Cumitech 26: Laboratory Diagnosis of Viral Infections Producing Enteritis

Cumitech IA Blood Cultures II June 1982 Cumitech 2A Laboratory Diagnosis of Urinary Tract Infections March 1987 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 Agent Susceptibility Te\ting September 1977 Cumitech 7A Laboratory Diagnosis of Lower Respiratory Tract Infection\ September 1987 Cumitech 8 Detection of Microbial Antigens by Counterimmunoelectrophoresis December 1978 Cumitech 9 Collection and Processing of Bacteriological Specimen5 * August 1979 Cumitech IO Laboratory Diagnosis of Upper Respiratory Tract Infection5 December 1979 Cumitech I I Practical Methods for Culture and Identification of Fungi m the Clinical MicrobIology Laboratory August 1980 Cumitech I2 Laboratory Diagnosis of Bacterial Diarrhea October I980 Cumitech I3 Laboratory Diagnosis of Ocular Infections May 1981 Cumitech I4 Laboratory Diagnosis of Central Nervous System Infections January 1982 Cumitech IS Laboratory Diagnosis of Viral Infections March 1982 Cumitech I6 Laboratory Diagnosis of the Mycobacteno\es March 1983 Cumitech I7 Laboratory Diagnosis of Female Genital Tract Infection\ August 1983 Cumitech I8 Laboratory Diagnosis of Hepatitis Viruses January 1984 Cumitech I9 Laboratory Diagnosis of Chlamydial and Mycoplasmal Infections 9 August 1984 Cumitech 20 Therapeutic Drug Monitoring: Antimicrobial Agents October 1984 Cumitech 21 Laboratory Diagnosis of Viral Respiratory Disease March 1986 Cumitech 22 Immunoserology of Staphylococcal Disease August 1987 Cumitech 23 Infections of the Skin and Subcutaneous Tissues June 1988 Cumitech 24 Rapid Detection of Viruses by lmmunofluorescence August 1988 Cumitech 25 Current Concepts and Approaches to Antimicrobial Agent Susceptibility Testing December 1988 l

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Cumitechs 1989. Cumitech Smith. American

should be cited as follows, 26. Laboratory diagnosis Society for Microbiology,

e.g.: Sherlock, C. H., C. J. Brandt, P. J. Middleton, and J. A. Smith. of viral infections producing enteritis Coordinating ed., J. A, Washington, DC

Editorial Board for ASM Cumitechs: Willlam J. Martone, John E. McGowan, Smith, Thomas J Tlnghltella, and Alice

Steven C Specter, Charman, Carl Abramson, Ellen Baron, Jr., Frederick S. Nolte. Glenn D Roberts, James W Smith, John A S. Weissfeld

The purpose of the Cumitech series is to provide consensus recommendations of-the-art operating procedures for clinical microbiology laboratories which routine or new methods. The procedures given are not proposed OS “standard” methods.

Copyrlgnt

may

by the author; as to appropriate statelack the facilities for fully evaluating

0 1989 American Society for MIcrobIology 1325 Massachuseiis Ave , NW Washlngton, DC 20005

LABORATORY CHRISTOPHER H. SHERLOCK, British Columbia, Vancouver,

DIAGNOSIS PRODUCING

Division of Medical Microbiology, British Columbia V5Z lM9, Canada

CARL J. BRANDT, Department of Pediatrics and George Washington University School PETER J. MIDDLETON, Columbia, Vancouver,

OF VIRAL ENTERITIS

INFECTIONS

St. Paul’s

Hospital

and

(Microbiology), Children’s Hospital, National of Medicine and Health Sciences, Washington,

Division of Medical Microbiology, Provincial British Columbia V5Z lM9, Canada

JOHN A. SMITH, Division of Medical Microbiology, Columbia, Vancouver, British Columbia V.52 IM9, COORDINATING JOHN A. SMITH, Division of Medical Microbiology, Columbia, Vancouver, British Columbia V5Z lM9,

INTRODUCTION AND EPIDEMIOLOGY Early studies employing isolation techniques, including those for enteroviruses and adenoviruses, generally failed to implicate viruses as the etiological agents in acute nonbacterial gastroenteritis (67). The modern era of laboratory study of diarrhea of viral etiology began in 1972 with the successful use of immune electron microscopy (IEM) by Kapikian et al. (37), which demonstrated the causative virus of a large outbreak in Norwalk, Ohio. Subsequent studies have documented that the Norwalk agent, caliciviruses, astroviruses (Fig. l), and several other enteric viruses of a similarly small (26- to 32-nm) size frequently cause outbreaks of enteritis that involve adults and school-age children (36). Such illnesses rarely require hospitalization and outbreaks often end in about a week. Waterborne, foodborne, and person-to-person transmission of these viruses have been described (23). In 1973, Bishop et al. (5) used electron microscopy (EM) of sectioned intestinal biopsy specimens of children with enteritis to detect what are now known as rotaviruses. Soon after, these 70-nm wheellike viruses (Fig. 1) were visualized in large numbers in diarrhea1 stools of children with acute, nonbacterial gastroenteritis (6). Rotaviruses are now known to be the leading worldwide cause of serious viral enteritis in infants and young children (32, 35) Rotaviruses are conventionally typed in three different ways: (i) by group, which identifies viruses with crossreacting antigens (groups A to F); (ii) by serotype, which identifies the antigenic specificity of the major capsid protein by neutralization assay;

