, and viral diseases including CMV) (56). As with recovery of the virus in cell cultures, CMV serologic results need to be interpreted in light of the clinical setting. Fourfold rises in specific IgG or initial elevation of specific IgM are considered diagnostic of recent infection, but this does not establish whether the infection is responsible for a given symptomatic illness. HSV, Specimens for HSV cultures can be obtained by swabbing open skin lesions or mucous-membrane ulcers with a cotton-tipped applicator. Fluid from fresh vesicles is also rich in virus. When there are no apparent oral or skin lesions, swabs or pharyngeal secretions may yield virus (17). This type of culture must be interpreted with caution since HSV may reactivate with stress or any illness. The relevance of such a finding thus depends on the clinical situation. In addition, specimens from diseased organs, cerebrospinal fluid, urine, and blood leukocytes may be cultured. HSV replicates readily in a number of primary and established cell lines. Primary or passaged cultures of cells from human embryonic tissues, Vero cells, and human diploid lines are all commercially available and sensitive to HSV. HSV generally grows rapidly in cell cultures, with typical CPE detected in 2 to 3 days. This is frequently sufficient for HSV identification, but in critical situations, such as visceral isolates, definitive typing is indicated. This can be achieved by indirect IF antibody or neutralization tests. HSV-1 can be differentiated from HSV-2 in these and other ways (solid-phase radioimmunoassay, inhibition of passive hemagglutination, immunoperoxidase assays), with indirect IF antibody being the most widely used (17) Antibodies to HSV-1 and HSV-2 are commercially available and permit direct detection of HSV antigen in clinical specimens by IF or immunoperoxidase techniques (53). This method with appropriate reagents is not as sensitive as viral isolation and not that much faster, but it can be employed in emergency situations. Kits which combine isolation and immunoperoxidase staining for rapid isolation and identification are commercially available but are generally less sensitive than standard culture techniques. In

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any event, culture “back-up” is mandatory for critical situations. Seroconversion in paired sera 2 to 4 weeks apart may provide evidence of primary HSV infection. The reagents for the CF test are readily available commercially. Other more sensitive serological methods can be used and have been modified to quantitate type-specific antibodies to HSV-1 and HSV-2. These include indirect IF, indirect hemagghttination, solidphase radioimmunoassay, and enzyme immunoassay procedures. Overall, serological diagnosis of HSV infection is of limited value since paired specimens are necessary, and if antiviral therapy is to be of value, it must be initiated before these results are available. Furthermore, significant titer rises do not occur only with primary infection but may also be seen in recurrent disease. Patients with VZV infections may have a rise in titer to HSV antigens due to cross-reacting antibodies; serologic testing, therefore, is not entirely specific. VZV. Direct examination of vesicular biopsy material provides the most rapid diagnosis of VZV. As for HSV, the Tzanck preparation can demonstrate the presence of multinucleated giant cells but cannot differentiate between the two agents. Electron microscopic examination of vesicular fluid can also demonstrate the presence of herpesvirus without specifically defining which one (52). Specific identification of VZV in vesicular scrapings or biopsy material can be established by IF with commercially available antisera, including monoclonal antibodies. With appropriate reagents and controls, IF staining for VZV is highly specific and more sensitive than culture (16, 52). VZV isolation in cell culture is best accomplishedin human diploid cell lines (e.g., embryonic lung or foreskin fibroblasts) or primary human cell cultures (e.g., human embryonic kidney) (39). As mentionedabove, culture techniquesare less sensitive than IF staining. Presumptiveidentification of VZV may be madeon the basisof typical CPE appearing3 to 14 days after inoculation. Initially the CPE consists of small discrete foci of rounded and swollen refractile cells, which slowly spread to involve most of the monolayer. Specific identification is accomplishedby IF staining of infected cell cultures. In contrast to HSV, VZV cannot replicate in rabbit or hamsterkidney cells. VZV can replicate in primary monkey kidney and human epithelialcells, which distinguishesit from CMV Go Serologicalprocedurescan be usedfor laboratory diagnosisof varicella or zoster infections, but their main role is in determining immunity status(presenceor absenceof VZV antibody) in high-risk individuals exposed to infection. For

