8.2 Clostridioides difficile, Shiga Toxin, and Serotyping

8.2 Clostridioides difficile, Shiga Toxin, and Serotyping

Quick Answer: Test unformed stool only for C. difficile with GDH, toxin A/B EIA, and/or NAAT; a positive NAAT is not a test of cure and does not by itself prove disease. Detect STEC with SMAC for O157 plus toxin EIA or PCR for non-O157. Serotype E. coli, Salmonella (O and H), and Shigella (A–D) and report isolates to public health.

Outline II.F groups antigen detection and molecular methods (Clostridioides difficile, Shiga toxin) with serotyping of Escherichia coli, Salmonella spp., and Shigella spp. Culture plates from Section 8.1 feed this section. A green HE colony is not a finished Shigella result until the serogroup is known, and a sorbitol-negative SMAC colony is not a finished STEC result until O157 and Shiga toxin are addressed.

Clostridioides difficile: specimen first, then the algorithm

C. difficile is an anaerobic Gram-positive spore-forming rod. Hospital-onset diarrhea after antibiotics, age, gastric-acid suppression, and inflammatory bowel disease raise pretest probability, but the laboratory gate is simpler and heavily tested: do not test formed stool. Formed stool means the patient is not having C. difficile diarrhea. The exception is ileus, when toxin can be present without liquid stool. Do not screen asymptomatic patients, do not test infants as a diarrhea workup (high colonization), and do not repeat NAAT during the same episode just because the first result was negative and the clinician is impatient.

Three assay classes appear on the outline:

1. GDH (glutamate dehydrogenase) EIA detects an enzyme found in toxigenic and nontoxigenic C. difficile. It is a sensitive organism screen, not a toxin test. GDH-negative, toxin-negative stools are reported negative.
2. Toxin A/B EIA detects free TcdA (enterotoxin) and TcdB (cytotoxin) in stool. It is more specific for toxin production but less sensitive than NAAT. GDH-positive / toxin-positive supports disease in the right host.
3. NAAT (PCR or other amplification of tcdB and sometimes tcdA or binary toxin) detects toxigenic C. difficile DNA. It does not measure toxin protein. A NAAT-positive result in a patient without diarrhea is colonization.

Common algorithms: GDH plus toxin EIA, with NAAT as the tie-breaker when GDH is positive and toxin is negative; or NAAT first, with a toxin EIA to sort colonization from toxin-positive disease. Either pathway is acceptable on the exam if you respect the clinical meaning. GDH-positive, toxin-negative, NAAT-negative is nontoxigenic C. difficile and is not CDI. NAAT-positive, toxin-negative is toxigenic organism without detected free toxin—correlate with symptoms; many stewardship programs do not treat that pattern as CDI.

Never use a single NAAT as a test of cure. Toxigenic DNA and spores persist for weeks after diarrhea stops. A formed specimen is the wrong sample, and a still-positive PCR does not mean treatment failed. Test of cure is clinical resolution, not a laboratory checkbox. Do not reculture or re-PCR to “clear” a patient for transfer.

Culture on cycloserine-cefoxitin-fructose agar (CCFA) is historical: yellow, ground-glass colonies with a horse-barn odor, anaerobic. Antigen/NAAT has replaced culture for diagnosis. If culture is used, it is for strain typing or outbreak work, not for day-to-day CDI calls.

Shiga toxin and STEC, including O157 and non-O157

STEC (Shiga toxin–producing E. coli), historically called EHEC when the disease is hemorrhagic colitis, includes O157:H7 and many non-O157 serogroups (O26, O45, O103, O111, O121, O145 are the usual U.S. six). All can cause bloody diarrhea and hemolytic uremic syndrome (HUS). Antibiotics and antimotility drugs can worsen HUS, which is why the laboratory must detect toxin, not just a lactose-positive E. coli that “looks like flora.”