Vancouver Canada

General

Laboratory

University

of

Medical Center, D.C. 20010

and University

of British

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and University

of British

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and University

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EDITOR Vancouver Canada

General

and (iii) by electropherotype, which identifies virus strains by the electrophoresis pattern of the segmented RNA. In most clinical situations these procedures are not necessary, but they can be useful in investigations of epidemics. It should also be remembered that group B rotaviruses have been identified in the United States and in recent epidemics of severe rotavirus gastroenteritis in adults in the People’s Republic of China. These non-group A viruses are unlikely to be identified by current commercially available immunoassays. There are at least four epidemiologically important human serotypes and a large numb.er of animal rotaviruses, but most human infections apparently derive from human sources. In temperate climates, rotavirus outbreaks occur annually, particularly in winter (10). Rotavirus is endemic in the tropics (8). Also, asymptomatic endemics occur in some newborn nurseries (28). Most rotavirus transmission probably occurs relatively directly by the fecaloral route. Waterborne outbreaks can occur (41, 55). Rotaviruses spread by aerosols among mice and probably also among humans, especially under conditions of low indoor relative humidity (8, 33). Adenoviruses, particularly enteric types 40 and 41 (19), apparently are second only to rotaviruses in causing viral diarrhea. These 80-nm viruses with icosahedral morphology can be seen in diarrhea1 stools by direct EM (Fig. 1). In contrast to other adenoviruses, enteric adenoviruses rarely grow in conventional cell cultures, and if these serotypes are to be cultured, special cell lines are needed (53). These adenoviruses

FIG. 1. Viruses associated with gastroenteritis (~300,000; courtesy of Maria Szymanski, Hospital for Sick Children, Adenovirus; (B) rotavirus; (C) calicivirus; (D) minireovirus; (E) astrovirus; (F) Norwalk-like virus.

Toronto,

Ontario, Canada). (A)

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epidemic gastroenteritis-rotaviruses, ruses, Norwalk and Norwalk-like civiruses, and astroviruses.

FIG. 2. Coronavirus. especially infect infants and tend to be present endemically in a community (10, 11). They are probably spread primarily by fecally contaminated objects and water. A number of other viruses, for example, influenza viruses in children (39, 42), may be associated with gastrointestinal symptoms although the gut is not the principal site of replication of these viruses. In other cases the gut is the principal site of replication but not the site of disease; this is certainly the case with the enteroviruses which cause a wide range of diseases but rarely, if ever, cause gastroenteritis except in newborns. All adenoviruses appear to replicate readily in the gastrointestinal tract and frequently cause gastroenteritis, particularly in young children. However, the nonenteric adenoviruses are frequently found also in the stools of asymptomatic individuals and may be shed for long periods (many months in some cases) without symptoms (26). This is in contrast to enteric types 40 and 41, which appear to be more frequently associated with symptoms. Other viruses replicate in the gastrointestinal tract but are not so far associated with disease, such as the nonrotavirus reoviruses. Others, such as the coronaviruses (Fig. 2), appear to have an association with enteritis, but much more work needs to be done to confirm or refute this. Abnormalities in the immune system of the host may extend the disease spectrum of many viruses so that diseases not normally encountered with these viruses occur. For example, cytomegalovirus frequently causes enteritis in severely immunocompromised individuals, such as those with acquired immunodeficiency syndrome or bone marrow transplant recipients, and enteroviruses may cause chronic enteritis in individuals with severe combined immune deficiency. This Cumitech will focus only on those viruses that are known to cause endemic and

3

adenoviviruses, cali-

PATHOGENESIS OF VIRAL ENTERITIS The pathophysiology and clinical features of viral enteritis, both in humans and in animals, have been reviewed by Hamilton and Gall (29) and by Bachmann and Hess (2). Columnar epithelial cells in the distal parts of the villi of the small intestine are the usual sites of viral replication. Light microscopy reveals shortening of the villi, elongation of crypts, and increased numbers of mononuclear inflammatory cells in the lamina propria. Irregularities of the brush border, changes in the villus tip cells from columnar to cuboidal, and vacuolation of the columnar epithelial cells are sometimes present. These pathological changes are usually patchy, with intervening areas of apparently normal mucosa. The presence of viral antigen and virions in the villus cells has been monitored, respectively, by immunofluorescence microscopy and thin-section EM. Vomiting may be accounted for by the stimulation of vagal and sympathetic afferent nerves that are connected to the vomiting center in the medulla oblongata. Diarrhea is characterized by profuse watery stools containing increased concentrations of electrolytes. Two mechanisms are advanced to explain why diarrhea occurs. By far the more important is the functional immaturity of villus epithelial cells; the other involves a reduction in the total absorptive area resulting from flattening and clumping of villi. Replacement of the epithelial cells in the villi is a constant process in which immature cells are generated in the crypts and migrate up the villi to be shed at the tips. Crypt cells are rich in thymidine kinase, while mature cells contain disaccharidases, alkaline phosphatase, and the active sodium pump (Na+-K+)ATPase. A glucose-stimulated sodium transport is located in the brush border. Crypt cells are secretory in nature, while mature villus enterocytes are functionally absorptive. Immature “secretory” cells migrate up the villus at an accelerated rate but are incapable of adequate water and electrolyte uptake-hence the expanded luminal contents of both water and electrolytes. Virus-infected cells are shed from the villi. In viral infection, leukocytes usually are not seen in the stool, although the bowel wall may show a mixed inflammatory infiltrate. CLINICAL SYNDROMES The triad of nausea, vomiting, and diarrhea is common to all of the viral enteritides. The symptom complexes are as varied as they are nonspecific and range from asymptomatic infection to severe vomiting and diarrhea, abdominal

