Cumitech 12A: Laboratory Diagnosis of Bacterial Diarrhea

T

Cumitech 1A Blood Cultures II June 1982 Cumitech 2A Laboratory Diagnosis of Urinary Tract Infections March 1987 Cumitech 3A Quality Control and Quality Assurance Practices in Clinical Microbiology May 1990 Cumitcch 4 Laboratory Diagnosis of Gonorrhea October 1976 Cumitech 5A Practical Anaerobic Bacteriology December 1991 Cumitech 6A New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide February 1991 Cumitech 7A Laboratory Diagnosis of Lower Respiratory Tract Infections September 1987 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 for 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 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 Immunofluorescence August 1988 Cumitech 25 Current Concepts and Approaches to Antimicrobial Agent Susceptibility Testing December 1988 Cumitech 26 Laboratory Diagnosis of Viral Infections Producing Enteritis September 1989 l

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Cumitechsshould be cited as follows, e,g,: Gilligan, P. H., J. M. Janda, M. A. Karmali, and J. M, Miller. 1992. Cumitech 12A, Laboratory diagnosis of bacterial diarrhea Coordinating ed, F. S. Nolte. American Society for Microbiology, Washington, DC

Editorial Board for A!%4 Cumitechs: Mary J R Gilchrist, William J. Martone, Tinghitella, and Alice S Weissfeld

Steven

Frederick

C

Specter,

S Nolte,

Chairman, Carl Abramson, Ellen Jo Baron Arne C Rodloff, John A Smith, Thomas J

The purpose of the Cumiiech series is to provide conserxus recommendations state-of-the-art operating procedures for clinical microbiology laboratories which evaluating routine or new methods. The procedures given are not proposed as “standard” methods.

Copyright

0 1992 Amerrcan Society for Mrcroblology 1325 Massachusetts Ave , NW Washington, DC 20005

by the authors as to appropriate may lack the facilities for fully

LABORATORY

DIAGNOSIS OF BACTERIAL

PETER H. GILLIGAN, Clinical Microbiology-Immunology Hospitals, Chapel Hill, North Carolina 27514 J. MICHAEL JANDA, Microbial Berkeley, California 94704

Diseases Laboratory,

Laboratoties,

DIARRHEA

University of North Carolina

State of California Deparhnent

MOHAMED A. KARMALI, Department of Microbiology, Toronto, Toronto, Ontario M5S IAI, Canada

The Hospital for Sick Children, University of

J. MICHAEL MILLER, Diagnostic Microbiology Section, Hospital Infections Prowam, Diseases, Centers for Disease Control, Atlanta, Geo@a 30333 COORDINATING FRZDERICK S. NOLTE, Clinical Microbiology Atlanta, Georgia 30322

of Health Services,

Center for Infectious

EDITOR

Laboratory,

Emory University Hospital, Emory University,

richia coli can cause diarrhea have also been discovered. Recent information concerning the agentsand diagnosisof viral diarrheas hasbeen summarizedin Cumitech 26. Two factors have refocused interest on protozoan diarrhea1pathogens. First, chronic diarrhea due to either Cryptosporidium sp. or Isospora sp. has been recognized asa major problem in AIDS patients. Second, outbreaks of diarrhea1 diseasedue to contamination of municipal water supplieswith Giardia Zamblia increasingly are being recognized. It is almost certain that during the next decade, additional infectious agents that cause diarrhea1diseaseswill be discovered. The detection of enteric bacterial pathogensis greatly complicated by the presenceof a complex and abundant normal fecal microflora. Present soon after birth, the normal intestinal microflora evolves over the first year of life mainly in responseto a changingdietary intake (37). By the first birthday, the gut microflora is essentially establishedfor the remainder of the individual’s life unlessmajor changesare induced by antimicrobial therapy. Fecal microflora obtained from normal adults contains between 1011and 101’ organismsper g, of which 99% are anaerobes, predominantly speciesof Bacteroides, Fusobacterium, Bifidobacterium, Eubacterium, and Peptostreptococcus (52). Compared with the anaerobic fecal microflora, the facultative fecal flora is more modest in number but still found in abundance,with lo* to 10’ organisms present per g of feces. The challenge for the clinical microbiologist is to attempt to detect various diarrhea1 pathogens in this incredibly complex milieu. The objectives of this Cumitech are threefold. First, it will update information on the causesof

Diarrhea1diseasecontinues to be a major humanhealth problem. It hasbeenestimatedthat 1 billion episodesof diarrheaoccur worldwide each year in children lessthan 5 years old, resultingin 5 million deaths(35,144). Diarrhea is particularly devastating in children who concurrently suffer from measlesor protein malnutrition, an all too frequent occurrencein the developingworld (95). Children in the developingworld are estimatedto have three to five times more casesof diarrhea per year than do children who live in countries with highly developed sanitary and water supply systems and adequate, hygienic food supplies (60,144). In the United States, children lessthan 5 years old each have approximately two episodesof diarrhea per year which result in 500 deathsannually. Although morbidity and mortality due to diarrhea are most important in infants and children, diarrhea1diseasehas an important impact on adults as well. Adults, on average, suffer one to two episodesof diarrheayearly (61). This results in significant economiccostsdue to utilization of health care resourcesand lost productivity. Like the young, the elderly, especially those institutionalized in long-term care facilities, are at increasedrisk for serious consequencesresulting from gastrointestinalinfections (60). Since the publication of Cumitech 12, “Laboratory Diagnosisof Bacterial Diarrhea,” in 1980 (149), our knowledge of the various bacterial, viral, and protozoan agentsand mechanismsby which diarrhea is induced hasexpanded greatly. Bacterial agentsof diarrhea1diseaserecognized in the past decadeinclude new speciesof Campylobacter (39) (C. fennelliae, C. lari, and C. cinaedi) and vibrio (68) (V: fluvialis and K hoksae). New mechanismsby which Esche1

2

GILLIGAN

ET AL.

bacterial diarrhea and techniques for their detection presented in Cumitech 12. Second, it will describe new agents of bacterial diarrhea and the means to isolate and identify them. Detailed procedures for identification of each species are found in the Manual of Clinical Microbiolom (9). Third, it will outline cost-effective strategies for obtaining the highest possible yield from stool examinations. Particular emphasis will be placed on recognizing appropriate clinical situations in which various agents should be sought. We are indebted to R. B. Sack, R. C. Tilton, A. S. Weissfeld, and the late S. J. Rubin for their valuable contributions to Cumitech 12 (149). CLINICAL ASPECTS AND PATHOGENESIS Knowledge about the clinical features and pathogenetic aspects of specific enteric infections can provide valuable information about the likely etiology and can be helpful to laboratory workers in channeling their energies toward the detection of relevant enteropathogens, particularly those that are not routinely sought. A history of diarrhea in contacts of an affected patient provides good evidence for an infectious etiology. The length of the incubation period (Table 1) can suggest a particular etiology in outbreaks. A history of recent antibiotic usage should direct attention toward toxigenic Clostridium dificile as an etiology. A history of recent travel may be a clue to enteropathogens such as vibrio cholerae and enterotoxigenic E. coli (ETEC) or to vzbrio parahaemolyticus in individuals returning from coastal regions. It is usually not possible to establish an accurate correlation between clinical features and microbial etiology because the spectrum of clinical manifestations is wide and variable. On the other hand, certain classic features can be suggestive of a particular etiology; examples include dysentery (amebiasis, shigellosis, enteroinvasive E. coli [EIEC]), bloody diarrhea (salmonellosis, campylobacteriosis, shigellosis, EIEC, and verocytotoxin [VT]-producing E. coli VW)9 “rice water” stools (cholera), significant diarrhea and vomiting in a young infant (rotavirus), hemorrhagic colitis or HUS (hemolytic-uremic syndrome) (VTEC), subacute or chronic diarrhea or flatulence (giardiasis), appendicular syndromes (yersiniosis), and shortterm incubation with vomiting as a predominant feature (staphylococcal food poisoning). In situations such as staphylococcal food poisoning, the illness results directly from the ingestion of enterotoxin preformed in food contaminated with enterotoxigenic Staphylococcus aureus (14). However, the vast majority of enteric bacterial infections result from the ingestion of a sufficient dose of organisms in either contaminated food or water. With highly infectious or-

CUMITECH

12A

ganisms such as shigellae, transmission may occur via contaminated fingers or for&es; the low infectious dose in this situation facilitates personto-person spread and explains why shigellosis occurs commonly in institutional settings and is associated with unsanitary conditions (14, 133). The incubation period is typically 24 to 48 h for most enteric bacterial pathogens but significantly longer for others such as Campylobacter jejuni (3 to 11 days) and VTEC (3 to 5 days) (14). During this time, the organism establishes itself in the bowel, colonizes the mucosa, multiplies to high numbers, and utilizes specific strategies of virulence (including adherence, production of specific enterotoxins or cytotoxins, and invasiveness) to initiate the disease process. Successful colonization requires the pathogen to overcome a number of protective barriers in the host such as the gastric acid barrier, intestinal mucus blanket, intestinal motility, the indigenous microbial flora, the secretory, humoral, and cell-mediated immune systems, and the gutassociated lymphoid tissues. Symptoms may be extremely severe if one of these barriers is compromised, as occurs in patients with gastrectomy or patients receiving antidiarrheal (antimotility) drugs or antibiotics which suppress the indigenous flora. Probably a variety of bacterial characteristics, including adhesins such as pili, motility, chemotactic factors, and bacteriocins, facilitate colonization. The precise mechanisms of colonization are not understood for most enteric pathogens and have been worked out in detail only for ETEC, which adhere to cells via specific surface structures known as pili or colonization factors. The discovery of specific virulence factors in certain pathogens has revolutionized our concepts about the widely different mechanisms of pathogenic@ in diarrhea and also allows correlations to be made between these mechanisms and clinicopathological features. The four beststudied specific virulence strategies are enterotoxin production, invasiveness, cytotoxin production, and adherence. Elucidation of the mode of action of cholera toxin (SO), the prototype enterotoxin, made it possible for the first time to explain the symptoms of a specific diarrhea1 disease and devise strategies for treating it. Cholera toxin acts on the mucosal epithelial cell through a specific receptor, GM1 ganglioside, to stimulate adenylate cyclase, which leads to the increased production of cyclic AMP. This results in massive fluid secretion with a net outflow of sodium, chloride, and bicarbonate ions and is manifested clinically by severe watery diarrhea. It was shown experimentally that this process can be slowed or reversed by isotonic glucose, and this finding led to the development of oral rehydra-

spp.

perjfiingens

Clostri&um

Invasive

Escherichia coli Enterotoxigenic

d@cik

Clostridium

Bacillus cereus Diarrheal Emetic

Aerwnonas

Organism

1.

’

Food

Food, water

>

Meat, meat products, gravy

Antimicrobial therapy

Po!Yz!z?k poultry

1

106-lo8

log-lolo

unknown

Enterotoxins (LT and STa) Invasion

Enterotoxin

Enterotoxin (cytotoxin)

Invasion(?) Toxins( ?)

unknown

Water, animals,

Enterotoxins(?) Depends on whether diarrheal or emetic toxin was ingested

unknown

of and isolation Predominant pathogenic mechanism

Meats, vegetables Toxins Fried rice 1

Water

dose

characteristics

Infecting C-J

General

Common source Of Pr&dis&o$rg

TABLE

8-24 h

4-24 h

8-16 h

4-9 days

3-11 days

6-24 h l-6 h }

unknown

procedures usual incubation period

>

Enterotoxin: Mix stool with buffer; test suspension directly or after centrifugation. Cytotoxin: Mix stool with PBS; centrifuge; filter supematant and prepare 10 -fold dilutions in PBS. Quantitative cultures of food and stools, incubated in an anaerobic environment

Enrichment

agents

Enteric agar media

Blood agar, egg yolk a!P

Human diploid fibroblast or other susceptible cell line

Cycloserinecefoxitin-fructose atw

Campylobacter blood agar, Skirrow’s medium or modification, charcoal-based selective medium

Blood agar, phenylethyl alcohol blood agar

Blood agar, CIN asw

lines

diarrhea” Media/cell

Procedure

of infectious

Chopped meat, heated to 80°C for 10 min and incubated at 35°C for 48 h

bacterial

Should include food, stools, and swabs of kitchen utensils and surfaces; quantitative cultures Incubation of cultures in microaerophilic environment at 42°C; membrane filtration Culture: alcohol or heat shock

Processing

for common

Continued

on next page

Test isolates for LT and STa production. Test isolates with DNA probes for invasiveness genes.

Serotype stool and food isolates. Direct detection of enterotoxin in feces (EIA, tissue culture).

Con&m specificity of positive result by neutralization test.

Several EIAs are commercially available.

Test isolates for enterotoxin production. Determine serotypes of isolates.

Other

“Abbreviations:

water,

unknown

106-lo8

108

Toxin

CIN, cefsulodin-irgasan-novobiocin;

Probably food

Yeminia entemcolitica

Water, shellfkh

Meat, dairy products

10-200

102-108, depending on age and g~a~~il%

Food (powdered milk, eggs, msgoducts),

Water Food, water

unknown

unknown

VW

Infecting dose

Water, shellfish

Meat, milk

Shellfish

cholerae

source

Of P~nggo”p

Viirio parahaemolyticus

V&i0

Staphylococcus

Shigella spp. S. dysenteriae Other species

amus

shigellodes

Salmonella sp . (nontyphoi&)

Plesiomonas

Verocytotoxigenic

Organism

Common

15-24 h

Toxins

PBS, phosphate-buffered

Should include food, open skin lesions on food handlers; quantitative cultures

PBS at 4-5°C for 3 weeks; usually not necessary

Alkaline peptone water

Direct inoculation on a ar preferred; GN %roth

Gram-negative, selenite F, or tetrathionate broth

Enrichment

saline; EIA, enzyme immunoassay.

16-48 h

l-5 days

Cholera toxin

Invasion

l-6 h

8-72 h

Invasion and multiplication; Shiga toxin Enterotoxin

Invasion and multi iication Toxins( Y.)

l-2 days

unknown

Processing

Free fecal toxin: Mix stool in PBS; centrifuge and filter supematant; test dilutions of filtrate.

l-Continued usual incubation period 3-5 days

TABLE

Verocytotoxins (Shiga-like toxins)

Predominant pathogenic mechanism lines

agar

agar

Salmonella-shigella and CIN agar media

TCBS agar

Marmitol-salt agar, staphylococcus 110 agar

Enteric and z;rtial

Enteric and differential media

Blood agar

Vero cells

Sorbitol Mac&key ahw

Media/cell

Procedure Other

isolates.

isolates.

K cholerae isolates should be tested with 01 antisera and biotyped if positive. Test isolates for cholera toxin production.

Test food or isolates for enterotoxin. Determine phage type of isolates.

