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Feces and, in some cases, rectal swab specimens are useful for determining the etiologic agent of infectious diarrhea or food poisoning, confirming the diagnosis of botulism, and diagnosing infections caused by adenoviruses, enteroviruses, some sexually transmitted pathogens, intestinal protozoa and helminths, and, in some instances, helminths of the respiratory and biliary tracts. Collection, transport, and processing of these specimens are different for viruses, bacteria, and parasites and are discussed separately for each group.
Viruses
Stool is preferred for detection of adenoviruses, enteroviruses, and the viruses responsible for gastroenteritis. Specimens should be collected in a clean container with a tight lid. If feces cannot be obtained, a swab is inserted beyond the anal sphincter, rotated, withdrawn, and placed in M4 transport medium. Deliver specimens promptly to the laboratory; if not, refrigerate for a short time and transport on wet ice. If the specimen must be mailed to a reference laboratory, store at -70°C and ship on dry ice.
Specimen processing
Cell culture is recommended for detection of adenoviruses and enteroviruses, although an ELISA for detection of adenoviruses is available. Rotaviruses are responsible for most cases of viral gastroenteritis. Most commonly, rotavirus is detected by ELISA; latex agglutination kits also are available but appear to be less sensitive ( Christensen, 1989 ). Specimens are processed according to the manufacturer's directions. Direct examination of stool specimens by electron microscopy is the reference method for detection of rotavirus and is useful for detection of caliciviruses, astroviruses, and noroviruses. Noroviruses also can be detected by PCR.
Bacterial Pathogens
Stool also is preferred for detection of bacteria responsible for infectious diarrhea but a rectal swab specimen is an acceptable alternative. Stool cultures for patients who have been hospitalized for more than 3 days are inappropriate. Stool specimens should be collected in a clean container with a tight lid, and the specimen should not be contaminated with urine, barium, or toilet paper. Rectal swab specimens, obtained as described earlier for viruses, are placed in a tube transport system containing modified Stuart's medium. Both specimen types should be transported promptly (within 2 hours) to the laboratory and processed as soon as possible, because the drop in pH that occurs as the stool cools may inhibit the growth of some pathogens, especially Shigella. If a delay in processing is unavoidable, or if the specimen must be mailed to a reference laboratory, transport of an aliquot of the specimen in transport media such as Cary Blair is required. For C. difficile culture or toxin testing, 20–50 mL of liquid stool should be submitted in a sterile container. The stool specimen is stable for 2 days, refrigerated, or frozen for 1 week.
Specimen Processing
Processing stool or rectal swab specimens for detection of bacteria is based on the organism or group of organisms expected to be present. Specimens received for ‘routine' bacterial culture should be processed to allow recovery of Shigella, Salmonella, Shiga-like toxin producing E. coli (STEC), Campylobacter jejuni/coli, andAeromonas species by plating to appropriate differential, inhibitory and non-inhibitory media (see Ch. 56 ). The prevalence of gastroenteritis caused by Yersinia enterocolitica, Vibrio cholerae or other Vibrio species, or Plesiomonas shigelloides is low in most parts of the United States; therefore, specific requests for their detection are most cost effective.
To detect STEC, the stool specimen is inoculated onto sorbitol–MacConkey agar (containing 1% D-sorbitol instead of lactose), a medium that differentiates isolates of STEC, which do not ferment sorbitol, from almost all other E. coli, which are sorbitol-positive. A more sensitive method than culture for detection of STEC is detection of toxin in the stool or stool filtrate by enzyme immunoassay or PCR ( Gavin, 2004 ; Pulz, 2003 ).
When isolation of Y. enterocolitica is requested, CIN agar is inoculated and incubated at room temperature. The organism also can be recovered by inoculating media typically used for ‘routine' bacterial culture (see Ch. 56 ). A MacConkey plate may be incubated at room temperature for 48 hours. Colonies of Y. enterocolitica are purple and pinhead in size.
Species of Vibrio frequently grow on the media used for ‘routine' stool culture but, for their optimal recovery, thiosulfate citrate bile salts sucrose (TCBS) agar is inoculated.Plesiomonas shigelloides also grows on media used for ‘routine' culture; but because up to 30% of P. shigelloides isolates ferment lactose, their colonies do not appear sufficiently distinct to be recognized on these media, and screening all colonies for Plesiomonas is not cost-effective. For this reason, culture of stool or rectal swab specimens for P. shigelloides should be requested specifically. Use of the selective–differential medium inositol brilliant green bile salts agar has been suggested but is not essential.
Rectal swab specimens submitted for detection of C. trachomatis are placed in transport medium and delivered promptly to the laboratory, or are refrigerated for a short time. Rectal swab specimens collected to diagnose gonorrhea are treated as discussed earlier for endocervical specimens.