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26

rhea is rare and suggestsa bacterial cause. In a smallminority of patients, dual enteric infection with a second virus or with a bacterium may occur (58). Fever is seen in more than 80% of Range of duration (days) cases, and malaiseor listlessnessoften is eviVirus Incubation Virus Vomiting Diarrhea dent. Abdominal pain occurs in fewer than 20% period shedding of cases. Rotavirus l-2 2-3 5-8 8-10 Dehydration is the key feature of rotavirus Adenovirus S-10 2-3 4-12 8-13 infection and is the principal reason for admisl-2 0.05-l l-2 3 Norwalk, sion to the hospital. This is a consequenceof Norwalk-like rapid fluid loss in a short space of time, a characteristic of those children who experience frequent vomiting and diarrhea during the first cramps, fever, myalgia, malaise,significant de- few days of illness. This dehydration is usually hydration, and, rarely, bloody stools(58,65). In mild and isotonic, with compensatedmetabolic most cases, epidemiologicalfeatures of infec- acidosis. Fatalities in developed countries are tion, particularly the age of the patient, the rare and are due to severe, sometimeshyperseasonof the year in temperate climates, the tonic, dehydration, often with acidosis. Maximal virus excretion occurs at about 3 to 5 duration of symptoms, and the severity of disease,allow an educatedguessto be madeabout days after the onset of symptoms. Shedding the infectious agent. This is important in direct- often persistsfor about 8 to 10 days, and the ing the approachto laboratory diagnosis.Enteri- amount of virus tends to diminish as symptoms tis in underdeveloped countries is frequently subside.In a minority of children who are not complicated by multiple infectious agents, mal- immunocompromised,sheddingcan persist for nutrition, and malabsorption and consequently as long as 2 to 3 weeks after diarrhea has may present a clinical picture different from that stopped (60). Immunocompromised children seenin developed countries. shedvirus for longer time periods (64). Table 1 summarizessomeof the typical feaADENOVIRUSES tures of enteric viral infections. As with the rotaviruses, gastroenteritiscaused ROTAVIRUSES by adenovirusestends to becomeless common The ageof the patient hasa profound effect on with increasingage (9-12). The diseaseis parthe presentation of the disease.Infants lessthan ticularly associatedwith serotypes40 and 41. 3 monthsof age, aswell asadults, frequently are Adenoviral gastroenteritis usually is milder infected but may be only mildly ill or asymptom- than its rotavirus counterpart. The onset tends atic (3, 4, 28, 63). Symptomatic diseaseespe- to be lessabrupt, with vomiting and fever that cially affects children 6 through 24 months of are milder and of shorter duration, averaging age. The diseasealsotends to becomemilder as about 2 days. However, adenoviral diarrhea the age of the child increases,possibly because may be more persistentthan that associatedwith acquiredantibody to the different serotypespro- other viral agents,at timeslasting for 12days or vides protection, including crossprotection, and more with adenovirus 41 and 8 days or more alsobecauseof increasingmaturation of intesti- with adenovirus 40 (59, 61). Respiratory symptoms are not common in patients infected with nal epithelial cells. Considerableconsistency has been recorded the enteric adenoviruses, although respiratory in most studiesof the duration of symptomsand carriagecan occur (34). In contrast, gastrointesthe percentages of individuals experiencing tinal symptoms associatedwith other adenovithese symptoms. The incubation period has rus serotypes,in which upper respiratory sympbeenestimatedat 24 to 48 h (38). Characteristic toms tend to be prominent, are of shorter duraof rotavirus infection is an abrupt onset of vom- tion (59). iting which may be quite pronounced and may be the major symptomwithin the first 1 to 3 days NORWALK AND NORWALK-LIKE VIRUSES after the onsetof symptoms(58,65). Santosham The characteristic feature of symptomatic inet al. (48)found rotavirus particlesin respiratory fection with Norwalk and Norwalk-like viruses, secretionsof someinfected patients. However, as exemplified by the prototype outbreak in probably fewer than 50% of patients have respi- Norwalk, Ohio, is the short duration of sympratory symptoms, and concomitant infection by toms: asshort as 12h and rarely more than 48 h. respiratory pathogensmay also be involved (9). The clinical features associatedwith these viWatery diarrhea with mucusis an almost invari- ruseshave been well studied both in outbreaks able feature of infection in young children, and and in volunteer studies.The symptom complex this often persistsfor 5 to 8 days. Bloody diar- is difficult to differentiate from that of the other TABLE 1. Most frequently observed ranges of duration of clinical and virological features of enteric virus infections