Serotype

Serotype

Test sorbitol-negative isolates with 0157 and H7 antisera or for verocytotoxin production. Confirm specificity of positives by neutralization with VTlandVT2 antisera.

P

CUMITECH

12A

LABORATORY

tion solutions which have revolutionized the treatment of this condition. The heat-labile and heat-stable enterotoxins (LT and ST, respectively) of ETEC act in a similar manner and result in choleralike symptoms. Other enteric pathogens that cause diarrhea through the production of enterotoxins include Bacillus cereus and Clostridium perjkingens. In contrast to the enterotoxigenic organisms that produce little or no histological damage to the mucosa, enteroinvasive organisms such as shigellae and EIEC have the ability to penetrate and multiply within colonic mucosal epithelial cells, leading to their destruction (59, 133). This process is associated with acute inflammation, ulceration, and, in severe cases, necrosis of the colonic mucosa, features that are characteristic of severe bacillary dysentery. The invasiveness properties of these organisms are encoded by large plasmids. The invasiveness genes have now been cloned and shown to produce several polypeptides which are probably involved in stimulating endocytosis of these organisms by epithelial cells (155). C. dificile strains associated with antibioticassociated colitis elaborate two toxins: toxin A, which has enterotoxic activity in experimental systems, and toxin B, a cytotoxin that is toxic to a wide variety of cultured cells. Toxin A is thought to have a major role in the pathophysiology of the disease. The precise mode of action of toxin A remains poorly understood. Some strains of E. coli also produce cytotoxins (VTs and Shiga-like toxins [SLTs]) whose etiologic relationship to the diarrhea associated with these organisms remains controversial. Conversely, there is increasing evidence that these cytotoxins are etiologically associated with the systemic complications of this infection, hemorrhagic colitis and HUS (83). In contrast to ETEC, which adhere to the mucosal epithelial cells by means of pili and cause no histological damage, a very characteristic pathology is seen at the mucosal attachment sites of E. coli strains belonging to the classical enteropathogenic serotypes. They show a specific type of attaching and effacing (AE) adherence to intestinal mucosal epithelial cells which is characterized by the destruction of microvilli and an intimate effacing adherence of the bacterium to the epithelial cell membrane which forms cups or pedestals at the base of the attached bacterial cell (92, 143). The AE lesion has been postulated to be responsible for the diarrhea associated with enteropathogenic E. coli (EPEC), but the precise mechanism and genetic basis of the phenomenon remain to be established. The mechanisms of pathogenic@ of a variety of other enteric bacterial pathogens such as Cam-

DIAGNOSIS

pylobacter lae remain fascinating nisms will

OF BACTERIAL

DIARRHEA

5

spp. and even nontyphoidal salmonelto be fully elucidated, and undoubtedly, stories about these and other orgaemerge over the next decade.

SPECIMEN

COLLECTION TRANSPORT

AND

The specimenof choice for isolating bacterial agentsof diarrhea is the diarrhea1stool collected during the acute stageof the disease.Pathogens are more likely to be present in large numbers during active or progressive disease. Formed stools are less likely to yield etiologic agents. Duodenal contents, vomitus, blood, and bone marrow may be submitted for culture of certain enteric pathogens (149). Blood cultures should be collected from patients with colitis and fever. Rectal swabs should be restricted to patients with active disease,to infants and children from whom feces may be difficult to obtain, and to epidemiologic surveys. The swab must be passedbeyond the anal sphincter and into the rectum for best results. Feces shouldbe seenon the swab. Swabs of rectal lesionsor lesionsof the sigmoidcolon visualized by proctoscopy or sigmoidoscopyare preferred over routine rectal swabs. Feces should be submitted directly to the laboratory in a clean, waxed cardboard or plastic container with a tight-fitting lid, and the container should be labeled and submitted with the laboratory requisition. Screw-capped glass or plastic containers may be appropriate for liquid stools. If the specimenis to be shippedor if a delay in processing of more than 2 h is anticipated, a transport medium will be necessary. The Cary-Blair formulation is suitable for protecting most bacterial pathogens (149). Another transport method, 0.03 M phosphate-buffered glycerol (pH 7.0), alsohasbeen used, but it is not satisfactory for K parahaemolyticus, K cholerae, or C. jejuni. A OS- to 2-g sampleof feces is adequatefor culture, and portions containing blood or mucusshould be cultured since these portions usually contain the highest numbers of enteric pathogens. If feces are not cultured within 2 h of receipt, the specimenshould be placed into Cary-Blair transport medium and refrigerated. Shigella spp. may not survive the change in pH that occurs in unbuffered stool with a drop in temperature unless a buffered transport medium is used. A single, negative stool culture may not be adequateto completely rule out bacterial infection, especially in light of persistent symptoms. Sometimes, up to three specimens may be needed. More importantly, a specimensubmitted without complete patient information may prevent the laboratory from modifying its routine procedure in order to search for other

6

GILLIGAN

ET AL.

appropriate agents suggested by the patient’s history. Direct microscopic examination of the stool may be useful in determining the presence of leukocytes in inflammatory bowel disease (160). Loeffler’s methylene blue stain is mixed with equal amounts of fecal suspension on a microscope slide, covered with a coverslip, and examined by using a light microscope with a 40x objective for the presence of fecal leukocytes (149). The presence of large numbers of leukocytes often indicates an inflammatory process. Polymorphonuclear leukocytes are the predominant inflammatory cell type in the stool of patients with bacillary dysentery and with colitis due to most invasive enteric pathogens; however, mononuclear leukocytes may predominate in patients with typhoid fever or amebic dysentery. Toxin-mediated diarrhea1 illness usually will not produce large numbers of leukocytes. The examination of Gram-stained smears of feces for the presence of specific enteric pathogens is generally not very useful. However, phase-contrast or dark-field microscopy of stool may be helpful in establishing an early diagnosis of Campylobacter enterocolitis (84, 165). PRIMARY CULTURE MEDIA The choice of the primary culture media to be inoculated for the cultivation of specific pathogens is dependent on the clinical profile of the patient. Decisions concerning the pathogens to be sought routinely in selected populations will be discussed below under Guidelines for Stool Examinations. The preparation and use of most media for isolation of enteric pathogens are described in the Manual of Clinical Microbiology (126). For outpatients, selective media for the isolation of Salmonella, Shigella, and Campylobacter spp. should be inoculated. In addition, enrichment broth may be used to increase the yield from specimens with low numbers of Salmonella and Shigella isolates. Selection of the isolation medium to be used for recovery of Salmonella and Shigella spp. is somewhat dependent on the preference of each laboratory. However, two selective and differential media, one of which is comparatively highly selective, are usually inoculated per stool. Two highly selective media generally useful in the isolation of these enteric pathogens from stool are Hektoen and xyloselysine-deoxycholate agars. Both contain H,S indicators, thus simplifying the recognition of Salmonella spp. Other highly selective enteric media such as salmonella-shigella, deoxycholate, bismuth sulfite, or brilliant green may inhibit some strains of these two genera. Because the recovery of Shigella spp. on these more selective media is not always reliable, a less selective, differential enteric medium also is

CUMITECH

12A

inoculated, usually either MacConkey or eosinmethylene blue agar. Careful examination of MacConkey agar may also reveal Yersinia enterocolitica. Isolation procedures for Campylobatter spp, will be described below. In addition to selective media, a nonselective medium, usually 5% sheep blood agar, should be inoculated. This medium is useful in detecting bowel overgrowth with organisms such as yeasts, Staphylococcus aureus, or .Pseudomonas aeruginosa. It is also useful in isolating B. cereus, Aeromonas spp., and Plesiomonas shigelloides. Notification of physicians regarding bowel overgrowths, especially in immunocompromised patients, may be helpful in their management. In regions with a high prevalence of vlbrio, Yersinia, or VTEC strains, selective and differential media for these bacteria should be included as part of the routine stool culture. More detailed information on techniques and media for recovery of specific pathogens is found in the sections that follow. Enrichment broths have two specific functions in the detection of enteric pathogens. Foremost, these broths are designed to detect small numbers of specific bacteria by allowing the growth of the specified organisms while inhibiting the normal flora component of the feces. A number of enrichment broths have been designed for the isolation of specific pathogens and will be described below. Guidelines for the use of each enrichment broth have been developed and should be followed if the use of these broths is to be successful. For example, GN broth, an enrichment broth for Salmonella and Shigella spp., was designed to be subcultured at 6 to 8 h. If incubated beyond that time, normal fecal flora organisms such as E. coli will begin to overgrow any pathogens (41). Each laboratory should carefully evaluate recovery rates of Salmonella and Shigella spp. before and after enrichment to determine the value of the enrichment broth (106). The second use is the direct detection of Salmonella and Shigella spp. in enrichment broth by using latex particles sensitized with specific antibodies to different Salmonella serogroups or Shigella serotypes. The advantage of this technique is mainly in its ability to screen out negative specimens within 24 h. Latex agglutination had an overall sensitivity of 86% for Salmonella spp. and 85% for Shigella spp. when compared with conventional methods for over 3,500 specimens. The specificity of the technique for both genera was in excess of 95% (118, 119). These data indicate that this technique cannot be used as a replacement for conventional techniques, but it may be useful in clinical laboratories with high recovery rates of Salmonella and Shigella spp. for screening enrichment broths.

CUMITECH

12A

LABORATORY

SPECIFIC PATHOGENS Aeromonas spp. A group of bacteria increasingly being implicated as an important causeof bacterial gastroenteritis is Aeromonas spp. (75). Mesophilic membersof this genus commonly inhabit most freshwater sources (lakes, rivers, streams, and ponds) and colonize many different cold- and warm-blooded vertebrates. During the warmer months of the year, aeromonad concentrations rise dramatically in environmental sourcessuch that they can be isolated from drinking fountains, grocery produce, and a wide array of meat products. This seasonalincrease in mesophilic aeromonad numbers coincides with the peak incidenceof aeromonad-associated gastroenteritis observed in the United States (late spring to early fall). Children under the age of 2 years seemespecially prone to developingAeromonas diarrhea, with the majority of casesreported to date falling into this age bracket (153). Most cases of Aeromonas gastroenteritis occur in normal individuals, although in some studies older adults with physiologic, anatomical, or immunologic problems in the gastrointestinal tract also have developed disease (56). One major study haslinked the development ofAeromonas gastroenteritis to the consumption of untreated well water (69). Gastrointestinal disease presumably caused byAeromonas spp. can presentin one of several forms, including a secretory (watery) diarrhea, a dysenterylike illness, a chronic (subacute) gastroenteritis of greater than 14 days’ duration, and a choleralike syndrome (rare). Aeromonadassociatedgastroenteritislinked to foreign travel alsohasbeendescribed(75). Although aeromonads are strongly associatedwith diarrhea, neither an animal model faithfully reproducing the disease nor any defined gastrointestinal outbreaks caused by mesophilic aeromonadshave been reported. However, a number of epidemiologic lines of evidence support their role in intestinal disease;such evidence includes casecontrolled studies, serologic evidence, resolution of diarrhea subsequentto specific antimicrobial therapy directed against Aeromonas spp., and identification of classicvirulence factors produced by aeromonadswhich might be operative in the bowel. Further support for the role of these organismsin gastrointestinal diseasestemsfrom the recent finding of Jiang et al. (78) that 11of 12 studentswho developedAeromonashydrophila- or A. sobria-associateddiarrhea while in Mexico mounted a fourfold or greater rise in surfaceimmunoglobulinA titer to the infecting strain. Besidesbeing involved in gastroenteritis,aeromonadsare involved etiologically in wound infec-

DIAGNOSIS

OF BACTERIAL

DIARRHEA

7

tions, bacteremia, osteomyelitis, peritonitis, urinary tract infections, and hepatobiliary illnesses (75,89). Wound infections(cellulitisor myonecrosis) occur primarily in individuals who have sustained a traumatic injury while in an aquatic environment containing aeromonads;bacteremic infections are most often reported in patientswith hematologicmalignanciesor cirrhosis. Because of the multiplicity of diarrhea1syndromes associatedwith Aeromonas spp., it is likely that several different virulence factors are operative in various forms of Aeromonas disease. These factors include a beta-hemolysin (cytolysin) with enterotoxigenic activity and a less frequently observed cytotonic enterotoxin (6, 31); both products have been associated primarily with A. hydrophila and A. sobria (as opposedto A. caviae). Other enzymes or structures which may be involved in Aeromonas gastroenteritis include invasins, serum resistance, hemagglutinins,and fimbriae (102). Aeromonas spp. can grow and be recovered on most enteric media used without significant reductions in colony-forming numbers. Although many strains are lactose negative (and sucrosepositive), some, especially those designatedA. caviae, are lactose positive and would be overlooked on such media as MacConkey. The best approach for routine recovery of mesophilic aeromonads probably involves the screeningof gram-negativecolonies growing on sheepblood agar for indole and oxidase positivity, two biochemical traits usually positive for most strains of this genus. Some authors have had successwith the addition of ampicillin to sheepblood agar, which aids in the recovery of aeromonads,since these bacteria are normally resistant to this antibiotic (88). Hemolytic Aeromonasspp. such asA. hydrophila andA. sobria are additionally highlighted on blood agar. With the possibleexception of cefsulodin-irgasan-novobiocin (CIN) agar(seediscussionof Y. enterocolitica below), the use of selective media for Aeromonas spp. is not cost-effective, since not all strains are associatedwith diarrhea. As with vibrios, alkaline peptone water is a suitable enrichment broth for most Aeromonas strains (181). The genusAeromonas currently belongs to the family Aeromonadaceae, and its biochemical identification is usually uncomplicated and straightforward. Commercial kits, while highly accurate in identification to the genuslevel, are incapableof accurate speciesidentification. Regardless of whether or not a conventional or commercial system is usedin identifying mesophilic aeromonads,biochemical panels should be supplementedwith growth on nutrient agar without added salt and resistanceto the vibriostatic compound 2,4-diamino-6,7-diisopropyl-

8

GILLIGAN

ET AL.

pteridine (O/129). In the acute phase of diarrhea1 disease, aeromonads should be present in moderate to large numbers on primary plates if implicated in the disease process, since small numbers may reflect only transient colonization. Therefore, it is recommended that only fecal cultures yielding a substantial number of Aeromonas colonies be reported as positive, since the significance of a few colonies is doubtful. Strains reported should ‘be identified as either Aeromonas spp., A. hydrophila complex, or the appropriate species designation (A. hydrophila, A. sobtia, A. caviae, or A. veronii) on the basis of supplementary biochemical tests such as esculin hydrolysis and gas from glucose (77). An additional advantage of identification is that an inference can be drawn regarding the enterotoxigenic potential of the isolate in vitro, as most strains of A. hydrophila and A. sobria produce enterotoxins in the appropriate animal model. Specific antimicrobial therapy may be indicated for those patients with severe aeromonadassociated diarrhea. A. hydrophila strains are almost always resistant to ampicillin, carbenicillin, and cephalothin (154). Susceptibility to tetracycline is variable, and most strains are susceptible to broad-spectrum cephalosporins. A. hydrophila is usually susceptible to trimethoprim-sulfamethoxazole, fluoroquinolones, chloramphenicol, aminoglycosides, and aztreonam (123, 140). A. sobria is usually susceptible to cephalothin, gentamicin, tobramycin, trimethoprim-sulfamethoxazole, and fluoroquinolones but varies in its susceptibility to ampicillin, carbenicillin, chloramphenicol, and tetracycline (123). B. cereus