Sorbitol-MacConkey (SMAC) is the O157 plate. Ordinary E. coli ferments sorbitol and is pink; O157:H7 is sorbitol-negative and colorless at 24 hours. Confirm colorless colonies with O157 latex and then H7. CT-SMAC (cefixime-tellurite) increases selectivity. SMAC does not catch most non-O157 STEC, because those strains ferment sorbitol and look like commensal E. coli. That is the exam trap: SMAC alone is an O157 screen, not a STEC screen.

Shiga toxin EIA (Stx1/Stx2) or PCR for stx1/stx2 must be performed on bloody stools, HUS, and routinely on enteric workups per laboratory policy—typically from an overnight enrichment broth, not from a random pink colony. Toxin-positive, SMAC-negative specimens are non-O157 STEC until the public-health laboratory isolates and serotypes them. Report immediately: STEC is a critical public-health organism, and HUS is a clinical emergency. A multiplex GI PCR that flags STEC still requires an isolate; do not stop at the panel.

Exam scenario. Bloody diarrhea, colorless SMAC colonies, O157 latex-positive: report presumptive E. coli O157, test Shiga toxin if not already done, and refer for H typing and public health. Bloody diarrhea, pink SMAC, Shiga toxin EIA positive: non-O157 STEC is still in play—do not call the SMAC “negative for STEC.”

Serotyping E. coli, Salmonella, and Shigella

Serotyping uses somatic O (heat-stable LPS) and flagellar H antigens. E. coli and Salmonella have both; Shigella is nonmotile and is typed by O antigens grouped A–D. Capsular K or Vi antigens can mask O agglutination; boiling the suspension strips the capsule so O typing can proceed. That boiling step is the classic S. Typhi Vi trap.

Escherichia coli. O and H define STEC and other pathotypes (O157:H7 is the prototype). Clinical laboratories usually stop at O157 latex and send other toxin-positive isolates to the public-health laboratory for full O:H typing and whole-genome sequencing. Do not serotype random lactose-positive flora from non-bloody stools; serotyping is for pathogen isolates.

Salmonella spp. The Kauffmann–White scheme is O group then H phase. Slide agglutination starts with polyvalent O, then group (A, B, C1, C2, D, E, and others). S. Typhi is group D and may carry Vi; if polyvalent O is negative in a biochemically typical isolate, boil and retest, and include Vi antiserum. Flagellar typing uses phase 1 and phase 2; a Craigie tube or similar phase-reversal method is used when only one phase agglutinates. Report the serotype the public-health laboratory confirms (Salmonella Typhimurium, Enteritidis, Typhi). Do not invent a serotype from a multiplex PCR target that says only “Salmonella spp.”

Shigella spp. Subgroups are O serogroups, not H phases:

Plesiomonas can agglutinate in Shigella antiserum. Oxidase-positive “Shigella” is not Shigella. Live isolates are required; you cannot serogroup from a NAAT well.

Public-health reporting

Salmonella, Shigella, and STEC are reportable almost everywhere; Vibrio and often Yersinia and Campylobacter join that list by jurisdiction. The clinical laboratory’s job is a timely isolate plus a phone or electronic report, not a finished PulseNet name. Multiplex GI PCR that detects these targets still needs reflex culture. C. difficile is tracked as a healthcare-associated event in many hospitals (NHSN), which is infection-prevention reporting, not the same as submitting a stool isolate for serotyping. Do not wait for a “complete” serotype before notifying someone of O157 or a toxin-positive STEC—the HUS clock is already running.

Exam scenario. NAAT for C. difficile is still positive on day 10, the patient is formed and well, and the floor wants “proof of cure.” Refuse the test: formed stool is the wrong specimen and NAAT is not a test of cure. Second scenario: GI panel positive for Shigella on a bloody stool. Plate HE/XLD, identify the NLF, agglutinate groups A–D, and send the isolate; the PCR result alone is not a serotype and is not a public-health culture.