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Shiga toxin-producing Escherichia coli (STEC) is an enteropathogen of public health concern because of its ability to cause serious illness and outbreaks. In this prospective study, a diagnostic screening algorithm to categorize STEC infections into risk groups was evaluated. The algorithm consists of prescreening stool specimens with real-time PCR (qPCR) for the presence of stx genes. The qPCR-positive stool samples were cultured in enrichment broth and again screened for stx genes and additional virulence factors (escV, aggR, aat, bfpA) and O serogroups (O26, O103, O104, O111, O121, O145, O157). Also, PCR-guided culture was performed with sorbitol MacConkey agar (SMAC) and CHROMagar STEC medium. The presence of virulence factors and O serogroups was used for presumptive pathotype (PT) categorization in four PT groups. The potential risk for severe disease was categorized from high risk for PT group I to low risk for PT group III, whereas PT group IV consists of unconfirmed stx qPCR-positive samples. In total, 5,022 stool samples of patients with gastrointestinal symptoms were included. The qPCR detected stx genes in 1.8% of samples. Extensive screening for virulence factors and O serogroups was performed on 73 samples. After enrichment, the presence of stx genes was confirmed in 65 samples (89%). By culture on selective media, STEC was isolated in 36% (26/73 samples). Threshold cycle (CT) values for stx genes were significantly lower after enrichment compared to direct qPCR (P < 0.001). In total, 11 (15%), 19 (26%), 35 (48%), and 8 (11%) samples were categorized into PT groups I, II, III, and IV, respectively. Several virulence factors (stx2, stx2a, stx2f, toxB, eae, efa1, cif, espA, tccP, espP, nleA and/or nleB, tir cluster) were associated with PT groups I and II, while others (stx1, eaaA, mch cluster, ireA) were associated with PT group III. Furthermore, the number of virulence factors differed between PT groups (analysis of variance, P < 0.0001). In conclusion, a diagnostic algorithm enables fast discrimination of STEC infections associated with a high to moderate risk for severe disease (PT groups I and II) from less-virulent STEC (PT group III)., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Conventional diagnostic methods for the detection of Salmonella enterica and Campylobacter jejuni are laborious and time-consuming procedures, resulting in final results, for the majority of specimens, only after 3 to 4 days. Molecular detection can improve the time to reporting of the final results from several days to the next day. However, molecular assays for the detection of gastrointestinal pathogens directly from stool specimens have not made it into the routine clinical microbiology laboratory. In this study we have assessed the feasibility of a real-time PCR-based molecular screening method (MSM), aimed at S. enterica and C. jejuni, in the daily practice of a routine clinical microbiology laboratory. We have prospectively analyzed 2,067 stool specimens submitted for routine detection of gastrointestinal bacterial pathogens over a 7-month period. The MSM showed 98 to 100% sensitivity but routine culture showed only 77.8 to 86.8% sensitivity when an extended "gold standard" that included all culture-positive and all MSM-positive specimens, as confirmed by an independent secondary PCR of a different target gene, was used. An overall improvement in the rate of detection of both pathogens of 15 to 18% was observed. Both approaches performed nearly identically with regard to the specificity, positive predictive value, and negative predictive value, with the values for MSM being 99.7%, 93.1 to 96.6%, and 99.8 to 100%, respectively, and those for routine culture being 100%, 100%, and 97.6 to 99.5%, respectively. Finally, the final results were reported between 3 and 4 days earlier for negative specimens compared to the time of reporting of the results of routine culture. Positive specimens, on the other hand, required an additional 2 days to obtain a final result compared to the time required for routine culture, although preliminary MSM PCR-positive results were reported, on average, 2.9 to 3.8 days before the final routine culture results were reported. In conclusion, MSM can be incorporated into the daily practice of a routine clinical microbiology laboratory with ease. Furthermore, it provides an improvement in the screening for S. enterica and C. jejuni and substantially improves the time to the reporting of negative results.