B. cereus food poisoning is a clearly defined illness that is uncommon in the United States. The organism, however, is ubiquitous. It is an aerobic, sporeforming, gram-positive bacillus easily cultured in the laboratory. B. cereus producesseveral enterotoxins and two distinct syndromes. A diarrheal-type food poisoningis most commonly encountered and usually is associated with meats and sauces.The emetic type is almost always associatedwith rice dishes. The diarrhea1form has an incubation period of 6 to 24 h, whereas the incubation period for the emetic type varies from 1 to 6 h. The clinical pictures of both types are compatiblewith a food intoxication. Diagnosis can be confirmed by isolation of >l@ organismsper g of food, in addition to the isolation of the sameorganism from feces (93, 149). Confirmation by serotyping is recommended. Quantitative culture for B. cerezu can be accomplishedin most laboratories (93). However, in most cases,outbreaks of B. cereus food

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poisoningwill be handledby local or state public health officials. Campyihbacterspp. Campylobacters rival nontyphoidal salmonellae as the most frequent causes of bacterial diarrhea in industrialized countries (21, 31, 51, 166). The typical incubation period of about 2 to 5 days is significantly longer than that for most other enteric bacterial infections. The illness is characterized by fever, abdominal pain, and diarrhea which is often bloody (21, 31, 84, 166). Occasionally, the bloody diarrhea1illness is so severe that it is indistinguishablefrom ulcerative colitis. Vomiting, if it occurs, is usually mild, and dehydration is infrequent. Unusual features of Campylobacter enterocolitis include bacteremia, intussusception, postinfectious arthritis, erythema nodosum, pancreatitis, meningitis, and the Guillain-Barre syndrome. The association of a specific serotype (Penner 019) with Guillain-Barre syndrome in Japan has recently generated much interest (54). Campylobacter diarrhea is also common in the developing world, although significantly less so than ETEC. The illness is considerably milder in severity than in industrialized countries, and asymptomatic infection is common (21, 166). C. jejuni, the most common enteropathogenic species, is widespread in the animal kingdom and hasbeenisolated from a variety of domestic animals, poultry, and virtually every wild bird species.It is probably well adapted for survival in birds since their body temperature, 43°C is close to the organisms’optimal growth temperature. Birds may thus be the primary reservoirs for C. jejuni in nature. Infection is usually acquired through the ingestion of contaminated food or water. Major outbreaks of illness have been associatedwith the consumption of poultry, contaminated water, and unpasteurized milk. Since commercial frozen poultry samples tested for this organism show contamination rates ranging from 30 to 90% or more, eating undercooked poultry may be the causeof illness in most casesof sporadic infection (21, 166). A variety of useful typing systems,including serotyping, biotyping, and phage typing, are now available for studying the epidemiology of campylobacteriosis(21, 134, 135, 166). Key tests used for differentiating human enteric Campylobacter spp. include catalase, growth at 32 and 42”C, nitrate reduction, hippurate hydrolysis, susceptibility to nalidixic acid and cephalothin, H,S production, and the indoxyl acetate test. Detailed differential characteristics have been published elsewhere (134, 135, 166). In most regions, over 90% of enteric isolates are C. jejuni. Occasional C. jejuni iso-

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DIAGNOSIS

OF BACTERIAL

DIARRHEA

9

lates are nitrate negative, and these are referred incubation at 37°C under microaerophilic condito as C. jejuni subsp. doylei. tions. C. cryaerophila has been reclassified as C. coli accounts for about 5% of enteric Arcobacter cryaerophilus (177). Campylobacter isolates, although the proportion The pathogenesis of Campylobacter enteromay be higher in some European countries. colitis is under investigation in many laboratoHippurate hydrolysis is the only practical test ries. Toxin-mediated mechanisms and invasivefor distinguishing C. coli from C. jejuni. Howness are both thought to be involved (165, 166, ever, DNA hybridization studies have revealed 182) the presence of a few strains of hippurate-negaThe definitive diagnosis of enteric Campylotive C. jejuni. C. coli is commonly found in the batter infection is usually made by fecal culture. intestinal contents of pigs and, to a lesser extent, However, direct microscopic examination of poultry and other domestic animals. Pork prodstools (ideally by phase-contrast or dark-field ucts may thus be important in the transmission microscopy) may provide a rapid presumptive of C. coli to humans. diagnosis (84). With experience, the organisms C. Zati (formerly C. lark&s [MO]) strains, orig- can easily be identified by their curved, S-shape, inally isolated from seagulls, are thermophilic, or spiral morphology and rapid, darting, corklike C. jejuni and C. coli, but differ from the latter screwlike motility. in that they are negative for the indoxyl acetate Fecal cultures for campylobacters are usually test and resistant to nalidixic acid. It should be done by using selective media containing a comnoted that occasional strains of C. jejuni and C. bination of antimicrobial agents which suppress coZi are also nalidixic acid resistant. the growth of normal flora but are noninhibitory C. upsaliensis comprises a group of thermoto Campylobacter spp. The media most widely philic strains that differ from C. coli in that they used include those formulated by Skirrow, Butzhave a weak or negative catalase reaction and ler and colleagues, and Blaser and colleagues are susceptible to cephalothin. Also known as (134). Although generally satisfactory, these meCNW (catalase-negative or weakly positive) dia have all had problems with overgrowth by strains, these organisms are increasingly being contaminating fecal bacteria, and this drawback isolated from human feces largely through the may result in failure to isolate enteropathogenic use of filtration techniques. Recovery from camcampylobacters. Recent improvements in campylobacter selective media has been inconsistent pylobacter media include the development of because these strains are usually inhibited by blood-free, charcoal-containing media and the antibiotics (particularly cephalothin) that are use of more selective antibiotic supplements, present in various selective media. including cefoperazone (23, 62, 86). A bloodC. hyointestinalis resembles C. fetus subsp. free, charcoal-containing medium for campylofetus in a number of characteristics but differs batters is commercially available. The newer from the latter in that it grows at 43°C and media are superior to the earlier media for the produces hydrogen sulfide in triple sugar iron selective isolation of most enteropathogenic medium. It has been recovered occasionally Campylobacter spp., particularly C. jejuni, C. from patients with diarrhea and from a homocoli, and C. Sari. A Skirrow-type selective mesexual man with proctitis. dium containing polymyxin B (5,000 IU/liter), The precise relationship of C. Sari, C. upsatrimethoprim (10 mg/liter), vancomycin (10 lien&s, and C. hyointestinalis to human diarmg/liter), amphotericin B (2 mg/liter), and ceforhea1 disease remains to be elucidated. C. fetus perazone (10 mg/liter) has been recommended subsp. fetus is very infrequently isolated from for the optimal isolation of C. cinaedi and C. human feces and is not generally considered to fennelliae from feces (175). be an enteric pathogen. The use of membrane filters applied directly to C. cinaedi and C. fennelliae are slow-growing the surface of non-antibiotic-containing agar Campylobacter species that produce only pin- plates is becoming increasingly popular for the point colonies on blood agar plates after 48 h of selective recovery from feces of organisms such incubation under microaerobic conditions. They as C. upsaliensis that are susceptible to several are associated with diarrhea, abdominal pain, antibiotics that are present in various campyloand proctocolitis in homosexual men and have batter selective media (168). The technique incaused bacteremia in individuals with AIDS. volves placing 0.45 - or 0.65 -km-pore-size celluC. cryaerophila encompasses a group of aerolose acetate membrane filters (47.mm diameter) tolerant Campylobacter strains that were origionto the surface of a non-antibiotic-containing nally isolated from reproductive tracts and blood agar plate. Several drops of test fecal aborted fetuses of cattle and pigs. This organism suspensions are then applied onto the membrane was recovered from the feces of a homosexual filters. After 30 min, when all of the fluid has man with intermittent diarrhea upon growth on a passed through, the filters are removed and Skirrow-type selective medium after 3 days of discarded. The plates are then incubated under

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conditions optimal for Campylobacter isolation. The method relies on the principle that campylobatters are much more slender than coliforms and other potential contaminating fecal flora and are able, selectively, to passthrough the pores of the cellular acetate membranes. Although these media are somewhat less sensitive than antibiotic-containing selective agarsfor isolating C. jejuni and C. coli, the method is essentialfor the recovery of C. upsaliensisand other antibiotic-susceptible Campylobacter strains from feces, and it deserves consideration for routine use in associationwith an antibiotic-containing selective agar (23, 168). The use of selective enrichment broths for enhancingthe recovery of C. jejuni from stools has been controversial. It is likely that such a procedure is not cost-effective and addslittle, on a routine basis, to direct isolation on selective agar media (73). The optimal conditions for isolating most enteropathogenic campylobacters from selective media inoculated with stools include an incubation temperature of 42°C and a microaerobic environment (166). A temperature of 42°C is optimal for C. jejuni and C. coli and also allows most other enteropathogenic Campylobacter spp. to grow while, at the sametime, providing further selective suppressionfor somecontaminating flora that grow poorly at this temperature. On the other hand, an incubation temperature of 37°Cmay be more practical in somelaboratories (especially in developing countries) in that it avoids the useof a separate42°Cincubator, and it is also essentialfor the recovery of C. cinaedi and C. fennelliae from feces. Most enteropathogenic Campylobacter spp., including C. jejuni and C. co&, grow optimally in an atmospherethat contains about 7% oxygen (about one-third of normal atmospheric oqgen tension) and between 1 and 10% carbon dioxide (166). A variety of commercial systems using gas-generatingpacks to produce a microaerophilic environment are widely used. This environment can also be achieved by evacuating two-thirds of the air from an anaerobicjar (without catalyst) and replacing this with a carbon dioxide-hydrogen mixture. Media for isolating campylobacters shouldbe incubatedfor 48 h and examinedfor growth after 1 and 2 days of incubation. Cultures for C. cinaedi and C. fennelliae should be incubated for at least 5 days. Campylobacter colonies are typically effuse with a butyrous consistency and are salmon pink, especially on blood-containing media. Extensive swarming is a feature on moist media, especially with C. jejuni. Campylobacter colonies show a characteristically slow-positive oxidasereaction. A handful of tests are necessary

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for identifying the species. Latex agglutination tests have been used for culture confirmation of Campylobacter colonies to the genuslevel (66, 126). They are highly sensitive and specific for C. jejuni and C. coli but not for most other species. They probably offer little over direct microscopy to confirm an organism as a Campylobacter species. Similarly, a commercially available synthetic nucleic acid probe conjugated to alkaline phosphatasehas been evaluated for culture confirmation as well as for the direct detection of Campylobacter spp. in stools. This test can be used for sensitive and specific confirmation of C. jejuni and C. coli but not of other CampyZobacter species. It had a sensitivity of 82.6% and specificity of 100%for detecting C. jejuni and C. coli directly in fecal specimens(129). C. jejuni is susceptiblein vitro to a wide range of antimicrobial agents,including erythromycin, tetracyclines, aminoglycosides, chloramphenicol, fluoroquinolones, and clindamycin (85). Erythromycin is the treatment of choice. C. coli is more commonly resistantto erythromycin and tetracyclines but otherwise similar to C. jejuni in its antimicrobial susceptibility (166). Not all patients with Campylobacter infections require antimicrobial therapy; less than one-half of those ill enough to seek medical attention are candidates for treatment with antimicrobial agents. Early treatment of children with Campylobacter enterocolitis is beneficial (84). Antimicrobial therapy is also indicated for patients with high fever, bloody diarrhea, or more than eight stools per day or for patients with persistent diarrhea (> 1 week). Antibiotic susceptibility testing is usually performed by a reference laboratory, using an MIC method with MuellerHinton broth supplementedwith 5% lysed horse blood or an agar dilution method (85). Chtridium d@cile C. dificile causesa wide spectrum of clinical manifestationsranging from asyrnptomatic carriage through antibiotic-associateddiarrhea and nonspecific colitis to pseudomembranouscolitis and toxic megacolon(10, 107). Clinical disease due to this organismis almostalways associated with prior or concurrent antimicrobial therapy. Antimicrobial agents are believed to eliminate normal flora components which inhibit the in vivo growth of C. dificile (27, 145, 146, 186, 187).With inhibitory flora eliminated, C. dificile can proliferate and causedisease.In any discussion of antimicrobial agentsin the context of C. di!ciZe disease,the term must be broadly interpreted to include chemotherapeutic agents which might cause alteration in gut flora. This category includes not only the traditional antibacterial agentssuch as penicillins, cephalospo-

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rins, and clindamycin but also other agents which have antibacterial activity, e.g., azidothymidine and methotrexate (36, 164). The pathophysiology of disease caused by C. di!‘ciZe is currently believed to be due primarily to the activity of two protein exotoxins produced by this organism, one designated an enterotoxin (toxin A) and the other designated a cytotoxin (toxin B) (107, 171). C. di!ciZe strains have been shown to either be nontoxigenic or produce both toxins. No strains that produce a single toxin have been recovered. Animal studies indicate that enterotoxin has a major role in the pathophysiology of this disease. It causes an accumulation of hemorrhagic fluid in the gut of experimental animals. This fluid accumulation is associated with mucosal damage, including macroscopically visible hemorrhagic lesions (101). This toxin also can cause cytopathic changes in tissue culture cell lines. Cytotoxin appears to have a secondary role in C. di!cile disease. It causes minimal fluid secretion and no mucosal lesions in the same experimental animals (101, 110). As its name implies, the cytotoxin is extremely toxic to a variety of tissue culture cell lines, being 1,000 times more cytotoxic than the enterotoxin (108). Interestingly, the toxins have similar 50% lethal doses when injected intraperitoneally into mice (109). In other animal studies, it has been observed that small amounts of enterotoxin can potentiate the systemic toxicity of cytotoxin, suggesting that both toxins play a role in the pathophysiology of this disease. C. dificile is the major identifiable cause of nosocomial diarrhea in adults (163). The organism, as spores, has been shown to survive for extended periods (months) in the hospital environment (90). It has been recovered from a variety of sources in the hospital, including the hands of health care personnel, endoscopes, bed pans, commodes and the area surrounding them, and bed rails (90, 114, 124). Given these data, patient-to-patient spread almost certainly occurs, and studies support that contention (30, 122, 156). In addition, approximately 3% of healthy adults have been shown to be colonized with C. difcile (179), suggesting that some patients enter the hospital carrying the organisms while others may become nosocomially infected. The common factor for both groups is the use of antimicrobial agents during hospitalization, which alters the gut flora and allows the disease to develop. The microbiological diagnosis of C. dificileassociated disease is controversial. Cumitech 12 recommended that although the diagnosis was supported by culturing the organism from feces, the definitive diagnosis, i.e., “gold standard,” was the detection of C. dijgicile cytotoxin in feces by using a tissue culture cytotoxicity as-

