1. INTRODUCTION Assessment of a real-time PCR for the identification and characterisation of Verocytotoxigenic E. coli (VTEC) Claire Jenkins, Andy J. Lawson, Tom Cheasty & Geraldine A. Smith Microbiology Services, Colindale, HPA Block-based PCR has been used at the Laboratory of Gastrointestinal Pathogens (LGP) for reference microbiology and primary diagnosis of verocytotoxigenic E. coli (VTEC) since the early 1990s (Figure 1). Recently, we have validated and evaluated a real-time PCR from the EU Community Reference Laboratory (CRL) for the identification of VTEC. The assay detects: Verocytotoxin genes (vtx1& vtx2) encoding the toxin that characterises the VTEC group (Figure 2) Intimin (eae) a protein involved in the intimate attachment of E. coli to the gut mucosa and found in strains causing serious illness (Figure 2) rfbE encoding the lipopolysaccharide O antigen of VTEC O157. Figure 2. Intimate attachment of E. coli to the gut mucosa involving intimin encoded by the eae gene (above) and phages encoding vtx (right) Figure 1. Block-based PCR for the detection of VTEC METHODS Bacterial strains and stool samples 169 strains of VTEC O157 from England and Wales 310 VTEC of other serogroups from the UK and abroad archived by LGP 66 VTEC with a range of vtx gene variants supplied for EQA tests 500 stool samples from patients with HUS, renal failure or bloody diarrhoea of diarrhoea. 25 of the 500 samples were from asymptomatic contacts Target detection by PCR Two duplex PCRs based on the CRL VTEC real-time PCR (Table 1) on a 5-plex Rotorgene-Q (Qiagen UK). Amplification parameters were 95 o C for 5m, followed by 95 o C for 15s and 60 o C for 60s. The cycle threshold was 0.05 for all targets except vtx1 for which it was 0.025. Bacterial culture & DNA extraction Faeces plated on MacConkey, CT- SMAC and SMAC agar Pure broth cultures of VTEC diluted 1:50 and boiled Faeces enriched (6h/18h) in modified tryptone soya broth (mTSB); DNA extracted with Instagene. Table 1. Duplex VTEC PCRs. eae/vtx2 probes are labelled with joe, vtx1 labelled with Cy5 and O157 is labelled with FAM. VALIDATION USING PURE CULTURES Tables 2 and 3 show comparisons of the real-time and block-based PCRs for vtx1and vtx2 and the detection of known vtx sequence variants. Common vtx subtypes include vtx1a, vtx2a and vtx2c. Other subtypes are rare. Generic vtx2 PCRs do not detect vtx2f for which a separate, specific PCR is required. RESULTS Table 2. Comparison of block-based PCR results with real time (RT) assay Table 3. vtx variants detected by the real time (RT) PCR compared to the block-based PCR. Important subtypes are highlighted in red. SUMMARY OF THE VALIDATION USING CULTURES Of 545 strains, 541, including all VTEC O157, gave concordant results with a routine block-based vtx1/2 PCR assay. CRL assay detected four additional vtx1d isolates. The assay was 99.3% sensitive for the detection of vtx genes. vtx2f, a rare subtype, was not detected. Most vtx2f strains produce intimin, so all eae- positive, vtx-negative strains can be tested with a specific vtx2f-PCR. 100% of the E. coli O157 strains were detected in the O157 rfbE PCR None of the E. coli of other serogroups were positive in the O157 rfbE PCR Real-time eae PCR detected 100% isolates positive by block-based assay VALIDATION USING FAECAL SAMPLES The PCR detected vtx genes in 62 of 500 (12.4%) faecal specimens and VTEC was successfully cultured from 36 of the 62 stools (58%) representing 7.2% of the total samples. Of these 36 cultures, 23 were VTEC O157, of which 19 were isolated by direct plating on CT-SMAC and 4 were isolated using IMS for E. coli O157. The 13 non-O157 VTEC were isolated by picking 10-20 colonies from MacConkey agar and retesting each colony with the PCR to identify any VTEC present (Table 4). SerogroupNumberGenotypeClinical symptomsTravel O262vtx1&2 + eaeHUSNo travel O1031vtx1 + eaeBDGermany O1045vtx2 + aggRHUSGermany O1111vtx1&2 + eaeBDEgypt O1172vtx1No dataUganda O1862vtx1 + eaeHUS/Renal failureBrazil Table 4. Characteristics of non-O157 serogroups isolated during the prospective study Figure 3. Results of eae/vtx2 PCRs cycling on the yellow channel (above) and vtx1 PCR on the red channel (below) DISCUSSION & CONCLUSIONS SUMMARY OF THE VALIDATION USING FEACAL SAMPLES Of the 62 PCR-positive specimens, 36 had Ct values in the real-time PCR ranging from 20-32 and VTEC were recovered from 32 of these by culture VTEC were isolated from 4 of 27 samples with Ct values of 32-39 Of the 26 cases with specimens that were PCR-positive but culture-negative, 8 had HUS, 3 had acute renal failure, 5 had BD, 3 had diarrhoea and 7 were asymptomatic The seven asymptomatic contacts had been exposed to VTEC O157 and low levels of VTEC, undetectable by current culture methods, may have been present in their stools. CONCLUSIONS The real-time assay showed (i) a substantial saving in time over the block-based assay (80 minutes compared to approximately five hours), (ii) ease of use and (iii) reduced risk of contamination. For isolates submitted to the reference laboratory, confirmation of VTEC and indication of the O157 serogroup were available on the day of receipt. A real-time PCR-based approach for the detection of VTEC from stool specimens is more sensitive than current methods and would enable frontline diagnostic laboratories to detect all VTEC serogroups, including those other than serogroup O157 (Figure 4). Figure 4. VTEC O104:H4 courtesy of Dr Henrik Chart & Dr Matthew Hannah Acknowledgements: We would like to acknowledge the EU Community Reference Laboratory for the their role in the design and development of the assay described in this study. We would also like to thank Marie Chattaway, Neil Perry, Vivienne Dos Nascimento, Yoshini Weerasinghe, Dawn Hedges and Carolyn Smith for processing the specimens and acknowledge the contribution of the frontline diagnostic laboratories in the England & Wales for submitting faecal samples to the LGP for the detection of VTEC.