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Detection of STEC by clinical laboratories

Detection of STEC by clinical laboratories. Dr Susan Taylor Clinical Microbiologist. Detection methods commonly used by clinical laboratories Isolation of E. coli O157 Direct detection of shiga-toxin Which samples to test. Culture.

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Detection of STEC by clinical laboratories

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  1. Detection of STEC by clinical laboratories Dr Susan Taylor Clinical Microbiologist

  2. Detection methods commonly used by clinical laboratories • Isolation of E. coli O157 • Direct detection of shiga-toxin • Which samples to test

  3. Culture • how to distinguish STEC from commensal E. coli in faeces • by the time a patient develops HUS, <1% of E. coli isolated from faeces may be STEC

  4. Culture • selective media aimed largely at detecting E.coli O157 • 2001- 2006 annual surveillance summaries from ESR • 94% of STEC recovered from clinical specimens were serotype O157 • 20- 50% of STEC infections are due to non-O157 E. coli in e.g. U.S., Canada, UK

  5. majority of E. coli O157 do not ferment sorbitol in contrast to most faecal E. coli lactose in routine MacConkey agar is substituted for sorbitol Sorbitol-MacConkey agar (SMAC)

  6. SMAC • any sorbitol-negative (colourless) colonies • confirm as E. coli biochemically • serological testing to confirm O157 +/- H7 serotype • confirm STX production or containing stx genes

  7. anti-O157 anti-H7

  8. modification of SMAC • supplemented with cefixime and tellurite (CT-SMAC) • suppress other sorbitol non-fermenting organisms • sorbitol-fermenting E. coli O157 have caused HUS in Europe • may be sensitive to tellurite • most non-O157 STEC ferment sorbitol

  9. Direct Laboratory Notification • sorbitol-negative colony confirmed as E. coli report as “sorbitol-negative E. coli” notify and refer isolate for detection of STX/stx • +/- perform latex agglutination with O157 report as “E. coli O157” notify & refer isolate for detection of STX/stx • +/- perform toxin testing report as “shiga-toxin producing E. coli” notify & refer isolate to ESR

  10. Chromogenic agar • several brands available • aimed at detecting O157 serotypes • can detect sorbitol-fermenting E. coli O157 • evaluation of BBL agar • regional lab in Calgary • compared against SMAC over 3 months • 27/3,116 (0.9%) samples positive • 26 from chromogenic vs 23 from SMAC • cost of chromogenic agar 5.6 times higher • 75% reduction in colonies that needed further work-up, 21% decrease in labour cost, 64% decrease in reagent costs J Clin Microbiol 2007;45:3098-3100

  11. Enterohaemolysin (EHEC) agar • most O157 STEC and many non-O157 STEC produce enterohemolysin (EHEC-Hly) • distinguished from α-haemolysin expressed by other E. coli by slower haemolytic action and inhibited in vitro by serum • small hemolytic zones on washed sheep rbc agar supplemented with Ca2+ after 18 - 24h incubation • subculture on routine blood agar or washed sheep rbc agar • if no haemolysis after 4-6 hours, potentially EHEC-Hly positive • confirm as E. coli biochemically • confirm STX production or containing stx genes

  12. Purity plate of E. coli O157 Example of direct faecal culture

  13. EHEC • less selective • in one study of children with bloody diarrhoea • 32% of faeces samples yielded haemolytic colonies after overnight incubation • 12% of these exhibited delayed haemolysis and were confirmed as STEC • used to detect STEC from samples that are positive for STX/stx

  14. Enrichment methods • STEC recovery reduced over time • in a group of patients with HUS • 100% recovery of STEC if culture performed within 2 days of onset of diarrhoea • recovery rate decreased to 92% at 3-6 days and 33% >6 days after onset of diarrhoea

  15. Enrichment methods • magnetic beads coated with anti-O157 antibodies (immunomagnetic separation, IMS) • incubation, then magnetic separation • the bead-bacterium complex is plated onto selective agar e.g. SMAC, CT-SMAC • used in food, water, bovine faeces testing • commercially available beads (Dynabeads) for isolating E. coli O157 or other specific serotypes • have been used in outbreak settings when implicated serotype known

  16. IMS • children with HUS in Germany • pre-enrichment step in GN broth • 18 samples +ve after IMS • 7 samples +ve after direct culture on SMAC or CT-SMAC • E. coli O157 detected at 102 CFU/g of stool in the presence of 107 coliforms from spiked samples J Clin Microbiol 1996;34:516-519

  17. IMS • UK • 25 samples +ve after IMS vs 15 +ve on CT-SMAC • difference in sensitivity was most marked on screening repeat samples from known cases and samples from asymptomatic contacts J Med Microbiol 1996;44:267-71 • 33 samples +ve with IMS vs 20 +ve on SMAC J Med Microbiol 1996;44:219-22

  18. Direct detection of shiga-toxin • a variety of immunoassays marketed for detection of STX 1 and 2 • EIA, OIA, ICT

  19. EIA • sensitivity and specificity improved when performed on stool incubated in broth overnight rather than directly on faeces • published evaluations of Premier EHEC EIA • from broth • sensitivity reported as 91-100% • specificity reported as 98% • EIA used at MMH 2003-7 • overnight incubation in MacConkey broth • 40 EIA reactive • 8 STEC confirmed (0-4/year) • 1 non-O157

  20. OIA • one published evaluation • similar performance to Premier EHEC EIA • detected 100% of STEC from overnight broth cultures • also used to directly test ‘colony sweeps’ from XLD or SMAC • ~15 min test vs 2.5hr for Premier EHEC EIA J Clin Microbiol 2007;45:3377-3380

  21. OIA • 5 E. coli O157 in the year to date at MMH • testing faeces after incubation in MacConkey broth • 3 positive by OIA • testing sweep of colonies off CT-SMAC plate • all 5 positive by OIA

  22. reactive results from immunoassay • attempt at isolation of STEC still required • isolate available for further characterisation • false-positive results do occur

  23. Testing strategies • Will vary • Rural vs urban populations • Community vs hospital patient • Size of laboratory • Rely on clinical details

  24. - processing 1-3 faeces/day • - primarily GP-generated requests • - staff inoculating plates may not be the microbiology plate reader • - include CT-SMAC on all samples having culture • don’t keep anti-sera Kaitaia • refer any sorbitol-negative • E. coli for STX/stx testing • refer faeces and sweep of • colonies if HUS or STEC • infection suspected Dargaville

  25. MMH Laboratory • process ~ 11 faeces culture per day • primarily ED or inpatients • use ‘3-day rule’ for culture • CT-SMAC + shiga-toxin OIA • selective testing: HUS, history of bloody diarrhoea, <13 yrs old, macroscopic or microscopic rbc in sample • test all samples having culture during periods of enhanced surveillance • carry O157 latex • refer any E. coli O157 isolates or OIA positive/culture negative samples

  26. Diagnostic Medlab - process ~ 120 faeces culture per day - primarily GP- requests - CT-SMAC - selective testing: HUS, history of bloody diarrhoea, liquid samples, macroscopic or microscopic rbc in sample - tested all samples from children during periods of enhanced surveillance - carry O157 latex - refer any E. coli O157 isolates

  27. Faecal Cultures: DML 2007 15% of requests for culture detect a bacterial pathogen *Notifiable

  28. detection of E. coli O157 is achievable early in illness • detection of STEC at the time of HUS requires additional testing for STX/stx

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