DIAGNOSIS

OF BACTERIAL

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11

say. The data supporting this viewpoint were solid. Over 95% of patients with antibioticassociated pseudomembranous colitis had cytotoxin present in their feces, while a smaller percentage of patients with antibiotic-associated diarrhea and nonspecific colitis also had cytotoxin present. It was also known that approximately 2 to 3% of normal individuals were culture positive but cytotoxin negative. Subsequent studies showed that in hospitalized patients without diarrhea who were being treated with antibiotics, culture was positive in 21% but cytotoxin was positive in only 2% (179). These data further supported the idea that results of cytotoxin testing were more closely associated with disease than were results of culture testing. Tissue culture cytotoxicity testing, however, is problematic. It requires 24 to 48 h of incubation, is labor-intensive, and requires tissue culture facilities and expertise. As a result, this testing is usually available only in larger hospitals or through reference laboratories. The observation that enterotoxin rather than cytotoxin was primarily responsible for the pathogenic@ associated with this organism in experimental animals suggested the need for an alternative test for the detection of C. dificile-associated disease. A latex agglutination test (Culturette Brand, CDT Latex Test, Becton Dickinson Microbiology Systems) which purported to detect toxin A was marketed. It had many advantages over tissue culture cytotoxicity in that it was easy to do, had a rapid turnaround time, and did not require special facilities or expertise. These factors ensured that the test would be widely used. Early studies showed that this test correlated more closely with culture results than did tissue culture cytotoxicity (87, 136). Because of this, several investigators questioned the suitability of cytotoxicity as the reference method and suggested instead that clinical criteria for the presence of C. di$iciZe disease be used to judge the performance of the different detection methods, including latex agglutination, culture, and fecal cytotoxicity. The clinical criteria included presence of diarrhea, prior or concurrent antimicrobial therapy, absence of other etiologic agents, clinical response to appropriate antimicrobial therapy, and pseudomembranes visualized by endoscopy. However, with the exception of the presence of pseudomembranes, the clinical criteria for C. dificile-associated disease are imprecise. The explanation for the close correlation between culture and the latex agglutination test results became clearer when it was found that the antigen being detected by the latex test was a cell-associated antigen which copurified with enterotoxin and not, as claimed by the manufac-

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turer, enterotoxin (112). This observation was confirmed by Borriello et al, (24). It also has been reported that this latex reagent cross-reacted with other organisms which could be present in feces, including C. sporogenes, Bacteroides asaccharolyticus, and Peptostreptococcus anaerobius (120). Several clinical evaluations of the latex test have been published. Four groups have used cytotoxicity as a reference method (16, 72, 159, 188). These groups tested over 1,100 clinical specimensand found latex to have a sensitivity of 89% and a specificity of 95%. The predictive value of a positive latex test was only 72%. The reasonfor the low positive predictive value was due in part to the detection of nontoxigenic strainsof C. diflcile by this technique. A clinical review of casesof patients with nontoxigenic strainsindicated that C. dificile was not responsible for their diarrhea. Other patients had positive latex tests and were negative by both culture and the cytotoxin assay. These falsepositive latex testsmay represent cross-reaction with other componentsof the fecal flora. When clinical criteria were used to define C. dificile diseasein two studies, sensitivity was greatest for culture, with the cytotoxin assay and latex test sensitivity being similar (87, 136). However, culture (4 and 7%) and the latex test (5 and 8%) had much higher rates of false positives than did the cytotoxin assay (1.4 and 2%). Becauseof the imprecision of the clinical criteria and the comparatively high rates of false positivity with both the latex test and culture, due in part to the presenceof nontoxigenic strains, the tissue culture cytotoxicity assay currently appears to be the method of choice for the laboratory diagnosisof C. dificile disease.For laboratories usinglatex agglutination as the diagnostic test, positive results must be interpreted cautiously in light of false positives due to nontoxigenic strains of C. di!ciZe and other crossreacting organismspresent in feces. In patients with repeatedly negative latex tests, when the physician still suspectsC. dijj’icile disease,cytotoxin testing shouldbe performed. Culture techniques for the recovery of C. dicfficile are dependent on the use of selective medium. George et al. (57) described the first selective agar for C. dijjkile, cycloserine-cefoxitin-fructose agar (CCFA). Wilson et al. (185) modified this medium by substituting taurocholate for eggyolk. The taurocholate-modified CCFA was shown to be superior for the recovery of environmental isolates becauseof better spore germination on this medium. Levett (98) has advocated decreasingthe concentrations of both cycloserine and cefoxitin by one-half (from 500 to 250 pg/ml and from 16 to 8 pg/ml, respectively) in this medium, since the higher

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concentrations of antimicrobial agents inhibit someC. dificile isolates. Alcohol or heat shock prior to culture, as well as enrichment broths, has been suggestedas a meansof improving the recovery of this organism.This organismcan be reliably identified on the basis of (i) its typical colonial morphology and fluorescencewhen exposedto UV light when grown on CCFA and (ii) its ability to produce the branched-chain fatty acids isocaproateand isovalerate. Culture should not be used as the primary diagnostic test for the presence of C. di!$ciZe disease.As previously discussed,the carriage rate of this organismcan be high, and nontoxigenie strains can be recovered, although the percentageof these isolatescomparedwith toxigenic strains is probably small. However, culture may be useful in assessingrelapse and is obviously important in epidemiologic studiesof this organism. Tissue culture cytotoxicity testing can be done efficiently by using a wide variety of cell lines grown in flat-bottom 96-well microtiter plates. Human diploid fibroblast cell lines are commonly usedbecausethe cytopathic changes induced by the C. dificile toxins are dramatic, but other cell lines may be used (115). Because feces can contain toxic substancesthat cause morphologic changessimilar to those produced by C. dificile toxin, an integral step in the cytotoxin assay is neutralization of C. dz$JWe toxin by specific antitoxin. The presenceof C. dificile toxin can be reported only if neutralization is demonstratedby using C. di!ciZe toxinspecific antiserum. Interestingly, C. sordellii antitoxin also has neutralizing activity against C. di!ciZe toxin and can be substituted in the cytotoxin assay (12). A method for performing this assayis describedin the Manual of Clinical Microbiology

(3).

Attempts to develop more rapid techniques for detecting C. dijkile toxins are continuing, with a particular emphasison the enterotoxin. Enzyme immunosorbent assayshave been developed for the detection of this toxin. Early studiesindicated that this test was not nearly as sensitive as the cytotoxin assay, with sensitivities as low as 56% and as high as 91% (94, 97, 111). However, one of the difficulties with the early studieswas the failure to recognize interfering substancesin stools. Of particular importance was the presencein feces of proteolytic activity which causedthe desorption of capture antibodiesfrom the solid phase.The addition of 50% bovine calf serum to specimen buffer greatly reduced this desorption (178). Several commercially available enzyme immunoassays for C. dificile enterotoxin detection have been licensed by the Food and Drug Administration for diagnostic use. One of these tests has been

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reported to have a sensitivity of 84.1% and a specificity of 98.9% in comparison with tissue culture cytotoxicity tests (43). C. dificile diarrhea often respondssimply to discontinuation of the inducing antimicrobial agents. Oral administration of vancomycin or metronidazole is indicated for patients with persistent symptoms or severe disease. Most C. di!ciZe isolates are very susceptibleto vancomycin, metronidazole, rifampin, and bacitracin in vitro; however, metronidazole resistance does occur (11). Antimicrobial susceptibility testing is generally not useful in guiding therapy. CkMridium perj+ingens Enterotoxin-producing strains of C. pe#ngens type A are commoncausesof food poisoning and are an uncommoncauseof sporadiccases of diarrhea that may or may not be associated with antibiotic use (96). Beta-toxin-producing strains of C. perj%n.ens type C rarely cause a severeand sometimesfatal necrotizing infection of the smallbowel, enteritis necroticans. The enterotoxin produced by C. pe@ingens type A and some strains of types C and D is a heat-labile polypeptide of 35,000Da that is produced during sporulation. In the small bowel, the enterotoxin binds to a membranereceptor and inducesa calcium-dependentbreakdown of colloid-osmotic equilibrium, resulting in a sizedependent loss of intracellular substancesand eventual cell death. Analogous effects are seen in vitro with Vero cells. Most casesof C. perjkingens diarrhea occur in the setting of an outbreak due to exposure to a commonfood source, usually meat. The incubation period following exposure is usually brief (8 to 16 h), and the usual symptoms include diarrhea and abdominalcramps. Nausea, vomiting, and fever occur less frequently, and nearly all patients have spontaneousresolution of symptoms in 6 to 24 h. Patients with sporadic, nonfood-borne C. perjkingens diarrhea have a more prolongedillnesswhich may resembleCampylobatter or Salmonella infection. Antibiotic therapy is usually not indicated. C. perjkingens is a relatively commoncauseof food-borne disease outbreaks in the United States. Data from the Centers for DiseaseControl indicate that C. perjkgens accounts for about 7% of food-borne outbreaks, with an average of 24 victims per outbreak. Many outbreaks are probably not reported becauseof the relatively mild symptomsand speciallaboratory tests necessaryto confirm the diagnosis. C. pe@ingens food poisoningshould be consideredin any outbreak of diarrheawith abdominal crampsoccurring soonafter consumptionof a common food source. Diagnostic criteria include the detection of > lo5 C. perfringens per g

DIAGNOSIS

OF BACTERIAL

DIARRHEA

13

in a common food source, a medianfecal spore count of >106/g in ill persons, and identification of the sameserotype of C. perjkingens in most ill persons but not in well persons (32, 65). In addition, the direct detection of enterotoxin in fecal sampleswill help confirm the etiology of the illness(17). A reverse passive latex agglutination assay,enzyme-linked immunosorbentassay (ELISA), and tissue culture assay using Vero cells and specific antibody to neutralize cytopathic effects have all been used for direct detection of enterotoxin (17). EPEC EPEC refers to specific serotypes of E. coli that were historically associatedwith outbreaks of infantile diarrhea particularly during the 1940s to 1960s;the best known of the traditional EPEC 0 serogroupsinclude 26, 55, 86, 111, 114, 119, 125, 126, 127, 128, 142, and 158 (59, 99, 143, 148). Although outbreaks of EPEC diarrhea declined considerably in frequency during the 197Os,they still occur occasionally, and outbreaks of infantile diarrhea should be investigated for EPEC serotypes. Current serotyping schemesfor pathogenicE. coli, including EPEC, are basedon the 0 and H antigens only. Contemporary lists of the more common EPEC 0 :H serotypes are available (48, 49). Investigation into the mechanismsof pathogenicity of EPEC has a confusing but colorful history, and a variety of different nomenclatures have emerged to distinguish putative virulent subgroupsof EPEC. However, the past decade haswitnessedsomeexciting new developments in this area (91, 92, 99, 143). Many EPEC serotype strains may be distinguished from non-EPEC strains by their ability to adhereto HEp-2 cells in vitro (40). Two types of adherence,localized and diffuse, may be seen (157). Localized adherenceis mediatedby plasmids of about 50 to 70 MDa in size which are referred to as EPEC adherence(EAF) plasmids (8). A 1-kb segmentof DNA from a reference EAF plasmidhasnow been cloned and shownto be useful as a DNA probe for detecting EPEC strains that have the ability to adhere to HEp-2 cells in a localized fashion (99, 100). EPEC strains show a specific type of AE adherenceto intestinal mucosal epithelial cells in vivo (92, 143), as described above under Clinical Aspects and Pathogenesis.It should be noted that EPEC do not produce the classic LT or ST, although some EPEC serotype strains produce VTs. There is somecontroversy as to whether the plasmid-encoded EAF-associated localized HEp-2 cell adherencein vitro correlateswith the AE adherenceseenin vivo. Knutton et al. (92) provide compelling evidence that localized ad-

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herence to HEp-2 cells and AE adherence are distinct phenomena, with the HEp-2 adhesin being of fimbrial origin and plasmid mediated, whereas AE adherence is encoded by genes located on the chromosome. They propose a two-stage model of EPEC adhesion, with a fimbria-mediated initiation step and a subsequent phase of AE adherence. Knutton et al. (91) have shown that actin filaments accumulate at sites of bacterial adhesion to cultured cells and can be detected by using fluorescein-labeled phallotoxin. When developed, a nonradiolabeled DNA probe assay specific for the genes encoding AE adherence will offer another attractive option for the detection of AE E. coli and EPEC in feces. The pathogenic&y of another candidate EPEC group that is characterized by its ability to attach to HEp-2 cells in an aggregativepattern remainsto be established(15). Lack of knowledge about specific virulence factors and mechanisms of pathogenic@ of EPEC diarrhea and the attendant problem of distinguishingvirulent from nonvirulent strains led to conflicting views about the need to search for EPEC serotypes in casesof sporadic diarrhea. Most authorities do not recommendinvestigation for EPEC serotypesin sporadic casesof diarrhea, although this view may change with the advent of specific tests to detect virulent strains. ETEC ETEC are major causesof bacterial diarrhea in developing countries, especially in children, and of traveler’s diarrhea (59,99, 148). They are distinguishedby their ability to elaborate one or both of two plasmid-encodedenterotoxins, the choleralike LT and ST. Like EPEC strains, human ETEC strains belong to a restricted group of 0: H serotypes. The mechanism of pathogenic@ of ETEC, now well known, involves attachment of the bacteria to small-bowel enterocytes by means of specific fimbriae, the best characterized of which are referred to as colonization factors I and II. The specific action of LT or ST on the enterocyte at the subcellular level, through the stimulation of adenylate cyclaseor guanylate cyclase, respectively, leadsto fluid secretion which results in watery diarrhea. The dose of ETEC required to cause symptomatic diseaseis high (lo* organisms). Infection is transmitted chiefly by consumption of contaminatedwater and food and is a problem in areaswith inadequatepublic health control measures(14,148). ETEC are not commoncausesof bacterial diarrhea in industrialized countries, and routine identification in sporadiccasesis not necessary. However, outbreaks of diarrhea should be investigated for ETEC.