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serodiagnosisof infection, acute- and convalescent-phasesera2 to 3 weeks apart are needed.A fourfold or greater increasein CF antibody titer in the absenceof a fourfold rise to HSV antigen is diagnostically significant. Up to one-third of individuals with primary HSV infections who experienced a prior VZV infection show a heterotypic CF antibody response to the VZV antigen.‘An enzyme immunoassaytest for VZV which is more sensitivethan CF is alsoavailable (52) For determining immune status to VZV in situationsas noted above,. CF is not a sensitive enough technique. Fluorescent antibody to membrane antigen in VZV-infected cells has been used clinically with good predictability of immune status for VZV (61). However, this method is cumbersomeand not always available. Anticomplement IF and enzyme immunoassaytechniquesare alsoof adequatesensitivity in this regard and may be usedif available (52). Detectionof Orthomyxoviruses Paramyxoviruses General. RSV can be detected with a high degree of sensitivity by direct examination of secretions using excellent commercially available antisera, a method which is at least as sensitiveasviral isolation in experiencedclinical laboratories (21). For influenza and parainfluenza viruses, direct testing is not as sensitive as viral isolation (42,43) but may be attempted. An ELISA for examination of secretionsfor RSV hasbeen describedas well and is commercially available. Virus isolation in cell culture is the diagnostic method of choice for most myxoviruses (6). Proceduresfor viral isolation are described in detail in the Manual of Clinical Microbiology, 4th edition (42, 43). Serologicaltests for all theseagentsare available but are generally consideredsecondary to direct viral isolation or detection for a numberof reasons.Once isolated, these agentsare considered relevant since asymptomatic carriage is rarely reported. Second, it requires2 to 3 weeks to obtain the acute- and convalescent-phase specimensneededto show a fourfold titer rise. Third, young children may fail to show rises in serum antibody, especially with RSV. Furthermore, for parainfluenza and influenza viruses, serology offers no information as to the type of virus involved since cross-reacting antibodies and anamnesticresponsescan be obtained. Influenza A and B. Influenza A and B can be isolated in primary rhesusor cynomolgus monkey kidney cell cultures or in the Madin-Darby canine kidney (MDCK) cell line (20). Cultures are observed microscopically for CPE and tested to detect hemadsorption with guinea pig

CUMITECH

21

erythrocytes. Hemadsorption may be detected in advance of CPE, and influenza A is usually detectable after 3 days of incubation (4). Influenza viruses can also be grown after inoculation into the amniotic and allantoic cavities of lo- to 1l-day-old embryonated hen eggs. Influenza viruses are the only human viral pathogens which commonly produce hemagglutinating activity in eggs, and detectable titers are obtained as early as 2 to 3 days after inoculation (42). If good influenza antisera are available, serotyping can be done by IF staining of cell culture material. This method can determine the viral strain (A or B) but will not distinguish the subtype unless antibodies to the hemagglutinin are used. Subtypes are usually distinguished by HI or hemadsorption inhibition. Influenza viruses may be typed as A, B, or C by a CF test as well (42). Because influenza undergoes continual antigenic drift, commercial antisera for identifying subtypes may not always be available. The Centers for Disease Control make antisera to current strains available through state health department laboratories. When used, available serological methods for influenza include type-specific CF tests and HI tests. The latter are more sensitive, but the former may be more readily available in many laboratories. These tests differentiate influenza strains. A single radial hemolysis assay to characterize the subtype response for hemagglutinin and neuraminidase antibodies has been described (42). Barainfiuenza. Direct fluorescent-antibody detection of parainfluenza viruses 1 and 3 is an effective method for establishing infection with these agents. For parainfluenza 2, reagents for antigen detection are not as generally available and are not as sensitive as viral isolation (43). IF staining is the best method described for direct examination of respiratory secretions for these agents (23). Viral isolation is possible for each of these agents, using cells such as human embryonic kidney and rhesus or cynomolgus monkey kidney cell cultures. Cultures are observed microscopically for CPE and tested to detect hemadsorption with guinea pig erythrocytes. Parainfluenza viruses do not always produce apparent CPE, and detection of viral growth is frequently made by hemadsorption with guinea pig erythrocytes. Hemadsorption may be detected in advance or in the absence of CPE. The peak period of isolation is 4 to ‘7 days after inoculation. Definitive identification of isolates is made by IF or hemadsorption inhibition assays using specific antisera. Serologic tests for parainfluenza viruses are available but are generally considered secondary to direct viral isolation or detection, for a number of reasons. Once isolated. these agents are

