Suitability of stx‐PCR directly from fecal samples in clinical diagnostics of STEC

Abstract

PCR-based testing for Shiga toxin producing Escherichia coli (STEC) directly from fecal samples is increasingly being implemented in routine diagnostic laboratories. These methods aim to detect clinically relevant amounts of microbes and not stx-carrying phages or low backgrounds of STEC. We present a diagnostic procedure and results from 1 year of stx-targeted real-time PCR of fecal samples from patients with gastrointestinal symptoms in Norway. A rapid stx2 subtyping strategy is described, which aims to quickly reveal the virulence potential of the microbe. stx was detected in 22 of 3320 samples, corresponding to a PCR positive rate of 0.66%. STEC were cultured from 72% of the PCR positive samples. Four stx1 isolates, eight stx2 isolates, and four isolates with both stx1 and stx2 were identified. With the method presented, stx-carrying phages are not commonly detected. Our results support the use of molecular testing combined with classical culture techniques for routine diagnostic purposes.

Implications of stx loss for clinical diagnostics of Shiga toxin-producing Escherichia coli

“…Stool samples were investigated for the presence of STEC and other gastrointestinal pathogens using a commercial CElabeled PCR kit (RIDA®GENE EHEC/EPEC real-time PCR, R-Biopharm AG, Darmstadt, Germany) [13]. Samples that tested positive for stx were cultured to recover the STEC isolate, which was then verified, serotyped, and further characterized at the National Reference Laboratory for Enteropathogenic Bacteria (NRL) at the Norwegian Institute of Public Health.…”

“…Lactose agar, an in-house medium for detection of Enterobacteriaceae, contains tryptose agar base, lactose, sodium chloride, and bromothymol blue. For each sample, 16-48 different colonies growing on the agars were examined for stx, eae, and the corresponding O-serogroup (target genes wzx or wzy), H-serogroup (target gene fliC), or lysozyme P (lysP) using real-time PCR [13,[15][16][17][18][19]. Primers and probes are described in Table 2.…”

“…To investigate if stx was lost during the in vitro culture step, the amount of stx relative to its corresponding STEC Oserogroup target gene (alternatively H-serogroup/lysP) was determined before and after culture on lactose agar. DNA was extracted from stool samples (100 μl) in Cary-Blair transport medium (Copan Italia S.P.A, Brescia, Italy) or RNAlater™ stabilization reagent (Qiagen, Hilden, Germany) using QIAsymphony (Qiagen) as previously described [13]. Following stool culture (100 μl) on lactose agar, all the colonies on the plate were suspended in 5 ml PBS, and 200 μl of this suspension was used for DNA extraction using the QIAsymphony protocol.…”

“…An indication of the presence of STEC in a patient sample may be the patient's clinical presentation or a positive stx PCR-result from stool samples. A routine procedure for STEC detection used at many hospital laboratories is to test 2-8 colonies isolated from stool samples for the presence of stx [13,31]. It can be technically challenging to culture low quantities of STEC in samples with co-occurring STEC-LST and other E. coli.…”

Experiences from multiplex PCR diagnostics of faeces in hospitalised patients: clinical significance of Enteropathogenic Escherichia coli (EPEC) and culture negative campylobacter

“…During the study period and for purposes of quality control after the introduction of a PCR based diagnostic, positive samples were also systematically cultured and pathogens identified according to routine microbiological methods. For the PCR assay, nucleic acids were extracted using Qiasymphony DSP Virus/Pathogen Kit (Qiagen) as described before [3]. EPEC and campylobacter were identified with the commercial Ridagene EHEC/EPEC kit and Ridagene Bacterial Stool (R-biopharm) panel respectively which detects eae gene and campylobacter 16 s .…”