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The diagnosisof ETEC diarrhea involves testing 10lactose-positive colonies(or colony pools) from a primary agar culture for LT and ST (59). The traditional method for detecting LT involves testing culture filtrates of colonieson Y-l adrenal or Chinese hamster ovary (CHO) cells. Exposure of these cells to LT results in an increase in the intracellular concentration of cyclic AMP and leads to a reversible “cytotonic” morphologic responsewhich can be seen by microscopy. CHO cells respond by elongation, and Y-l cells become rounded. ST is a low-molecular-weight protein which is nonantigenic and not detectable in cell culture. Two types of ST, STa (human) and STb (porcine), are recognized. STa traditionally hasbeen detected by using an infant mouseassay. A variety of immunospecific tests (including ELISA, receptor ELISA, latex, and coagglutination) have been developed to detect both STa and LT (29,42,152,169). At least two assaysare available commercially. Both LT and ST can be identified by meansof DNA hybridization techniques using synthetic oligonucleotide probesspecific for genesencoding the toxins (47, 59). Echeverria and colleagues(47) have usedreplicate blots and DNA hybridization techniques to detect LT-positive and ST-positive E. coli and alsoto detect strains expressingother virulence factors such as VTs (SLTs), invasiveness,and EAF. Trimethoprim-sulfamethoxazole and antibiotics in the quinolone group are commonly used for the treatment of ETEC diarrhea and also as prophylaxis for traveler’s diarrhea (59). EIEC EIEC refers to a group of E. coli belonging to a restricted number of 0 : H serotypes (including 0124:NM, 0124:H7, 0126:H30, 0135:NM, 0136:NM, 0143:NM, 0144:NM, 0152:NM, and a few others 148, 491) that are associated with a diarrhea1illness that resemblesbacillary dysentery. EIEC have been associated with several outbreaks of diarrhea (many of them food borne), and someserotypes, notably 0124, have been isolated from sporadic casesof diarrhea and dysentery (59, 148). Like shigellae,EIEC produce keratoconjunctivitis in rabbits and are invasive for cultured cell lines such as HEp-2. Also like shigellae,EIEC proliferate within cells, causing their destruction. In both shigellaeand EIEC, invasivenessis associatedwith the presenceof a high-molecular-weight plasmid. Several plasmid-encoded outer membrane proteins (closely related in shigellae and EIEC) are thought to mediate invasion (59, 155). While outbreaks of diarrhea are normally investigated for evidence of EIEC infection, the

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lack of a simple, cost-effective procedure for the detection of invasiveness has made it difficult for laboratories to screen stools for EIEC in sporadic cases of diarrhea. However, biotinylated DNA probes specific for the invasiveness genes have now been used successfully to detect EIEC and shigellae in epidemiologic studies and should become more widely used (22, 46). An ELISA method has been developed for identifying EIEC by detecting outer membrane proteins possibly associated with invasiveness (132). EIEC are often atypical; many strains are nonmotile, lactose negative, or lysine decarboxylase negative (48, 49, 174). VTEC and EHEC Culture supematants of some E. coli strains contain one or two of at least three closely related bacteriophage-mediatedtoxins that produce an irreversible cytopathic effect in a restricted number of cultured cell lines, including Vero and HeLa. These toxins have been referred to interchangeably as VTs or SLTs becauseof their close structure-function relationship to Shigatoxin produced by Shigella dysenteriae type 1 (83, 128). The toxins associated with human diseaseinclude VT1 (SLTI) and VT2 (SLTII). A number of VT2 variants are now recognized (151). VTEC are associatedwith a wide spectrum of illness that includes asymptomatic infection, mild uncomplicated diarrhea, hemorrhagic colitis, and classicalHUS. Since bloody stools are often produced as a result of infection with these strains, they are also referred to as enterohemorrhagic E. cob (EHEC) (99). HUS is defined by the triad of features that consistsof acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia. In the most common form of HUS, these features occur typically about a week after the onset of a nonfebrile, usually bloody diarrhea1 illness. HUS is a leading causeof acute renal failure in childhood and is associatedwith significant morbidity and mortality (83). Hemorrhagic colitis is a distinct clinical syndrome that presents typically with abdominal crampsand watery diarrhea followed by a hemorrhagic dischargeresemblinglower gastrointestinal bleeding(141, 184). It is distinguishedfrom inflammatory colitis in that typically there is lack of significant fever and an absenceof an inflammatory exudate in the stools. Studies on the pathogenesisof VTEC infection suggestthat after ingestion, VTEC colonize the bowel and associatewith the mucosaby an AE mechanismsimilar to that seenwith EPEC serotypes (83, 143). This may be responsiblefor the diarrhea1illness, although the precise mechanism remains to be clarified. Colonization is

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OF BACTERIAL

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15

followed by a systemic VT toxemia that is thought to be responsiblefor the systemic manifestations such ashemorrhagic colitis and HUS through action on the endothelial cells of capillaries and other small blood vessels supplying the kidney and bowel (83). The main reservoirs of VTEC, like those of other E. coli strains, appear to be the intestinal tracts of animals,and foods of animal origin are probably the most important sourcesfor human infection. Outbreaks have been linked to the consumption of improperly cooked hamburger meat and unpasteurized milk, and VTEC have been recovered from cheeseand from about 1 to 4% of retail samplesof beef, pork, poultry, and lamb (44, 83). Most of our knowledge about human VTEC infection comesfrom studiesof a singleserotype, 0157: H7. This is largely becausethis sero-type has a biochemicalproperty (lack of sorbitol fermentation) that allows it to be readily distinguished from other fecal E. ~024 which makes recovery of this organismpractical in most clinical laboratories. However, over 50 different 0 :H serotypeshave now beenassociatedwith human disease, and more information is beginning to emergeon the non-0157 serotypes (83). The optimal strategy for the diagnosis of VTEC infection involves the demonstration of VT in fecal filtrates and the culture of VTEC from feces (49,82,83). The test for free fecal VT (FVT) is often positive even if VTEC cannot be cultured because of low relative numbers of VTEC in mixed fecal flora, particularly if fecal specimensare collected more than 5 days after the onset of symptoms (82). The detection of FVT requires a cell culture assay and is therefore not practical in many routine laboratories. However, the availability of immunospecific methodsmay make the direct detection of FVT a practical option in the future. The isolation of VTEC by culture involves the testing of up to 20 isolatedE. coli colonies from primary media for VT production, typically in a cell culture system. VTEC can alsobe detected in fecal cultures by DNA hybridization techniques using DNA probes specifying genes for the different VTs (47). Methods using the polymerase chain reaction are also being developed to detect VTs (138). ELISAs (44) and receptor ELISAs (13) have also been developed but are not yet commercially available. Many laboratories that do not have tissue culture facilities use a sorbitol-containing medium to detect the predominant VTEC serotype, 0157 :H7. This serotype doesnot ferment sorbito1 after 24 h of incubation, whereas 95% of other E. coli serotypes are sorbitol fermenters under these conditions. A sorbitol MacConkey agar (similar to standard MacConkey medium

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except that it contains 1% D-sorbitol instead of lactose) is a useful selective medium for this purpose (116). After inoculation with feces, this medium is incubated for 18 to 24 h at 37°C and examined for colorless (D-sorbitol-negative) colonies. Such colonies should then be tested by slide agglutination tests against 0157 and H7 antisera, and the results, if positive, should be confirmed by tube agglutination tests. If the culture is serotyped as 0157: H7, it is highly likely to be VT positive, but this assumption should be confirmed by having the culture tested by an appropriate reference laboratory. Good commercial antisera to detect 0157 antigen are now available (49). A false-positive serologic identification of E. coli 0157 may result with Escherichia hermanii. The latter resemblesE. coli biochemically, is sorbitol negative, gives a positive agglutination reaction with 0157 antiserum, but is VT negative. These bacteria are distinguished from E. coli becauseunlike the latter, they ferment cellobiose, grow in the presence of potassiumcyanate, and produce a yellow pigment (83). The frequency of diarrhea associated with VTEC 0157: H7 rivals that associated with Campylobacter and Salmonella spp. in some centers (131). This finding suggeststhat this organism should be sought routinely in stool cultures. Further work is required to develop techniquesfor detection of non-0157 VTEC in clinical laboratories. Pksiomonasshigelbides One of the lessfrequently encountered bacteria involved in gastrointestinal disease is P. shigelloides, a gram-negative, oxidase-positive bacillus that is currently placed in the family Vibrionaceae along with the genusAeromonas. Plesiomonads commonly inhabit freshwater sourcesand are often found colonizing or infecting reptiles and cold-bloodedvertebrates such as lizards, snakes, and frogs. In humans, the asymptomatic carriage rate for this microbe appears to be extremely low, and its isolation is usually associatedwith disease(28). In contrast to the phenotypically similar Aeromonas spp., two well-described outbreaks of diarrhea1diseasein Japan have been attributed to this bacterium (176). However, as with Aeromonas spp., no animal model that faithfully reproduces the diseaseis available, leaving Koch’s postulates unfulfilled. In addition, defined virulence factors that might explain the gastrointestinal symptomsproduced by this organismare generally lacking (121). With respect to gastrointestinal disturbances, both a typical secretory diarrhea and a colitislike infection have been documented (28). A recent major epidemiologicinvestigation conducted by

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the Centers for DiseaseControl on this organism identified foreign travel (to Mexico) and ingestion of undercooked shellfish(oysters) as significant risk factors associatedwith plesiomonad diarrhea (70). The only major clinical syndrome associatedwith P. shigelloides other than gastroenteritis is bacteremia with or without meningitis. Reported cases of meningitis involve primarily either newborns or those who are severely immunocompromised,and in someseries the fatality rate approached 80%. Besides septicemia, only one extraintestinal P. ShigeZloides infection (cholecystitis) has been described in the literature, for which there was good serologic evidence of infection (28). A number of reputed virulence factors produced by P. shigelloides have been reported, including the ability to invade epithelial cells, cytotoxin activity, a large plasmid potentially involved in uptake by or invasion through the gastrointestinal tract, and an extensive glycocalyx (1, 121). Most of thesereputed factors have only recently beendescribed, occur in only rare strains, or are not reproducible in other investigators’ hands. Two recent additions to this list are a choleralike activity produced in vitro by plesiomonadsand a heat-stableenterotoxin which is detectable only by serial passageof P. shigelloides strains in vivo (55, 117). P. shigelloides is easy to isolate and identify by virtue of its unusualbiochemical profile (positive ornithine decarboxcylase,lysine decarboxylase, and arginine dihydrolase tests and fermentation of inositol). Because the asymptomatic carriage rate is so low, the isolation of P. shigelloides from a diarrhetic stool in the absenceof other enteric pathogenscan be considered diagnostic. The easiestmediumfrom which to isolate the bacterium is blood agar following a screening protocol similar to that outlined for Aeromonas spp. Becausethe frequency of plesiomonad-associated gastroenteritis is very low, use of a selective agar is not warranted. Antimicrobial therapy is indicated in severeor prolonged gastroenteritis due to P. shigelloides. P. shigelloides is usually susceptibleto chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, aminoglycosides, fluoroquinolones, imipenem, and broad-spectrum cephalosporins (140). However, each isolate should be tested becauseof strain-to-strain variation in antibiotic susceptibility. Salmonellae Salmonellaeare a leadingcauseof food-borne gastroenteritis on a worldwide basisand a major public health concern, being responsible for significant morbidity and mortality in both industrialized and developing countries (173). In the United States, it is estimated that approxi-

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mately 3 million cases of food-borne salmonellosis occur annually. Many vehicles can serve as the source of salmonellosis; however, most infections appear to be transmitted via improperly cooked or handled eggs, poultry, dairy, and beef products, which almost invariably harbor endogenous salmonellae. In studies of defined outbreaks, the infectious dose for some serotypes may be as low as < lo3 CFU (19). A problem of increasing concern is Salmonella food-borne outbreaks caused by strains that are resistant to many antibiotics; in some instances, the source of these resistant isolates appears to be endogenous salmonellae in cattle that have been exposed to grain feed containing antibiotics (167). Currently, over 2,000 Salmonella serotypes are known to exist. Many of these serotypes, such as S. typhi (humans), S. pullorum (chickens), S. choleraesuis (swine), and S. dublin (bovines), exhibit a particular tropism for a given host. There are several clinical syndromes that result from Salmonella infections, including enterocolitis, enteric fever, bacteremia, focal infection, and a chronic fecal or urinary carrier state. Salmonellae penetrate and invade the gastrointestinal mucosa with intra- and submucosal spread. After penetration, acute enterocolitis is the common clinical manifestation. In most cases the stools are loose, of moderate volume, and without blood. However, in exceptional cases the stools may be watery and voluminous or, in other instances, of small volume and associated with tenesmus and gross blood. Salmonellae may be transported to a variety of organs in the reticuloendothelial system via macrophages that phagocytize but do not kill the bacteria. In many individuals at the extremes of life, with poor nutritional status, or with severe underlying immune deficiencies, more severe sequelae can result as a consequence of gastrointestinal colonization and infection. Of particular note is the recent upsurge in cases of nontyphoidal Salmonella bacteremia in patients with AIDS, in which only 20 to 60% of cases display gastrointestinal symptoms prior to sepsis. Many other extraintestinal manifestations of Salmonella infection, including osteomyelitis, endovascular disease, arthritis, orchitis, and hepatitis, have been described. As with most other enteric pathogens, virulence in salmonellae is probably multifactorial, requiring a variety of extracellular and cellassociated factors to produce the full complement of disease-producing capabilities (63). A number of virulence factors have been defined; these factors include the ability to penetrate and replicate in epithelial cells (invasins), a choleralike enterotoxin, a cytotoxin, resistance to killing by serum, motility, and endotoxin (lipopolysaccharide). The exact contribution of each of

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17

these factors to overt disease manifestations remains to be defined. In some salmonellae (S. typhimurium and S. dublin), virulence is partially regulated by plasmids 80 to 100 kb in size. These plasmids are thought to promote infection of mesenteric lymph nodes and to be involved in mouse virulence, serum resistance, and HeLa cell adhesion and invasion. Other strains, such as S. enteritidik and S. choleraesuis, harbor plasmids of characteristic molecular mass ranging from 30 to 37 MDa, while some groups, such as S. infant& are almost always plasmid free. In general, salmonellae can easily be recovered from gastrointestinal specimens since these microorganisms grow quite readily on both differential media and selective agars designed specifically for their isolation. During the acute phase of an illness, if an appropriate specimen is obtained, enrichment procedures (tetrathionate broth or selenite broth) should not be required since large numbers of the etiologic agent should be present. However, under suboptimal conditions or when one is trying to identify Salmonella carriers, enrichment and selective agars are warranted. One recent advance in this area has been the introduction of latex beads coated with polyclonal Salmonella antibodies to screen enrichment broths for the presence of salmonellae (118, 119) (Wampole Laboratories, Cranbury, N. J.; Wellcome Diagnostics, Research Triangle Park, N.C.). Once the organism is isolated, other rapid screening systems can be used (Analytab Products, Plainview, N.Y.). After isolation and identification, Salmonella isolates should be screened against the major grouping sera A, B, C,, C,, D, and E. Greater than 95% of all clinical isolates belong to these six serogroups. Once grouping has been accomplished, strains should be forwarded to local or state public health laboratories for culture confirmation and complete serotype determination. Because of the increasingly large number of major food-borne outbreaks attributed to salmonellae, the clinical laboratory must be aware of the potential emergence of epidemiologically related strains. Although both the serogroup and antimicrobial susceptibility profile may be helpful, in many instances more sophisticated methods are required to trace and monitor such outbreaks (172). Plasmid analysis can be useful in many instances, although some serotypes normally fail to contain plasmids while others have identical plasmid profiles. More recent advances that have great promise along these lines include chromosomal fingerprinting (genomic digests) and ribotyping. These techniques are usually performed by reference or research laboratories. Antimicrobial therapy of Salmonella gastroenteritis usuallv does not shorten the duration of