Stool specimens or gastric contents collected from persons with short-incubation food poisoning should be evaluated for S. aureus and Bacillus cereus. Processing includes preparation and examination of smears stained with Gram's stain, and culture. Because both organisms may be present normally in food, quantitative cultures must be performed. A series of dilutions (10-1, 10-2, 10-3, 10-4, and 10-5) of the sample are prepared in buffered gelatin diluent, and 0.1 mL of the undiluted specimen and each one of the dilutions are plated on to colistin–nalidixic acid or phenylethyl alcohol blood agar (selective for Gram-positive organisms). In addition, to demonstrate endospore production by B. cereus, 1 mL of the original specimen is mixed with 1 mL of absolute ethanol and the mixture is allowed to stand for 1 hour at room temperature. Dilutions of the mixture are prepared as described above, and 0.1 mL of each dilution and of the undiluted mixture are plated onto sheep blood agar. All plates are incubated for 18–24 hours at 35–37°C in ambient air, and the colonies are counted. The presence of 105CFU or more of S. aureus or B. cereus organisms per gram of specimen has potential significance, especially if found in samples from the majority of affected individuals. Testing for agents causing bacterial food poisoning as described above is normally conducted at public health laboratories and not in the clinical laboratory.
The clinical diagnoses of food-borne botulism and infant botulism may be confirmed by detecting botulinal toxin, Clostridium botulinum, or both in feces. Optimally, 25–50 mL of stool, 15–20 mL of serum, and a sample of the suspect food should be collected. Most clinical laboratories are not properly equipped to process specimens from persons with suspected botulism. In the United States, when a case of botulism is identified, investigators at the CDC should be notified to ensure appropriate diagnosis, treatment, and investigation of the potential outbreak.
Diseases associated with C. difficile, such as pseudomembranous colitis and antibiotic-associated diarrhea, are caused by the toxins produced by the organism and are diagnosed by detecting toxin A, B, or both in feces. The reference method for detection of the cytotoxin is cell culture assay. To extract toxin, the stool specimen is clarified by centrifugation at 2000×g for 20 minutes or 10 000×g for 10 minutes and filtered through a 0.45 μm membrane filter. Serial dilutions are prepared and inoculated to cell monolayers, which are incubated for 24–48 hours. Alternatively, toxin A or both toxins A and B may be detected in stool samples by ELISA. This technique appears to be almost as sensitive as cell culture, and provides results within a few hours ( Whittier, 1993 ; Merz, 1994 ). The sensitivity of different commercial ELISAs, however, varies ( Lyerly, 1998 ; Fedorko, 1999 ; Turgeon, 2003 ; Wilkins, 2003 ). Tests for detection of the common antigen glutamate dehydrogenase (GDH) are also commercially available ( Wilkins, 2003 ). These tests, like bacterial culture, do not distinguish isolates that produce toxin from those that do not. However, despite this, the common antigen, which has a turnaround time of 15–45 minutes, has been shown to be a useful screening test in certain institutions. Specimens yielding negative results require no further testing, whereas GDH-positive specimens should be tested for toxins.
For epidemiologic studies, C. difficile may be isolated from stool or from rectal swab specimens placed in an anaerobic transport system. Because many bacteria are present in stool, procedures that select for C. difficile must be used. The most effective medium for this purpose is cycloserine cefoxitin fructose egg yolk agar (CCFA), with or without horse blood. The organism grows more quickly and luxuriantly on formulations containing horse blood. The medium is incubated anaerobically for 48 hours and the plates are examined for colonies that have a peripheral fringe and a ‘horse stable' odor. Alternatively, C. difficile may be isolated by using an alcohol spore selection procedure, where 1 mL of the original specimen is mixed with 1 mL of absolute ethanol. The mixture is allowed to stand for 1 hour at room temperature, followed by subculture to CCFA and incubation anaerobically.
Clostridium perfringens is one cause of long-incubation food poisoning (7–15 hours after eating contaminated food). It also may cause antibiotic-associated diarrhea, typically in hospitalized elderly patients, and enteritis necroticans. To diagnose food poisoning caused by C. perfringens, quantitative anaerobic culture of a stool specimen that was transported in an anaerobic transport collection system is performed. Ethanol-treated and untreated fecal material are diluted as described earlier for detection of bacteria associated with short-incubation food poisoning. Phenylethyl alcohol agar plates are inoculated and incubated anaerobically for 48 hours. A spore count of 105 or more per gram of stool may be significant, especially if demonstrated in samples from the majority of affected individuals. As previously noted, testing for agents of food poisoning is conducted primarily in public health laboratories and not in the clinical laboratory. The toxin may be detected in the original stool specimen but this test is usually performed in reference laboratories. Similar criteria are used to diagnose diarrhea produced by C. perfringens during antibiotic therapy.
In regard to mycobacterial culture, stool specimens usually are submitted for isolation of Mycobacterium avium complex (primarily from patients with AIDS), but M. tuberculosis and other species of Mycobacterium also may be recovered. Processing the specimen (1–2 g of formed stool or 5 mL of liquid stool) involves decontamination and concentration, preparation of smears, and inoculation of media as discussed in
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