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enteric viruses in that vomiting, diarrhea, fever, malaise, etc., may all be present; however, these systemic symptoms tend to be absent or mild. It is usually only an epidemic outbreak that draws medical attention owing to the mild, selflimited nature of endemic disease. In outbreaks and in volunteer studies, viral shedding in adequate amount for detection by EM has only occurred in the symptomatic phase of infection; the virus is only detectable up to about 72 h after the onset of illness (22, 57). CALICIVIRUSES, ASTROVIRUSES, AND SMALL, ROUND VIRUSES Symptoms are generally milder in calicivirus enteric infections than in Norwalk and rotavirus infections. However, these infections are difficult to differentiate clinically. Calicivirus outbreaks tend to last 3 to 5 days longer than those of Norwalk virus (14). In this sense, calicivirus disease may mimic rotavirus infection but is generally milder. Less well characterized are the clinical characteristics of astrovirus infection and the symptoms and signs associated with the other small, round viruses found in diarrhea1 stools. All may cause nausea, vomiting, and diarrhea, but their pathogenicity appears to be low (43). INFECTIONS AND IMMUNOCOMPROMISED PATIENTS Diarrhea often is seen in individuals who are immunocompromised or immunosuppressed, and there is increasing evidence that viruses can play a significant role in producing such diarrhea. Also, various causes of the immunocompromised state tend to be associated with the shedding of enteric viruses in unusually large quantity, infection for unusually long periods, and even infection by as many as five different enteric viruses at one time (15, 17, SO, 64, 66). Enteric viral infections appear to lead to increased mortality among those who are immunocompromised and are a potential source of infection to susceptible contacts. ROLE OF DIAGNOSIS IN PATIENT MANAGEMENT As with most bacterial enteric infections, the course of these viral enteritides is almost always self-limited in well-nourished, immunologically normal individuals, and in many cases they require little or no medical intervention. However, a significant minority of children become dehydrated, particularly with rotavirus infection. Worldwide, many young children die from this complication (32, 35). As many as 50% of young children with serious acute gastroenteritis are admitted to a hospital, resulting in major health care costs. Thus,

VIRAL

INFECTIONS

5

the role of specific diagnosis is critical in directing appropriate treatment, such as exclusion of unnecessary antibiotics and initiation of infection control measures, in providing a prognosis, and in keeping down costs. In addition, specific diagnosis is essential in assessing an epidemic of gastroenteritis such as that caused by contaminated food (especially shellfish) or water. Viral contamination of water is not predicted by fecal coliform bacterial counts, so it is important to identify the etiological agent of an outbreak in order to locate the source and eliminate its spread (62). PATIENT MANAGEMENT No specific antiviral agent is presently effective against the gastroenteritis viruses. However, since some bacterial enteritides require antibiotic treatment, it is important to identify these viruses to distinguish them from bacterial agents. The single most important management modality is fluid replacement. This generally can be achieved easily at home with proprietary preparations. In cases in which rapid fluid loss is occurring from severe vomiting and diarrhea, parenteral fluid replacement is necessary, requiring admission to a hospital. A specific diagnosis of viral infection at this point will achieve important gains: unnecessary antibiotic treatment can be avoided, additional expensive enteric bacterial cultures or examination for parasites may not need to be done, and spread of the virus to other patients and staff can be prevented by appropriate infection control measures. In the vast majority of pediatric cases, the cause of the diarrhea will be rotavirus (particularly in winter) or enteric adenovirus. As detailed below, these can be diagnosed rapidly and accurately in the laboratory, whereas most bacterial pathogens require 2 to 3 days for specific identification. Also, a specific diagnosis and knowledge of the prognosis will allow early discharge of the patient from the hospital once the fluid and electrolyte status has been stabilized. An important diagnostic decision time is at 48 h of illness when it will generally be clear whether the disease is resolving. Also, virus excretion will then be maximal, affording the best chance of laboratory diagnosis. If the disease is resolving, in most cases there is no need for a diagnosis. Two important exceptions to this general rule are the following: (i) when it becomes apparent that an epidemic outbreak is occurring, in which case selected patients should be investigated early in the course of their illness to establish an etiology and probable source; and (ii) when early, rapid fluid loss has occurred, requiring admission to an hospital. A key point is that admission to the hospital may be prevented in many cases, once the diagnosis

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is established and if oral rehydration can be achieved. When sporadic, community-acquired gastroenteritis persistsbeyond 2 to 3 days, a diagnosis should be sought to exclude treatable causes (e.g., Clostridium difzcile colitis and giardiasis) and noninfectious causes.With children, testing for rotavirus can be achieved rapidly, and in the majority this will obviate more lengthy and expensive tests for bacteria and parasites. A caveat here is that newbornsand infants may be shedding rotavirus asymptomatically (27) and their symptoms may be caused by another pathogen. In immunologically compromisedpatients, a diagnosisshould be attempted without delay since, in general, all infectious diseases are more severe and progressmore rapidly in thesehosts, SPECIMENS

Regardlessof the ultimate method used for detecting evidence of infection by any of the enteritis-producing viruses, a fecal sampleof at least 1 g is the specimenof choice. Submission of rectal swabsshould be discouragedbecause the amount of virus in the specimen may be insufficient to allow a reliable laboratory diagnosis. In fatal rotavirus and adenovirus infections, virus can also be detected in duodenal aspiratesandjejunal biopsies(45). Transport to the laboratory by mail should be in accordance with the Wsrld Health Organization recommendations (44). A leakproof vial wrapped in absorbent material is placed in a sealedplastic bag. This is insertedinto an appropriatelylabeled metal canister. The requisition shouldbe placed outsidethe container,to avoid further problemsin caseof leakage.For transportation within an institution, the sample principles apply, namely avoidance of leakageand contamination of the requisition. Nonenveloped virions and virus antigens in feces are relatively resistant to destruction; thus, continuous refrigeration is not necessary. In a World Health Organization survey in which 12laboratoriesworldwide exchangedstool samples containing virus, by meansof unrefrigerated air freight, good agreementwas observed amongdifferent laboratoriesusingEM detection for the more structurally robust viruses such as rotavirus and adenovirus. On the other hand, the receiving laboratories found that it was difficult to detect astrovirus, calicivirus, minireovirus, and coronavirus, presumablybecausethese virions readily lose their structura1integrity (C. R. Madeley , personal communication), For longterm preservation, -70°C is recommended.Cycles of freezing and thawing mustbe avoided, as thev lead to disintegration of virions.