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symptoms and may prolong the carrier state. Antibiotic therapy is limited to neonates and patients with AIDS, sickle cell disease, lymphoma, leukemia, and systemic salmonellosis. Chloramphenicol, ampicillin, and trimethoprimsulfamethoxazole are useful agentsfor bacteremia and enteric fever. Many of the broad-spectrum cephalosporins(cefoperazoneand ceftriaxone) and new fluoroquinolones (ciprofloxacin) demonstratebactericidal activity againstsalmonellae. The antimicrobial therapy for systemic diseaseshould be basedon susceptibility testing of the isolate becauseof the emergenceof multiply resistant strains. Shigellae Members of the genusShigella are associated classically with bacillary dysentery, a local diseaseof the large bowel associatedwith acute inflammation, ulceration, and, in severe cases, necrosisof the colonic mucosa (133). The virulence of shigellae is based on their ability to penetrate and multiply within epithelial cells and the lamina propria (133, 155). Virulent strains causekeratoconjunctivitis in rabbits and invade epithelial cells such as HEp-2 in vitro. The invasive property is associated with large (= 140.MDa) plasmids(155). The incubation period for shigellosisis usually 24 to 48 h (14). The diseaseis characterized by fever, abdominal cramps, and watery diarrhea, followed in many casesby classic dysentery in which the stoolscontain blood, pus, and mucus. The symptoms vary widely from mild self-limited diarrhea with or without classic dysentery to an illnessassociatedwith severe dehydration, constitutional symptoms,and convulsions(133). Shigellosisis associatedtypically with poor sanitation and overcrowding, and the infection is spread chiefly via person-to-person transmission, which is facilitated by the very low infectious dose(10to 100organisms).In North America, the infection occurs most commonly in institutionalized populations, day-care centers, military recruits, Indian reservations, and slum populations. Numerous food- and waterborne outbreaks have been described(14, 20, 133). The vast majority of isolatesin North America are S. sonnei, a speciesassociatedtypically with a mild illness.The most severecasesof bacillary dysentery are generally associatedwith Shiga’s bacillus (S. dysenteriae type 1). Seriousepidemics of Shiga dysentery have occurred in Asia during the past decade, and one of the major problems has been the emergence of strains multiply resistant to antibiotics; such strains may be isolated from immigrants or travelers returning from areas where the organism is endemic. S. dysenteriae type 1 is the only Shigella type that produces a potent exotoxin,

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Shiga toxin. This toxin probably contributes to the enhancedvirulence of S. dysentetie type 1 and is likely to be causally related to severe complications of Shiga dysentery such as HUS (83, 133). Microscopy of fresh dysentery stools reveals a large cellular exudate consisting of erythrocytes and, typically, sheets of polymorphonuclear leukocytes. The definitive laboratory diagnosis of shigellosisis achieved through direct culture of feces onto appropriate selective and nonselective agar media (46, 48, 49, 133). Standard broth enrichment media generally usedfor salmonellaeare mostly unsuitable for shigellae probably becausethey contain substancesinhibitory for E. co24 to which shigellaeare closely related. A variety of selective differential agar media, including xylose-lysine-deorrycholate, deoxycholate-citrate, salmonella-shigella,Hektoen, and MacConkey agars, are usedfor isolating shigellae from stools. Suspect colonies should be tested by biochemical and serologic methods to confirm the identification of shigellae. DNA probes specific for genes encoding invasivenessare being usedin somecenters for the diagnosisof shigellosisby using DNA hybridization assays(22, 46, 53). Antibiotics recommended for treating shigellosisinclude ampicillin, trimethoprim-sulfamethoxazole, tetracycline, and the quinolone group, although the latter two are not appropriate for children (133). It should be noted that multiple-antibiotic resistance has been frequently observedin Shigella strainsin south and east Asia. Antibiotic therapy of shigellosiswill shorten the period of fecal excretion and limit the clinical course of the illness. Since humans are the major reservoirs for shigellae, public health concerns dictate that each patient with a positive stool culture be treated. Fluoroquinolones, trimethoprim-sulfamethoxazole, and ampicillin are effective, but resistance to these antibiotics may be common in certain geographic areas. Specific antibiotic therapy should be guidedby antimicrobial susceptibility testing. Staphybcoccus

aureus

S. aureusmay occasionally be found aspart of the normal fecal flora, and its isolation from the stool may or may not be significant. Its role as a common cause of food poisoning has been known for many years. These organisms,growing in stored food products such as meat, dairy, or custard products, produce thermostable enterotoxins that cause vomiting and, occasionally, diarrhea within 1 to 6 h of ingestion of contaminated food. The diseaseis self-limiting and usually resolves within 12 to 18 h (79). Culture of the stool is not a sensitive method of confirming the etiologic agent of this illness.

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Isolates from specific food items and from food handlers should be matched by phage typing or other molecular typing methods. Toxin in food items or in culture fluids of suspected isolates can be detected by a reference laboratory. Local health departments should take the lead in resolving these food-borne outbreaks. S. aurew was thought to be the cause of antibiotic-associated pseudomembranous colitis up until the late 1970s. Although it is now established that C. difcile is almost always the cause of this disease, overgrowth of the bowel by the staphylococci does occur in patients receiving broad-spectrum antimicrobial agents (137). Coagulase-negative staphylococci have recently been suggested as a cause of necrotizing enterocolitis in premature infants; however, their etiologic role is not proven (158). Vibrio spp. Members of the genus vibrio are gram-negative, facultatively anaerobic, curved rods that normally inhabit marine and estuarine environments and can occasionally be isolated from freshwater streams, rivers, ponds, and lakes throughout the world. Of the over 30 reported or proposedspeciesfor inclusionwithin this genus, only 12 to date (including V: carchariae) have been proven or implicated to cause human infections (76). By far the greatest number of vibrio-associated casesof gastroenteritis in the United States and other countries are causedby either V. cholerae or K parahaemolyticus. In the United States, more than 75% of all vibrio intestinal infections are attributed to these two species(76). Although vibrios are infrequently isolated from diarrhetic stools in the United States, they are bacteria of major public health significance, sincemembersof the V: cholerae group have for centuries been responsiblefor outbreaks, epidemics, and pandemicsof cholera, primarily in the Eastern Hemisphere. However, epidemic cholera was reported from several locations in Peru in January 1991.Cholera spreadfrom Peru to seven other Western Hemispherecountries, including the United States. As of July 1991,this epidemic included 257,399 probable cholera casesand 2,697 cholera-associateddeaths (33). V: cholerae 01 is the preeminent pathogen of this genusand is the etiologic agent of the vast majority of casesof cholera, a devastating illness and one of the most severe diarrhea1diseasesknown. Acute cholera is a gastrointestinal infection characterized by the daily outpouring of large volumes of fluid and electrolytes (>15 stoolsper day) in the form of watery, opalescent stools. Classiccasesof cholera have been typically described as “rice water” stools, which consist of macroscopic flecks of mucus in the

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19

aqueousmilieu. If left untreated (by electrolyte replacement), severe casesof cholera can ultimately lead to death. An additional emerging problem associatedwith V: cholerae 01 concerns recent reports of strains multiply resistant to antibiotics, including tetracycline, from areas of Southeast Asia, Bangladesh,India, and Tanzania (161). These strains have been involved in both endemic and outbreak-associatedcasesof cholera. In contrast to 01, other serogroupsof V. cholerae (VI cholerae non-01) are rarely implicated as the causative agent of choleralike disease,although they are commonly associated with milder forms of gastroenteritis in the United States. Rare outbreaks of diarrhea1disease caused by V. cholerae non-01, however, have been documented.In addition, V: cholerae non-01 has often beenrecovered from a variety of extraintestinal infections, including ear infections, bacteremia, cellulitis, and cystitis. Of particular concern is the recent increase in the number of reported cases of sepsiscaused by these microorganisms, which occur predominantly in individuals with immunologicor physiologic deficiencies such as cancer, cirrhosis, and achlorhydria (150); often such infections result in death despite appropriate antimicrobic therapy and medical intervention. The other major intestinal pathogen, K parahaemolyticw, typically produces a self-limiting watery diarrhea that is characterized by abdominal discomfort, nausea, or vomiting, usually without significant fever. On rare occasions, a dysenteric or choleralike illness caused by K parahaemolyticus has been described. In contrast to V: cholerae non-01, systemic diseaseis a very rare occurrence, with most extraintestinal infections reported involving wounds. Three other Vzbrio spp. (K mimicus, v1 hoIZisae,and V: fluviaks) have been strongly implicated as causing bacterial gastroenteritis, although very little epidemiologic data are available on such illnesses. VI mimicus, a nonhalophilic member, occasionally causes choleralike diarrhea, and rare isolateshave been found to produce heat-labile cholera toxin. K hollisae gastroenteritis, on the other hand, appearsto be generally more severe, as published reports have documentedan intense, cramping, abdominalpain associatedwith this disease;this speciesgrows poorly (if at all) on thiosulfatecitrate-sucrose-bile salts agar (TCBS), making its laboratory diagnosis more difficult. Most cases of V: fluvialis diarrhea to date involve children from Bangladesh with vomiting and dehydration, although similar symptoms in adults have been noted. The small intestine is the major target organ for a majority of Wbrio gastrointestinal infections in which an enteritis syndrome is the chief

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clinical presentation. For both K cholerae 01 and non-01, the mucus coat surrounding the villi apparently serves as the initial site for colonization and attachment (189,190); adherence to this layer appears to be at least partially mediated through K cholerae cell-associated hemagglutinins in both 01 and non-01 strains. Other surface structures such as a toxin-coregulated pilus, outer membrane proteins, lipopolysaccharide, and flagella probably play important roles in the eventual adherence of V. cholerae to the intestinal microvillus. Once attachment is completed, choleralike illness produced by I? cholerae 01 is dependent on the elaboration of cholera toxin by 01 strains. After release from the bacterium, the cholera toxin enters susceptible intestinal cells, causing an increase in intracellular cyclic AMP levels. This activation produces a release of electrolytes (Na+ and Cl-) and water from the intestinal epithelium into the gastrointestinal lumen, thereby eliciting the classic choleralike illness (68). On rare occasions, 01 strains lacking the cholera toxin gene (CZA) have been detected, and associated cases of choleralike illness are usually much milder and of shorter duration (71). The presumed reason that K cholerae non-01 strains have not been associated with epidemic and pandemic cholera is that the vast majority of non-01 serogroups consistently lack the ctx gene. For most non-01 strains, other virulence factors involved in the diarrhea1 disease syndrome are probably operative; these factors include a heat-stable enterotoxin and a hemolysin/cytolysin that possesses enterotoxigenic activity (6). For V: parahaemoZyticus, there has been a long-standing association between clinical infections (vibrio-associated gastroenteritis) and the ability of individual strains to elaborate a thermostable direct hemolysin (76). Recent studies have implicated other extracellular factors as potentially important in V: parahaemotyticus-induced diarrhea; these factors include hemolysin related to the thermostable direct hemolysin and surface structures such as fimbriae and cell-associated hemagglutinins (191). Because of the sporadic nature of individual cases of vibrio-associated gastroenteritis in the United States, it has been demonstrated repeatedly that the routine addition of a selective agar, TCBS, to routine stool cultures for the recovery of vibrio spp. is not cost-effective (67). Rather, awareness of the possibility of a vibrio infection is dependent on the physician obtaining a good medical history from the patient and then relaying the appropriate information to the clinical laboratory. One should consider the possibility of a Wbrio infection whenever a medical history reveals the recent consumption of shellfish, exposure to aquatic environments or marine-asso-

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ciated products, foreign travel, or a diarrhea1 disease resembling cholera (104,105). By far the greatest predictor of potential vibrio-associated gastroenteritis is the recent consumption of raw shellfish, particularly oysters; other seafoods such as clams, shrimp, and crabs have also been implicated but to a far lesser extent (105). When such epidemiologic data are available, TCBS should be added to the primary plating medium. If the patient’s medical history is unavailable, vibrio spp. can still be recognized and identified if the laboratory includes a blood agar plate as part of its standard fecal culture workup. By use of blood agar, gram-negative colonies not resembling Pseudomonas aeruginosa can be screened for oxidase and indole positivity, and certain species such as K cholerae and K JEuviaZis are often hemolytic, which aids in their recognition. The isolates of V. cholerae 01 responsible for the Western Hemisphere outbreak in South and Central America are nonhemolytic, unlike the U.S. Gulf Coast isolates. On rare occasions when an enrichment procedure is felt necessary to recover a Vibrio species, alkaline peptone water (pH 8.5) is generally used for K cholerae and alkaline peptone water with 3% NaCl is used for V: parahaemoZyticus. After isolation of a possible Vzbrio species from a selective or nonselective agar, the presumptive identification should be confirmed biochemically. Ideally, vibrios should be identified by using a conventional panel of biochemical tests which includes susceptibility to the vibriostatic agent O/129, salt tolerance, and growth on TCBS (76). Salt tolerance for vibrio identification is extremely important as it separates the nonhalophilic species (VI cholerae and V: mimicus) from the halophilic species (all others). In addition, conventional media may require salt supplementation in order for halophilic species to grow and become metabolically active. If commercial systems are used for vibrio identification, the microbiologist should be aware that many kits commonly misidentify these bacteria at either the genus or species level (130). Other problems associated with kit use include lack of correlation between commercial and conventional test results, inability of the data base or test panel to identify Wbrio spp. from biochemically similar organisms such as Aeromonas spp., and the failure of some species to grow in the inoculated broth without addition of salt or some other supplement. If a presumptive identification of K cholerae is made, the isolate should be immediately forwarded to the nearest public health or reference laboratory for culture confirmation and for further evaluation because of clinical and epidemiologic significance of the 01 group. Reference laboratories should be able to determine