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considered pathogenic since asymptomatic carriage is rarely reported. As already emphasized, a period of 2 to 3 weeks is required to obtain the acute- and convalescent-phase specimens needed to show a fourfold titer rise, and young children may fail to show rises in serum antibody. Furthermore, serology offers limited information as to the type of parainfluenza virus involved since cross-reacting antibodies and anamnestic responses can be seen. The three antibody tests available for parainfluenza viruses are HI, neutralization, and CF. Due to its sensitivity and ease of performance, HI is preferred. RSV. As mentioned above, direct detection of RSV in respiratory samples by immunologic methods, e.g., IF, is equal if not superior to viral isolation. If culture is to be employed, RSV grows best in HEp-2 cells (33). These cells are commercially available, although individual lots or passages of cells can vary in their sensitivity to RSV. Thus, periodic testing with a stock RSV source or use of a frozen, known-sensitive line is advisable. Virus growth is detected by the characteristic syncytium formation. The IF test is the best method of definitive identification (5). Serological tests for RSV antibody are available but are generally considered secondary to direct viral isolation or detection for a number of reasons. It requires 2 to 3 weeks to obtain the acute- and convalescent-phase specimens needed to show a fourfold titer rise. Second, young children may fail to show rises in serum antibody to RSV. CF, tube neutralization, plaque reduction, and ELISA serological methods for detecting RSV antibody have all been described; the CF test is the most practical and available (43). CONCLUSION In this Cumiteck we have summarized current information on clinical and laboratory aspects of viral respiratory disease. Each individual laboratory will have to decide how far it wishes to go in viral diagnosis. Even small laboratories can provide meaningful diagnostic services by establishing a close working relationship with a laboratory already performing viral diagnostic procedures. Each week the “support” laboratory can provide a few cell culture tubes to the smaller laboratory, where specimens for virus culture can be inoculated promptly. After inoculation, the tubes can be returned to the support laboratory for daily inspection, hemadsorption, and other procedures. In time the small laboratory may wish to increase its involvement in the processing of these specimens by examining cultures for CPE and becoming familiar with the cellular changes induced by common viruses such as HSV. This program can be likened to the

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levels of tuberculosis services that have been established for clinical microbiology laboratories. As reagents for IF tests become more available, even small laboratories may wish to provide direct fluorescent-antibody examination for viruses such as RSV. A last point in support of performing viral studies is the impressive gains that have been made in the laboratory diagnosis of these infections. In the past the diagnostic yield of “viral studies” may have been approximately lO%, but in laboratories where closecommunicationis required between the technologist and the physician the yield is greatly increased. LITERATURE

CITED

1. Abdallah, P, S,, J. B. D. Mark, and T. C, Merlgan, 1976. Diagnosis of cytomegalovirus pneumonia in compromised hosts. Am. J. Med. 61:326332.

2. Alpert, G., RI,-C. Mazeroni, R. Coleman, and S, Plotkin. 1985. Rapid detection of human cytomegalovirus in the urine of humans. J. Infect. Dis. 152~631-633. 3.

Arvin, A, M., A, S. Yeager, F, W, Bruhn, and N. Grossmm. 1982. Neonatal herpes simplex in the absence of mucocutaneous lesions. J. Pediatr.

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Baxter, B. D., R, B. Couch, S. B, Greenberg, and J. A. Kasel. 1977. Maintenance of viability and comparison of identification methods for influenza and other respiratory viruses of humans. J. Clin. Microbial. 6: 19-22. Bell, D. M., E+ E, Walsh, J. F. Hruska, K, C. Schnabel, and C. l3. Hall. 1983, Rapid detection of respiratory syncytial virus with a monoclonal antibody. J. Clin. Microbial. 17:1099-l 101a Benj;amin, D. R., and C+ C. Ray. 1974. Use of immunoperoxidase for the rapid identification of human myxoviruses and paramyxoviruses in tissue culture. Appl, Microbiol. 28:47-5 1 Borkuwsky, W, 1984. Viral infections in immunocompromised children. Pediatr. Ann. 13:682-692, Boyer, K, M., and J. D. Cherry, 1981. Nonbacterial pneumonia, p, 186-196. In R. D. Feigin and J. D. Cherry (ed.), Textbook of pediatric infectious disease, W. B . Saunders Co., Philadelphia. Brtit, C. D., H. W. Kim, and A. J. Vargosko. 1969. Infections in 18,000 infants and children in a controlled study of respiratory tract disease. I. Adenovirus pathogenicity in relation to serologic type and illness syndrome. Am. J. Epidemiol.