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LABORATORY PROCEDURES FOR DIAGNOSIS EM

Direct EM is a usefuland rapid (lo- to 15min) meansfor detecting enteric viruses, particularly rotaviruses and adenoviruses,in the stools of patients with acute gastroenteritis(21). It is also the standardtest procedurewith which sensitivity and specificity of other methods,.includingimmunologicaltests,have traditionally beencompared. For direct EM, a 10%suspensionof stool can be prepared in water and mixed with an equal volume of 1.5 to 3% phosphotungstic acid (brought to pH 7.1 with sodiumhydroxide). One drop of the mixture is applied to a Formvar (or Formvar-carbon)-coated specimen grid. The grid is blotted dry with filter paper after 2 min and viewed with a transmissionelectron microscope at approximately X 50,000 magnification until found to be positive or for at least 6 min. Grids are bestread the sameday aspreparedbut often are useful for demonstrationsor diagnosis for weeksto months after preparation. Specimensfound to be negative by direct EM examination can be shown at times to be virus positive after ultracentrifugation or by IEM with high-speedcentrifugation to concentrate virions. Various manipulationsof the stool preceding EM and IEM have been reviewed (18, 30). The following techniquesmay be useful. (i) Make a 10 to 20% suspensionof feces in phosphate-bufferedsaline. Clarify by centrifugation at 1,500x g for 20 min. Next, centrifuge the supernatantfluid at 12,ooOx g for 30 min at 4”C, and centrifugethat supernatantfluid at lOO,0OO x g for 1 h at 4°C. Suspendthe pellet in phosphatebufferedsalineor distilledwater, sonicatefur 30s, placea drop on a preparedEM grid, and stain for 10 s with a drop of 1.5 to 3.0% potassiumphosphotungstatethat haspreviously been adjustedto pH 6.5 to 7.0 with sodiumhydroxide. (ii) Mix 4 ml of a clarified 20% fecal suspension with 6 ml of a saturated (NH&SO, solution. Stand the mixture at 4°C for 1 h, and ultracentrifuge at 50,000 x g for 2 h. Suspend the pellet in 4 drops of distilled water, and prepare negatively stained EM grids, using potassiumphosphotungstateas above. (iii) For IEM, 0.5 ml of clarified (1,500 x g, 20 min) fecal suspensionis added to 0.1 ml of undiluted, lo-fold, and 1O&fold dilutions in phosphate-bufferedsaline of either human immune globulin or specific antiserumwhich may be of humanor animal origin. The mixtures may be incubated overnight at 4°C or for 3 to 2 h at 37°C. The mixtures are subsequentlypelleted in the ultracentrifuge (35,000 x g for 90 min). Doane and Anderson (21) describe a serumin-anar method for performing IEM. The same

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authors detail a protein A-gold IEM procedure which is even more sensitive than conventional IEM methods for detecting virus in stools. Hammond et al. (30) employed an airfuge expeditiously to increase the yield of positive specimens from clinical specimens and virus isolates. The airfuge rotor takes six specimens at a time and is operated at 90,000 rpm for 30 min; this generates 178,000 x g at a maximum pressure of 30 lb/in2. Virus particles are deposited onto EM grids located at the periphery of the rotor. The processing of stool samples to detect small, round viruses calls for the use of salts containing divalent ions, e.g., CaCl, or MgCl,, in the stool suspension buffer and potassium tartrate-glycerol rather than cesium chloride to form a density gradient medium (1). If these guidelines are not followed, particle disintegration occurs. Croxson and Bellamy (16) describe a simplified extraction procedure for rotavirus and rotavirus RNA. In their protocol cold 1% lithium dodecyl sulfate at a neutral pH was used to treat fecal material before the low- and high-speed centrifugation steps. Those who lack an electron microscope should also note that it is quite feasible to prepare grids and then mail them to a facility equipped to perform EM. Alternatively, the stool specimen may be tested by an immunological method. ELISA Procedures Enzyme-linked immunosorbent assay (ELISA) techniques are the most sensitive methods routinely used for the detection of viruses that cause diarrhea (20). By virtue of the availability of a variety of commercially prepared kits, reagents, and equipment, these are now the method of choice in many laboratories for such diagnosis. However, users of ELISA procedures should be aware of the sensitivities and specificities of individual kits. Many ELISA results can be obtained within 2 to 4 h, and reading of endpoints without the use of a spectrophotometer often is possible. In its simplest form (and without mention of the necessary washing and incubation steps), ELISA procedures for antigen detection typically begin with a virus-specific antibody coated on a plastic bead or well. Homologous virus in a fecal specimen binds to this antibody. An enzyme-labeled specific antibody is then bound to the virus. The addition of the enzyme substrate results in a color-producing reaction, indicating a positive test. Latex Agglutination Procedures which utilize latex agglutination for antigen detection are both simple and rapid (OS h).- Typically, a stool specimen is mixed