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whether the isolate in question belongs to serogroup 01 by screening with polyclonal somatic antisera; if the results are positive, the individual biotype of V: cholerae 01 (classical or El Tor) should be determined. In addition, reference laboratories should be able to ascertain whether the V: cholerae strain (in very rare instances, V: mimicus) can produce cholera toxin. This can be accomplished by several simple techniques, including cell culture with toxin neutralization, ELISA (not commercially available), and latex agglutination (4). If toxigenic V: cholerae 01 is reported, the appropriate public health authorities should be immediately notified so that timely epidemiologic investigations can commence and the medical community can be alerted. For V: parahaemolyticus, laboratory identification is fairly straightforward with the exception that an increasing number of urea-hydrolyzing strains have been reported (2). This unusual biotype was almost never seen prior to 1980. Strains of V: parahaemolyticus implicated in causing gastroenteritis almost invariably produce a thermostable hemolysin that can be detected on a special high-salt-mannitol-containing agar, termed Wagatsuma’s agar (76). Strains expressing beta-hemolysis of human 0 erythrocytes on this medium are referred to as Kanagawa test positive; most environmental isolates recovered from shellfish, in contrast, are Kanagawa test negative. However, evidence is accumulating that some Kanagawa test-negative isolates of V. parahaemolyticus can produce disease in humans, although the epidemiologic significance is presently unclear. Intravenous or oral fluid replacement therapy is central to the management of patients with cholera; however, antibiotics will shorten the duration of diarrhea and thus reduce fluid losses. Most strains of K cholerae are susceptible to the drug of choice, tetracycline, but multipleantibiotic resistance seems to be a growing problem (76). Diarrhea due to V. parahaemolyticus is usually self-limited, and antimicrobial therapy shortens neither the duration of symptoms nor the excretion of the bacterium. V. parahaemoZyticus is usually resistant to ampicillin and carbenicillin but susceptible to colistin, characteristics that can be used to distinguish it from I? vulnificus. Vibrios, including the halophilic strains, usually grow well on Mueller-Hinton agar for disk diffusion antibiotic susceptibility testing. Yersinia enterocolitica The genus Yersinia is composed of a number of pathogenic and saprophytic members that inhabit a wide range of environmental niches and cause disease in numerous species of ani-

DIAGNOSIS

OF BACTERIAL

DIARRHEA

21

mals, including humans. Pathogenic species produce both zoonotic infections (Y pestis and Y pseudotuberculosis) and a number of intestinal and extraintestinal illnesses in humans, the most common of which is gastroenteritis (25). Yersinia-associated diarrhea is primarily caused by pathogenic strains belonging to the species Y enterocolitica and occurs on a worldwide basis, although higher rates appear to occur in northern Europe, Canada, and the United States. Y. enterocolitica can cause several diarrhea1 syndromes ranging from a mild self-limiting disease to an enteritis with severe abdominal pain (mesenteric lymphadenitis or terminal ileitis) which often mimics acute appendicitis (25). Pathogenic members of this species can also cause several extraintestinal illnesses, including septicemia, bacteremia via contaminated blood transfusions, arthritis, endocarditis, pyomyositis, and pharyngitis (18, 25, 74, 147). Because of its ability to survive and multiply at low temperatures, the bacterium has the capability of being transmitted through a wide range of refrigerated products, although the exact vehicle of transmission for most infections has not been determined. Some recent epidemiologic studies have implicated pasteurized and unpasteurized milk as the source of yersinia-associated gastroenteritis, while another investigation identified raw pork consumption as a major source in Belgium (170). Yersiniae produce gastrointestinal disease by an invasion process through the intestinal mucosa with intra- and submucosal proliferation (63). After penetration, Y, enterocolitica passes through the lamina propria to the lymphatic nodules (Peyer’s patches) of the intestine (terminal ileum), where replication occurs (26). Although the exact virulence determinants responsible for pathogenic@ in Y. enterocolitica are still not completely understood, chromosomal invasin genes, plasmid (40 to 50 MDa)-mediated outer membrane proteins (adherence), and a heat-stable enterotoxin are probably all involved (63); saprophytic and nonpathogenic strains of Y, enterocolitica apparently lack most or all of these factors. Y. enterocolitica can be recovered from diarrhea1 stools on a number of enteric media such as MacConkey and salmonella-shigella agars. In general, Y, enterocolitica appears as pinpoint, lactose-negative (though not invariably) colonies after 24 h of incubation at 22 to 25°C. An excellent selective agar for the recovery of Y. enterocolitica, CIN, is commercially available. Yersiniae appear as small colonies with a red center and transparent edge (“bull’s eye”) after 24 h of incubation at 25 to 35°C; however, some studies indicate a significant growth of contaminants on this agar (80). Because the incidence of

22

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yersiniosis is very low in most medical centers, the routine use of CIN is probably not justifiable; however, since this medium also recovers most Aeromonas strains, it might be considered for inclusionin the normal enteric battery becauseof its dual use(5). In the past, incubation of feces in phosphate-bufferedsaline(PBS) at 4°C for 14 to 21 days has been recommendedas a method of enhancingthe recovery of yersiniae from fecal specimenswhen the numbersare apparently low (cold enrichment). However, the recovery of Y. enterocolitica from an individual in the acute phase of gastroenteritis only upon enrichment raisesthe question of clinical relevance. Not all strains of Y. enterocolitica are virulent, and pathogenic strains can be separated from saprophytic isolateson the basisof somatic antigens(serogroup)and the presenceof certain phenotype markers. There are more than 50 recognized serogroups of Y. enterocolitica. However, >90% of virulent strains fall into one of five serogroups:0: 1,2a,3; 0 :3; O&27; 0: 8; and 0: 9 (38). Historically, most 0: 3 and 0: 9 isolates have been recovered from patients in Europe while 0: 8 and 0: $27 have predominated in the United States. However, since the early 198Os,a general decrease in 0: 8 and increase in 0: 3 have been reported in most locales throughout the United States (18). The exact epidemiologic significance of this serogroup shift is presently unknown. Unfortunately for the clinical laboratory, serogrouping is not practical since antisera are not commercially available and few reference laboratories have the capability of typing such isolates. Strains can, however, be assignedto pathogenicversus nonpathogenicgroupson the basisof a biotyping scheme(183) and the presenceof one or more markers associated with pathogenic@. Most pathogenic strains, irrespective of serogroup designation, are esculin and pyrazinamidase negative (81,183). In addition to determiningthe biotype of a given strain, the laboratory can supplement test results via one of several in vitro assaysassociatedwith virulence products of pathogenic Y. enterocolitica. These tests include autoagglutinationin broth, growth on magnesium-oxalate agar, and a variety of assays aimed at determining the relative hydrophobic nature of the surface of virulent strains (Congo red and crystal violet dye binding) (139, 142). Y. enterocolitica is usually susceptiblein vitro to aminoglycosides,chloramphenicol, tetracycline, and broad-spectrum cephalosporins.Isolates are usually resistant to ampicillin and narrow-spectrum cephalosporinsbut vary in susceptibility to carbenicillin (26). The value of antimicrobial therapy of uncomplicated enterocolitis andmesentericlymphadenitisis not clear. Antimicrobial therapy may be beneficial in cases

CUMITECH

12A

of chronic or fulminating disease.The agentsto be used should be selected on the basis of antimicrobial susceptibility test results. GUIDELINES FOR STOOL EXAMINATIONS The cost of doing a stool examination on every patient for all the potential pathogens described in this Cumitech is prohibitive. Strategiesshould be developed to ensurethe highest possible positivity rate, since stool culture has been shown to have an extremely high cost per positive result (61). It is particularly important to identify those casesof diarrhea1diseasecaused by agents that require therapy other than oral rehydration. It also is important to identify etiologic agents responsible for food-borne outbreaks so that food-handling techniques can be modified to prevent further transmissions. Several factors should be considered when one is determiningthe organismsfor which stool should be examined. Such factors include the age and hospitalization status of the patient, season,geographic locale, and histories of recent travel, diet, and underlying disease.Often only limited information is directly available to the laboratory unlessthere is good communication between the laboratory and the physician. Becauseage and hospitalization status are usually available, thesetwo factors can be usedasa starting point for designingstrategiesto obtain cost-effective stool examinations. Most casesof outpatient diarrhea in adults are inflammatory diarrheas, and stools may be screenedfor leukocytes with a methylene blue stain. However, the sensitivity of fecal leukocyte examinationsis lessthan 90%. The absence of leukocytes does not adequately rule out agentsof inflammatory diarrheas, but the presence of leukocytes may eliminate from consideration agents of noninflammatory diarrheas, including toxins produced by vibrios and ETEC, viral agents, and certain parasitic agents (64, 162) In’ most geographic locales, outpatient fecal screensshould include techniquesfor detecting Salmonella, Shigella, and Campylobacter spp. In certain geographic locales, positive yields may be increasedsignificantly by adding screening methods for vibrios (along the coast of the Gulf of Mexico), E. coli 0157: H7 (northwestern United States and Canada)(113), and Y, enterocolitica or ETEC (areas with high numbers of migrant workers from Mexico and Central America). Patient information may also help improve yield. C. di!‘ciZe mustbe consideredfor any patient who currently is receiving or recently hasreceived antimicrobial agentsand has developed diarrhea. E. coli 0157: H7 should be sought routinely in all patients in areaswith high endemicity and in patients with frankly bloody

CUMITECH

12A

LABORATORY

stools in areas of low endemicity. People who consume raw shellfish are at increased risk for vibrio infections. Children who have chronic malabsorptive diarrhea may be infected with G. lamblia or enteroadherent E. coli. In the winter months, children with diarrhea should first be screened for rotavirus, and only if that test is negative should testing be done for bacterial pathogens. Simple quality assurance studies such as screening stools for various agents for 3to 6-month periods and assessing the impact of these studies on stool yield will help determine what organisms should be included in outpatient screens at individual institutions. One other important role that the laboratory plays in management of outpatient diarrheas is in the detection of common-source outbreaks. The laboratory should notify public health officials whenever an unusual number of enteric pathogen isolates are recovered. For example, in many institutions, the isolation during 1 week of more than one isolate of Shigella sp. from children less than 5 years of age may suggest an outbreak of shigellosis in a day-care center. Nosocomial diarrhea is described in the literature as the onset of diarrhea more than 3 days after the patient enters the hospital (163). This definition is reasonable in that it recognizes that certain patients either will enter the hospital because of diarrhea1 symptoms (especially young children) or may have brief bouts of self-limited diarrhea, usually virally induced, at or near the time of admission. In children, rotavirus (discussed in detail in Cumitech 26) is the leading cause of nosocomial diarrhea, and this virus is the only agent for which stools from children with nosocomial diarrhea should be routinely screened. In adults, studies have shown that C. dijsf’icile is the only agent reliably detected in stools of patients with nosocomial diarrhea1 disease (163). As a result, stools from patients hospitalized for more than 3 days should be screened only for this organism. There are two exceptions to this rule. First, screening for pathogens which can cause food-borne outbreaks of diarrhea such as Salmonella sp. or E. coli 0157: H7 should be done when infection control personnel have recognized a possible outbreak of diarrhea1 disease. The other exception is diagnosing the etiologic agent of diarrhea in AIDS patients. The diagnosis of the etiologic agent of diarrhea in AIDS patients presents a special challenge to the clinical laboratory. Infectious diarrhea in these patients can be due to various bacterial pathogens, protozoans (including those recognized only in this population), helminths, and viral agents, including human immunodeficiency virus (58, 103, 127). It also may be the result of proctitis due to a variety of sexually

DIAGNOSIS

OF BACTERIAL

DIARRHEA

23

transmitted diseases, including those caused by Neisseria gonorrhoeae, Treponema pallidum, herpes simplex virus, or Chlamydia trachomatis. Diagnosis of diarrhea due to agents of proctitis is dependent upon physicians notifying laboratories of their suspicions, since none of these agents are routinely sought in feces. Establishing the etiology of diarrhea is important especially for patients who have treatable infections. Routine stool screens in these patients should include culture for Salmonella, Shigella, and Campylobacter spp., examination for ova and parasites (including an acid-fast stain for the detection of CIyptosporidium and Isospora spp.), and detection of C. dificile toxin. Two points should be made concerning this screen. Campylobacter species other than C. jejuni and C. coli have been recovered from homosexual men with diarrhea and perhaps should be considered. These other campylobacters may not grow at 42°C and may be susceptible to cephalosporins; therefore, this class of drugs should not be used in selective medium used to isolate campylobacters from these patients. Second, AIDS patients now receive, for life, a variety of drugs, including azidothymidine, which can induce C. difcile disease, and so testing for this organism should be included in stool examination in this patient population. It should also be understood that patients may be infected with more than one agent and that the finding of one infective agent does not exclude the possibility that other agents are present; thus, examinations should be carried to completion. The diagnosis of human immunodeficiency virus infections should be considered in adults less than 50 years old who present with bacteremia due to a diarrhea1 pathogen such as nontyphoid Salmonella sp. or Shigelra sp. and who do not have a known predisposing medical condition such as sickle cell anemia. Other agents should be sought if an appropriate history such as shellfish ingestion or frankly bloody diarrhea is obtained. Mycobacterium avium complex should be considered if initial screening for more common enteric pathogens is negative, since it is thought to cause diarrhea in this patient population. If extensive testing proves negative, then bowel biopsies to look for viral agents such as cytomegalovirus may also be performed (103). REPORTING RESULTS With the recognition of an increasing number of bacterial agents of diarrhea, it is important to specifically identify each organism for which stool specimens were examined. It is incorrect to issue a report “No enteric pathogen isolated” if the stool was cultured only for Salmonella, Shigella, and Campylobacter spp. Rather, this

24

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report should state “No Salmonella, Shigella, or Campylobacter isolated.” Protocols should also make provisions for reporting the absence of the normal gram-negative fecal flora and the presence of significant quantities of organisms such as Staphylococcus aureus, yeasts, and Pseudomonas aeruginosa. If stool specimens or isolates are sent to reference laboratories for further work, such as examination for the presence of C. di!cile toxin or serotyping of Salmonella isolates, the final laboratory report on such specimens must include the name of the reference laboratory, and the work performed, such as serotyping, must be indicated. REFERENCES 1. Abbott, S. L., R. P. Kokka, and J. M. Janda. 1991. Laboratory investigations on the low pathogenic potential of Plesiomonas shigelloides. J. Clin. Microbial. 29:148-153. 2. Abbott, S. L., C. Powers, C. A. Kaysner, Y. Takeda, M. Ishibashi, S. W. Joseph, and J. M. Janda. 1989. Emergence of a restricted bioserovar of Vibrioparahaemolyticus as the predominant cause of Vibrio-associated gastroenteritis on the West Coast of the United States and Mexico. J. Clin. Microbial. 27:2891-2893. 3. Allen, S. D., and E. J. Baron. 1991. Clostridium, p. 505-521. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 4. Almedia, R. P., F. W. Hickman-Brenner, E. G. Sowers, N. D. Puhr, J. J. Farmer III, and I. K. Wachsmuth. 1990. Comparison of a latex agglutination assay and an enzyme-linked immunosorbent assay for detecting cholera toxin. J. Clin. Microbial. 28:128-130. 5. Altorfer, R., M. Altwegg, J. Zollinger-Iten, and A, von Graevenitz. 1985. Growth of Aeromonas spp. on cefsulodin-irgasan-novobiocin agar selective for Yersiniu enterocolitica. J. Clin. Microbial. 22:478-480. 6. Arita, M., T. Takeda, T. Honda, and T. Miwatani. 1986. Purification and characterization of Vibrio cholerue non-01 heat-stable enterotoxin. Infect. Immun. 52:45-49. 7. Asao, T., Y. Kinoshita, S. Kozaki, T. Uemura, and G. Sakaguchi. 1984. Purification and some properties of Aeromonas hydrophila hemolysin. Infect. Immun. 46:122-127. 8. Baldini, M. M., J. B. Kaper, M. M. Levine, D. C. A. Candy, and H. W. Moon. 1983. Plasmid-mediated adhesion in enteropathogenic Escherichiu coli. J. Pediatr. Gastroenterol. Nutr. 2:534-538. 9. Balows, A., W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy @I.). 1991. Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 10. Bartlett, J. G. 1979. Antibiotic-associated pseudomembranous colitis. Rev. Infect. Dis. 1:530-539. 11. Bartlett, J. G. 1984. Treatment of antibiotic associated pseudomembranous colitis. Rev. Infect. Dis. 6=S235-S241. 12. Bartlett, J. G., N. Moon, T. W. Chang, N. Taylor, and A. B. Onderdonk. 1978. The role of Clostridium dificile in antibiotic-associated pseudomembranous colitis. Gastroenterology 75:778-782. 13. Basta, M., M. Karmali, and C. Lingwood. 1989. Sensitive receptor-specified enzyme-linked immunoassay for Escherichiu coli verocytotoxin. J. Clin. Microbial. 27:1617-1622. 14. Benenson, A. S. 1985. Control of communicable diseases in man. The American Public Health Association, Washington. D.C.

