A sensitive multiplex, real-time PCR assay for prospective detection of Shiga toxin-producing Escherichia coli from stool samples reveals similar incidences but variable severities of non-O157 and O157 infections in northern California

Abstract

Rapid and accurate detection of Shiga toxin-producing Escherichia coli (STEC) of all serotypes from patients with diarrhea is critical for medical management and for the prevention of ongoing transmission. In this prospective study, we assessed the performance of a multiplex, real-time PCR assay targeting stx1 and stx2 for the detection of O157 and non-O157 STEC in diarrheal stool samples enriched in Gram-negative broth. We show that the assay is 100% sensitive (95% confidence interval [CI], 89.1% to 100%) and 98.5% specific (95% CI, 90.6% to 99.9%) based on a panel of 40 known STEC-positive specimens and 65 known negative specimens. During a 2-year postvalidation period, the assay detected more positive samples from patients in northern California than did culture and PCR testing performed at a public health reference laboratory, with a positive predictive value of 95.6% (95% CI, 87.6% to 99.1%). Serotyping data showed an incidence rate of 51.2% for non-O157 STEC strains, with 5.8% of patients (1/17) with non-O157 strains and 42.9% (6/14) with O157 strains (P = 0.03) developing hemolytic-uremic syndrome. The findings from this study underscore the recommendations of the CDC for laboratories to test all diarrheal stool samples from patients with acute community-acquired diarrhea for non-O157 STEC in addition to the O157 serotype by using a sensitive assay. Additionally, a survey of 17 clinical laboratories in northern California demonstrated that nearly 50% did not screen all stool specimens for the presence of Shiga toxins, indicating that many clinical microbiology laboratories still do not routinely screen all stool specimens for the presence of Shiga toxins as recommended in the 2009 CDC guidelines.

Fig 1

Melting curve profiles of the SYBR green amplicons obtained in multiplex, real-time PCR assays with the screening and confirmatory primer mixes. Representative melting curves for E. coli O157 (ATCC 43895) are shown. Peaks corresponding to specific products are labeled. The two peaks for stx₁ and stx₂ with the screening primer mix have melting temperature (T_m) ranges of 77 to 80°C and 80 to 83°C, respectively. The stx₁ and stx₂ amplicons for the confirmatory primer mix have similar T_m values and appear as a single peak at 77 to 79°C.