VIRAL

INFECTIONS

with a small quantity of buffer solution and clarified by centrifugation at 1,500 x g for 20 min, and the supernatant fluid is tested for its ability to aggregate a suspension of latex particles coated with virus-specific antibody without aggregating a control latex suspension. A magnifying glass can be helpful in detecting minimal agglutination. Appropriately controlled positive reactions can be quite specific (7, 20). Firstgeneration latex agglutination methods were insensitive, and nonspecific agglutination reactions commonly occurred simultaneously with both the negative control suspension and the antibody-positive suspension. However, newer commercial kits in which monoclonal antibodies are used have shown considerable improvement in performance (56). Antibody Detection Measurements of antibody responses to diarrhea viruses are rarely attempted except for research into virus epidemiology or studies of experimental vaccines. Virus Cultivation Virus culture is not suitable for the routine diagnosis of diarrhea virus infection except in cases of adenovirus infection. For cultivation procedures applicable to research, see the agentspecific methodology below. ROTAVIRUS-SPECIFIC DIAGNOSTIC PROCEDURES EM Direct EM is the most rapid laboratory means for the diagnosis of rotavirus infection and, for laboratories that have the appropriate equipment, EM can be both an efficient and a reasonable practical (although expensive) means for diagnosing such infections. However, many laboratories do not have access to EM, and ELISA methods are acceptable alternatives. Moreover, although EM is a rapid method for small numbers of specimens, enzyme immunoassay methods are more appropriate for larger numbers of specimens. Three factors account for the utility of EM. First, very large numbers of virions (10’ to 109/ml) are regularly present in the stools of infants, children, and some adults who are acutely ill with rotavirus gastroenteritis; thus, 10 or more virions often are seen per minute of EM viewing of unconcentrated diarrhea specimens. Second, rotaviruses are easily recognized by virtue of their characteristic morphology, frequently resembling an approximately 70-nm spoked wheel with a large hubcap (Fig. 1). Third, infections by viruses other than (or in

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addition to) rotaviruses can be detected in the test specimens. Rotaviruses of differing groups, subgroups, and serotypes are indistinguishable by direct EM, although serotyping can be performed by IEM if appropriate antisera are available (28). Electropherotyping Rotaviruses can be recognized and identified by virtue of the patterns of migration of the 11 segments of their RNA upon polyacrylamide gel electrophoresis, a means of identification commonly called electropherotyping (35). The migration of RNA segments is strain specific and thus is especially useful for tracking the spread of infection in settings such as nurseries or day-care centers. Many different electropherotypes exist, and viruses from any one serotype commonly have different electropherotypes. However, viruses of different serotypes at times have the same electropherotype. Of diagnostic importance, type 2 rotaviruses can presumptively be distinguished from the other human serotypes by electrophoresis, since many serotype 2 rotaviruses have slow-migrating RNA segments 10 and 11 (short migration patterns). Electrophoretic procedures are too slow, complex, and expensive to be used for the routine diagnosis of rotavirus infection, but they can be very useful for research and epidemiological purposes. Enzyme Immunoassay In many laboratories, a rotavirus ELISA is the only test used to diagnose any diarrhea virus infection. This occurs not only because of a general realization that rotaviruses are the most important human diarrhea viruses and that ELISA procedures are the most sensitive of the readily available methods for detecting these agents, but also because licensed diagnostic kits are commercially available at modest prices and the tests (13, 20) are relatively rapid and simple to perform. The apparent sensitivity and specificity of rotavirus ELISA procedures are highly dependent on the quality of the standard against which they are compared, typically EM, IEM, electrophoretic analysis, or another ELISA procedure. It is important to note that rotavirus ELISA kits routinely detect group A rotaviruses that cause most human infections but typically miss nongroup A viruses which cause disease in humans, notably in China. In the most carefully controlled studies with the best of reagents and techniques and when used only to detect group A rotaviruses, ELISA sensitivity and specificity can exceed 95% (20). However, with disturbing frequency the re-

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ported sensitivity and specificity of individual rotavirus ELISA tests fall below 90%, and quite different results may be obtained when two or more ELISA procedures are used to test collections of positive and negative stool specimens from different patients or even to test successive stool specimens from the same patient. Thus, caution must be exercised when reporting critically important results, particularly unexpected or unusual results, which are based only on a rotavirus ELISA. Radioimmunoassay and Dot Blot Methods For rotavirus diagnosis, the best radioimmunoassay and dot blot nucleic acid hybridization methods tend to exceed ELISA procedures in sensitivity, while specificity is similar to that of ELISA. Detection by dot blot of as low as 8 pg of rotavirus RNA has been reported (24). Compared with ELISA, these methods are more complex, time-consuming, and expensive, require the use of radioisotopes, and are not commercially available in diagnostic kits. Essentially, then, the disadvantages of dot blot and radioimmunoassay procedures preclude their use for more than research applications. Latex Agglutination Latex agglutination methods seem particularly well suited for testing diarrhea1 stool in a physician’s office, as such tests require little equipment, are commercially available, and require less than 30 min to perform. Thus, results can potentially be obtained before the patient leaves the office. For such tests, stool specimens typically are mixed in a suspending buffer solution and clarified by centrifugation at 1,500 x g for 20 min. The supernatant fluid is added to rotaviruspositive (antibody-coated) and rotavirus-negative (nonreactive-serum) latex suspensions and gently agitated at room temperature on a black slide or card. Positive agglutination reactions commonly develop within 2 min. The specificity of appropriately controlled latex tests tends to be high. Accordingly, a positive test is likely to indicate infection. However, latex tests typically are the least sensitive of the routinely used methods for rotavirus diagnosis (7,20). Thus, rotaviruses in low concentrations, particularly those in rectal swab specimens or in stools taken late in the course of illness, are not likely to be detected. Latex tests have the additional disadvantage that indeterminate agglutination reactions (simultaneous agglutination with both the rotavirus-negative and rotavirus-positive latex suspension) commonly occur. Thus, a negative latex agglutination should be followed by a more sensitive technique.