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15. Bhan, M. K., P. Rqj, J. B. Kaper, N. Bhandari, R. Srivastara, P. Kumar, M. M. Levine, and S. Sazawal. 1989. Enteroaggregative Escherichiu coli associated with persistent diarrhea in a cohort of rural children in India. J. Infect. Dis. 159~1061-1064. 16. Biddle, W. L., J. L. Harms, N. J. Greenberger, and P. B. Miner, Jr. 1989. Evaluation of antibiotic associated diarrhea with a latex agglutination test and cell culture cytotoxicity assay for Clostridium difficile. Am. J. Gastroenterol. 84:379-382. 17. Birkhead, G., R. L. Vogt, E. M. Heun, J. T. Snyder, and B. A. M&lane. 1988. Characterization of an outbreak of Clostridium perjkingens food poisoning by quantitative fecal culture and fecal enterotoxin measurement. J. Clin. Microbial. 26~471-474. 18. Bissett, M. L., C. Powers, S. L. Abbott, and J. M. Janda. 1990. Epidemiologic investigations of Yersiniu enterocolitica and related species: sources, frequency, and serogroup distribution. J. Clin. Microbial. 28:910-912. 19. Blaser, M. J., and L. S. Newman. 1982. A review of human salmonellosis. I. Infective dose. Rev. Infect. Dis. 4: 1096-l 106. 20. Blaser, M. J., R. A. Pollard, and R. R. Feldman. 1983. Shigellu infections in the United States, 1974-1980. J. Infect. Dis. 147:771-775. 21. Blaser, M. J., and L. B. Reller. 1981. Cumpylobacter enteritis. N. Engl. J. Med. 305:1444-1452. 22. Boileau, C. R., H. M. d’aauteville, and P. J. Sansonetti. 1984. DNA hybridization technique to detect Shigellu species and enteroinvasive Escherichiu coli. J. Clin. Microbial. 20:959-961. 23. Bolton, F. J., and D. N. Hutchinson. 1988. Reassessment of selective agars and filtration techniques for isolation of Campylobucter species from faeces. Eur. J. Clin. Microbiol. Infect. Dis. 7:155-160. 24. Borriello, S. P., F. E. Barclay, P. J. Reed, A. R. Welch, J. D. Brown, and D. W. Burdon. 1987. Analysis of latex agglutination test for Clostridium difficile toxin A (D-l) and differentiation between C. difficile toxins A and B and latex reactive protein. J. Clin. Pathol. 40~573-580. 25. Bottone, E. J. 1977. Yersiniu enterocoliticu: a panoramic view of a charismatic microorganism. Crit. Rev. Microbiol. 5:211-241. 26. Bottone, E. J. 1984. Yersinia enterocolitica, p. 13-47. In P. D. Ellner (ed.), Infectious diarrhea1 diseases: current concepts and laboratory procedures. Marcel Dekker, New York. 27. Bowden, T. A., Jr., A. R. Mansberger, Jr., and L. E. Lykins. 1981. Pseudomembranous enterocolitis: mechanism of restoring flora homeostasis. Am. Surg. 47:178-183. 28. Brenden, R. A., M. A. Miller, and J. M. Janda. 1988. Clinical disease spectrum and pathogenic factors associated with Plesiomonas shigelloides infections in humans. Rev. Infect. Dis. 10~303-316. 29. Brill, B. M., B. L. Wasilauskas, and S. H. Richardson. 1979. Adaptation of the staphylococcal coagglutination technique for detection of heat-labile enterotoxin of Escherichiu coli. J. Clin. Microbial. 9=49-55. 30. But-don, D. W. 1982. Clostridium dificile: the epidemiology and prevention of hospital-acquired infection. Infection 10~203-204. 31. Butzler, J. P., and M. B. Skit-row. 1979. Cum&obacter enteritis. Clin. Gastroenterol. 8:737-765. 32. Centers for Disease Control. 1985. Foodborne disease outbreaks annual summary. 1982. Centers for Disease Control, Atlanta. 33. Centers for Disease Control. 1991. Cholera. New York, 1991. Morbid. Mortal. Weekly Rep. 40:516-518. 34. Chakraborty, T., M. A. Montenegro, S. C. Sanyal, R. Helmuth, E. Bulling, and K. N. Timmis. 1984. Cloning of enterotoxin gene from Aeromonas hydrophila provides conclusive evidence of production of a cytotonic enterotoxin. Infect. Immun. 46~435-441.

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35. Cohen, M. L. 1988. The epidemiology of diarrhea1 disease in the United States. Infect. Dis. Clin. North Am. 2:557-570. 36. Colarian, J. 1988. Clostridium difficile colitis following antiviral therapy in the acquired immunodeficiency syndrome. Am. J. Med. 84=1081. 37. Cooperstock, M. S., and A. J. Zedd. 1983. Intestinal flora in children, p. 79-99. In D. J. Hentges (ed.), Human intestinal microflora in health and disease. Academic Press, New York. 38. Comelis, G., Y. Laroche, G. Balligand, M. P. Sovy, and G. Wauters. 1987. Yersinia enterocolitica, a primary model for bacterial invasiveness. Rev. Infect. Dis. 9~64-87. 39. Comick, N. A., and S. L. Gorbach. 1988. Campylobacter. Infect. Dis. Clin. North Am. 2:643-654. 40. Cravioto, A., R. J. Gross, S. M. Scotland, and B. Rowe. 1979. An adhesive factor found in strains of Escherichia coli belonging to the traditional infantile enteropathogenie serotypes. Curr. Microbial. 3:95-99. 41. Croft, C. C., and M. J. Miller. 1956. Isolation of Shigella from rectal swabs with Hajna “Gn” broth. Am. J. Clin. Pathol. 26~411-417. 42. Cryan, B. 1990. Comparison of three assay systems for detection of enterotoxigenic Escherichia coli heat-stable enterotoxin. J. Clin. Microbial. 28:792-794. 43. DiPersio, J. R., F. J. Varga, D. L. Conwell, J. A. Kraft, K. J. Kozak, and D. H. Willis. 1991. Development of a rapid enzyme-linked immunoassay for Clostridium difitile toxin A and its use in the diagnosis of C. difficileassociated disease. J. Clin. Microbial. 29=2724-2730. 44. Donahue-Rolfe, A., N. A. Kelley, M. Bennish, and G. T. Keush. 1986. Enzyme-linked immunosorbent assay for Shigella toxin. J. Clin. Microbial. 24~65-68. 45. Doyle, M. P., and J. L. Schoeni. 1987. Isolation of Escherichia coli 0157:H7 from retail fresh meats and poultry. Appl. Environ. Microbial. 53:2394-2396. P., 0. Sethabutr, and C. Pitarangsi. 1991. 46. Echeverria, Microbiology and diagnosis of infection with Shigella and enteroinvasive Escherichia coli. Rev. Infect. Dis. 13(Suppl. 4):S22O-S225. 47. Echeverria, P., D. N. Taylor, J. Seriwatana, and J. E. Brown. 1989. Examination of colonies and stool blots for detection of enteropathogens by DNA hybridization with eight DNA probes. J. Clin. Microbial. 27~331-334. 48. Ewing, W. H. 1986. Edwards and Ewing’s identification of Enterobacteriaceae. Elsevier, New York. 49. Farmer, J. J., III, and M. T. Kelly. 1991. Enterobacteriaceae, p. 360-383. In A. Balows, W. J. Hausler, Jr., IS. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 50. Field, M. 1971. Intestinal secretion: effect of cyclic AMP and its role in cholera. N. Engl. J. Med. 284=1137-l 144. 51. Finch, M. J., and L. W. Riley. 1984. Campylobacter infections in the United States. Results of an 11-year surveillance. Arch. Intern. Med. 144:1610-1612. 52. Finegold, S. M., V. L. Sutter, and G. E. Mathisen. 1983. Normal indigenous intestinal flora, p. 3-31. In D. J. Hentges (ed.), Human intestinal microflora in health and disease. Academic Press, New York. 53. Frankel, G., L. Riley, J. A. Giron, J. Valmassoi, A. Friedmann, N. Stockbine, S. Falkow, and G. K. Schoolnik. 1990. Detection of Shigella in feces using DNA amplification. J. Infect. Dis. 161:1252-1256. 54. Fujimoto, S., N. Uki, T. Itoh, and K. Amako. 1991. Specific serotype of Cam~lobacter jejuni associated with Guillain-Barre syndrome. J. Infect. Dis. 165:183. 55. Gardner, S. E., S. E. Fowlston, and W. L. George. 1987. In vitro production of cholera toxin-like activity by Plesiomonas shigelloides. J. Infect. Dis. 156:720-722. 56. George, W. L., M. M. Nakata, J. Thompson, and M. L. White. 1985. Aeromonas related diarrhea in Intern. Med. 145:2207-2211.

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DIARRHEA

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57. George, W. L., V. L. Sutter, D. Citron, and S. M. Finegold. 1979. Selective and differential medium for isolation of Clostridium difficile. J. Clin. Microbial. 9~214-219. 58. Gillian, J. S., M. Shike, N. Alcock, C. Urmacher, S. Krowu, R. C. Kurtz, C. J. Lightdale, and S. J. Winawer. 1985. Malabsorption and mucosal abnormalities of the small intestine in the acquired immunodeficiency syndrome. Ann. Intern. Med. 102:619-622. 59. Gross, R. J. 1990. Escherichia coli diarrhea, p. 470-487. In M. T. Parker and L. H. Collier (ed.), Topley and Wilson’s principles of bacteriology, virology, and immunity. B. C. Decker, Inc., Philadelphia. 60. Guerrant, R. L., J. M. Hughes, N. L. Lima, and J. Crane. 1990. Diarrhea in developed and developing countries: magnitude, special settings, and etiologies. Rev. Infect. Dis. 12:S41-S50. 61. Guerrant, R. L., D. S. Shields, S. Thorson, J. B. Schorling, and D. H. M. Groschel. 1985. Evaluation and diagnosis of acute infectious diarrhea. Am. J. Med. 78(Suppl. 6B):91-98. 62. Gun-Munro, J., R. P. Rennie, J. H. Thomley, H. L. Richardson, D. Hodge, and J. Lynch. 1987. Laboratory and clinical evaluation of isolation media for Campylobatter jejuni. J. Clin. Microbial. 25:2274-2277. 63. Hale, T. L., and S. B. Formal. 1988. Virulence mechanisms of enteroinvasive pathogens, p. 61-69. In J. A. Roth (ed.), Virulence mechanisms of bacterial pathogens. American Society for Microbiology, Washington, D.C. 64. Harris, J. C., H. L. Dupont, and R. B. Homick. 1972. Fecal leukocytes in diarrhea1 illness. Ann. Intern. Med. 76:697-703. 65. Hauschild, A. H. W. 1975. Criteria and procedures for implicating Clostridium perjkingens in foodborne out1breaks. Can. J. Public Health 66~388-392. 66. Hodinka, R. L., and P. H. Gilligan. 1988. Evaluation of 4the Campyslide agglutination test for confirmatory iden1tification of selected Campylobacter species. J. Clin. Microbial. 26:47-49. 67. Hoge, C. UT., D. Watsky, R. N. Peeler, J. P. Libonati, E. Israel, and J. G. Morris, Jr. 1989. Epidemiology and spectrum of vibrio infections in a Chesapeake Bay community. J. Infect. Dis. 160~985-993. 68. Holmberg, S. D. 1988. Vibrios and Aeromonas. Infect. Dis. Clin. North Am. 2:655-677. 69. Holmberg, S. D., W. L. Schell, G. R. Fanning, I. K. Wachsmuth, F. W. Hickman-Brenner, P. A. Blake, D. J. Brenner, and J. J. Farmer III. 1986. Aeromonas intestinal infections in the United States. Ann. Intern. Med. 105:683-689. 70. Holmberg, S. D., I. K. Wachsmuth, F. W. HickmanBrenner, P. A. Blake, and J. J. Farmer III. 1986. Plesiomonas enteric infections in the United States. Ann. Intern. Med. 105:690-694. 71. Honda, S. I., K. Shimoirisa, A. Adachi, K. Saito, N. Asano, T. Taniguchi, T. Honda, and T. Miwatani. 1988. Clinical isolates of Hbrio cholerae 01 not producing cholera toxin. Lancet ii:1486. 72. Hussain-Qadri, S. M., J. Akhter, S. Ostrawski, S. G. M. Qadri, and B. A. Cunha. 1989. High incidence of false positives by a latex agglutination test for the diagnosis of Clostridium dificile associated colitis in compromised patients. Diagn. Microbial. Infect. Dis. 12:291-294. 73. Hutchinson, D. N., and F. J. Bolton. 1983. Is enrichment culture necessary for the isolation of Campylobacter jejuni from faeces? J. Clin. Pathol. 36~1350-1352. 74. Jacobs, J., D. Jamaer, J. Vandeven, M. Wouters, C. Vermylen, and J. Vandepitte. 1989. Yersinia enterocolitica in donor blood: a case report and review. J. Clin. Microbial. 27:1119-1121. 75. Janda, J. M., and P. S. Duffey. 1988. Mesophilic aeromonads in human disease: current taxonomy, laboratory

26

76.

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