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In R, D. Feigin and J. D. Cherry (ed.), Textbook of pediatric infectious diseases. W. B. Saunders Co,, Philadelphia. l3 Cherry, J. D. 1981. Upper airway infections-the common cold, p. 97-103. Ipt R. D. Feigin and J. D. Cherry (ed.), Textbook of pediatric infectious diseases. W. B. Saunders Co., Philadelphia. 14, Connor, J. D. 1970. Evidence of an etiological role of adenoviral infection in the pertussis syndrome. N. Engl. J. Med. 283:390-394. 15. Cooney, M. K. 1985. Adenoviruses, p* 701-704. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 16. Drew, W. L., and L. Mintz. 1979. Rapid diagnosis of varicella-zoster virus infections by direct immunofluorescence. Am. J. Clin. Pathol. 73:699-701. 17. Drew, W. L., and W. E. Rawls. 1985. Herpes simplex viruses, p. 705-710. In E. H. Lennette, A, Balows, W. J. Hausler, Jr., and H, J, Shadomy (ed.), Manual of clinical microbiology, 4th ed, American Society for Microbiology, Washington, DC 18. Evans, A. S., and E. C. Dick. 1964. Acute pharyngitis and tonsillitis in University of Wisconsin students, J. Am. Med. Assoc, 190:699-708. 19. Fox, J. P., and C. E, Hall (ed.). 1980. Viruses in families. Department of Epidemiology, School of Public Health and Community Medicine, University of Washington, Seattle. PSG Publishing Co., Inc., Littleton, Mass. 20. Frank, A, L., R. B. Couch, C, A, Eriffis, and B, D. Baxter. 1979. Comparison of different tissue cultures for isolation and quantitation of influenza and parainfluenza viruses, J, Clin. Microbial. l&32-36. 21, Friedman,

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Chanock, R., It. Chambon, W. Chang, F. G, Ferrlera, P. Gharpuri, IL. Grant, J. Hatem, I. Iman, S. Kalra, K. Lim, J. Madalengoitia, L. Spense, P. Teng, and W, Ferreira. 1967. WHO respiratory disease survey in children, A serological survey. Bull, WHO 37:363-369. 11, Chemesky, M. A., C. G, Ray, and T. F. Smith. 1982, Cumitech 15, Laboratory diagnosis of viral infections, Coordinating ed., W. L. Drew. American Society for Microbiology, Washington, D.C. 12. Cherry, J, D, 1981. Acute bronchitis, p* 174-177.

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H. M., and C, B, Ferrer, 1982. Diagnosis of viral respiratory infections in the 1980’s. Clin. Lab, Med. 2:383-391. Gardner, P. S, 1977. How etiologic, pathologic and clinical diagnoses can be made in a correlated fashion. Pediatr. Res. 11:254-261. Gardner, P, S., and J. McQuiIlin. 1974. Rapid virus diagnosis: application of immunofluorescence. Butterworth & Co., Ltd., London, Gleaves, C. A., T. F, Smith, E. A* Shuster, and G. R. Pearson. 1985. Comparison of standard tube and shell vial cell culture techniques for the detection of cytomegalovirus in clinical specimens. J. Clin. Microbial. 21:217-221. Glezen, W. P. 1983. Viral pneumonia as a cause and result of hospitalization. J. Infect. Dis. 147:765-770. Glezen, W. P., G. W. Fernold, and J. B. Lohr. 1975. Acute respiratory disease of university students with special reference to the etiologic role of Herpesvirus hominis. Am. J. Epidemiol. 101: 11l121. Goldstein, L. C., J. McDougall, R. Hackman, J. D. Meyers, E, D, Thomas, and R. C. Nowinski. 1982. Monoclonal antibodies to cytomegalovirus: rapid identification of clinical isolates and preliminary use in diagnosis of cytomegalovirus. Infect. Immun. 38:273-281. Gwdtiey, J. M,, Jr. 1982. Rhinoviruses, p. 491517. In A. S. Evans (ed.)., Viral infections of

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