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Rotavirus Groups Group A rotaviruses cause most human infections, but large human outbreaks caused by non-group A rotavirus have occurred (55). Nongroup A rotaviruses should be suspected when a reliable ELISA (most of which are group A specific) fails to detect virions which are seen in EM preparations. Rotavirus Subgroups Rotaviruses within group A can be subgrouped by ELISA, and most of these viruses are from subgroup I or II (35). Subgroup I viruses characteristically are from serotype 2. Subgrouping is rather infrequently performed and, with the increasing availability of serotyping methods, probably will be even less frequently performed in the future. Cultivation and Serotyping Human rotaviruses were first grown by Sato et al., using MA104 cell cultures in the presence of trypsin (49). Although much too slow and laborious a procedure for routinely detecting rotavirus infection, cultivation has enabled the use of neutralization procedures, including plaque reduction techniques (35), for rotavirus serotyping. Both virus protein (VP) 7 and VP3 virus components determine serotype specificity (46). Cross-reactivity between serotypes is rather common, particularly when polyclonal antisera are used. Monoclonal serotyping reagents are becoming increasingly available and, when used in ELISA procedures, often can be used to type rotaviruses directly from stool preparations without the need for virus cultivation (54). Serotyping of rotaviruses in stool preparations can also be performed by IEM (27) and dot hybridization (24). Serotype 1 rotaviruses appear to be the most prevalent of the epidemiologically important rotavirus serotypes. ADENOVIRUS-SPECIFIC DIAGNOSTIC PROCEDURES Many of the methods applicable to rotavirus diagnosis have been applied to detecting and identifying adenoviruses, particularly enteric adenoviruses 40 and 41 in diarrhea1 stools. However, experience with the diarrhea-associated adenoviruses is much less extensive than that for rotaviruses. Thus, the relative sensitivity and specificity of individual diagnostic methods for the adenoviruses are not always clear from the existing literature. EM In the acute phase of adenovirus enteritis, adenovirus vii-ions often can be seen in stool specimens bv direct EM. The finding of one or

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more such virions per minute of viewing provides a strong presumptive indication of the presence of adenovirus 40 or 41 (12). Virus Cultivation Adenoviruses 40 and 41 initially were considered to be “noncultivatable” as they usually do not grow serially in HEp-2 or human embryo kidney cells (cells in which previously recognized adenoviruses can be cultivated). However, Takiff et al. demonstrated that adenoviruses 40 and 41 do grow readily in Graham 293 cells (53). Other adenovirus serotypes also grow readily in 293 cells, and they are rather commonly found in stool specimens in the absence of apparent illness. Thus, adenovirus isolates in 293 cells should be specifically identified. Electrophoretic Identification Adenoviruses 40 and 41 from stool specimens or cell cultures can be distinguished from each other and from previously recognized adenoviruses by gel electrophoresis of their DNA after restriction endonuclease treatment (19). Electrophoretic identification is generally regarded as the standard method for distinguishing adenoviruses 40 and 41 and is likely to remain so until serologic means for identification of adenoviruses isolated from diarrhea1 stools are more widely available and more fully evaluated. These methods are relevant to research and epidemiological studies, but not to clinical practice. Dot Blot Hybridization Dot blot hybridization can detect less than 20 pg of adenovirus DNA in stool specimens, although this assay does not distinguish adenovirus 40 from adenovirus 41 (52). Serologic Identification Neutralization or IEM tests with polyclonal antisera can distinguish adenovirus 40 and 41 isolates from the lower-numbered adenoviruses (19). However, because of a marked antigenic similarity between the enteric adenoviruses, it is often not possible to distinguish between adenoviruses 40 and 41 with polyclonal antisera. Monoclonal serotyping reagents have been used to distinguish adenovirus 40 from adenovirus 41, particularly using ELISA technology (31, 51). Ultimately, ELISA procedures are likely to become the method of choice for both detecting and identifying adenoviruses in diarrhea1 stools. Commercially Available Tests Kits capable of detecting group-specific adenovirus antigens by latex agglutination, ELISA, and immunofluorescence have been marketed for in vitro diagnostic use. Adenoviruses in stool specimens and in cell cultures can be detected

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with these kits, but the lack of serotype specificity is an obvious limitation to the general application of these tests for the study of enteric adenoviruses. At least one monoclonal ELISA kit is sold for identifying adenoviruses 40 and 41 in stool specimens (Cambridge Bioscience, Worcester, Mass.). DIAGNOSIS

OF OTHER VIRUSES THAT CAUSE DIARRHEA Except for recognition by EM, viruses such as Norwalk-like agents, astroviruses, and caliciviruses are rarely identified by other than major research and public health laboratories. IEM and ELISA procedures are used for the identification of some of these viruses (36), but antisera for diagnostic purposes often are in extremely short supply. Kits and reagents for detection and identification of some of these viruses probably will not be available commercially for some years. CONCLUSION The viral agents of gastroenteritis are responsible for enormous morbidity worldwide and a high mortality rate in developing countries. Despite the present lack of specific therapy for these infections, it is essential for clinical virology laboratories to be able to make a diagnosis when called upon. There may be major public health implications in instances of food- or waterborne outbreaks of gastroenteritis, and it is essential to identify the agent in such cases. In sporadic gastroenteritis it is important to differentiate treatable from nontreatable infections so that anti-infective therapy is given only in appropriate cases and not applied indiscriminately. It is also critical to good infection control practice in health care institutions to be able to identify the agents causing gastroenteritis in order to control spread effectively. Recent advances in molecular biology, virology, and immunology have made available many diagnostic kits for the identification of rotaviruses and some reagents for other agents. Most of these techniques are within the scope of general microbiology laboratories and should probably be included in their menu if a virology laboratory is not available. More specialized techniques, such as EM and cell culture, will remain in the domain of the virology laboratory, however. For those laboratories that do not have access to an electron microscope, a mechanism should be put in place to refer specimens rapidly to a laboratory with expertise in this area. As clinical and laboratory investigations of the infectious enteritides proceed, one can expect new viral agents to be identified and, consequently, increased demands for specific diagnostic techniques.

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Advances in technology will also bring changes to laboratory practices for the known pathogens. A good example of this is the impact that nucleic acid hybridization and gene amplification techniques are making in virology, in particular for those viruses that are impossible or impracticable to culture (47). For example, a recent report describes a monoclonal antibody to rotavirus RNA which both extends the spectrum of detection to non-group A rotaviruses and increases the sensitivity of detection over that of conventional immunoassays (40). Such developments can only be expected to increase in number. LITERATURE

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1. Ashley, C. R., and E. 0. Gaul. 1982. Potassium tartrateglycerol as a density gradient substrate for separation of small, round viruses from human feces. J. Clin. Microbial. 16:377-381. 2. Bachmann, P. A., and R. G. Hess. 1982. Comparative aspects of pathogenesis and immunity in animals, p. 361-397. In: D. A. J. Tyrrell and A. Z. Kapikian (ed.), Virus infections of the gastrointestinal tract. Marcel Dekker, Inc., New York. 3. Barnett, B, B. 1986. Other viruses with etiologic roles in childhood gastroenteritis. Pediatr. Infect. Dis. S(Supp1.): 4 S75-S82, Bishop, R. F., G. L. Barnes, E. Cipriani, and J. S. Lund. ’ 1983. Clinical immunity after neonatal rotavirus: a prospective longitudinal study in young children. N. Engl. J. Med. 309:72-76. R. F., G. P. Davidson, I. H. Holmes, and B. J. 5* Bishop, Ruck. 1973. Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis. Lancet ii: 1281-1283. 6. Bishop, R. F., G. P. Davidson, I. H. Holmes, and B. J. Ruck. 1974. Detection of a new virus by electron microscopy of faecal extracts from children with acute gastroenteritis. Lancet i: 149-l 5 1. 7. Brand& C. D., C. W. Amdt, G. L. Evans, H. W. Kim, E. P. Stallings, W. J. Rodriguez, and R. H. Parrott. 1987. Evaluation of a latex test for rotavirus detection. J. Clin. Microbial. 25: 1800-1802. 8. Brand& C. D., H. W. Kim, W. J. Rodriguez, J. 0. Arrobio, B. C. Jeffries, and R. H. Parrott. 1982. Rotavirus gastroenteritis and weather. J. Clin. Microbial. 16: 9 478-482, Brandt, C. D., H. W. Kim, W. J. Rodriguez, J. 0. Arro* bio, B. C. Jeffries, and R. H. Parrott. 1986. Simultaneous infections with different enteric and respiratory tract viruses. J. Clin. Microbial. 23:177-179. Brandt, C. D., H. W. Kim, W. J. Rodriguez, J. 0. Arrolo* bio, B. C. Jeffries, E. P. Stallings, C. Lewis, A. J. Miles, R. M. Chanock, A. Z. Kapikian, and R. H. Parrott. 1983. Pediatric viral gastroenteritis during eight years of study. J. Clin. Microbial. l&71-78. Brandt, C. D., H. W. Kim, W. J. Rodriguez, J. 0. Arro*‘* bio, B. C. Jeffries, E. P. Stallings, C. Lewis, A. J. Miles, M. K. Gardner, and R. H. Parrott. 1985. Adenoviruses and pediatric gastroenteritis. J. Infect. Dis. 151~437-443. 12. Brandt, C. D., W. J. Rodriguez, H. W. Kim, J. 0. Arrobio, B. C. Jeffries, and R. H. Parrott. 1984. Rapid presumptive recognition of diarrhea-associated adenoviruses. J. Clin. Microbial. 20: 1008-1009. 13. Chernesky, M., S. Castriciano, J. Mahony, M. Spiewak, and L. Schaefer. 1988. Ability of TESTPAK ROTAVIRUS enzyme immunoassay to diagnose rotavirus gastroenteritis. J. Clin. Microbial. 26:2459-2461. 14. Chiba, G., Y. Sakumo, R. Kogasaka, M. Akihara, K. Horino, T. Nakao, and S. Fukiu. 1979. An outbreak of

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