Clinical Study
A screening algorithm for diagnosing bacterial gastroenteritis by real-time PCR in combination with guided culture

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Abstract

We have introduced a real-time PCR for the simultaneous detection of Campylobacter jejuni, Salmonella spp., Shigella spp./enteroinvasive Escherichia coli and Yersinia enterocolitica in fecal samples in our routine laboratory. This new approach showed consistent results, with minimal inter-sample variation. When compared to conventional culture, the hands-on time decreased by 13 h/wk, and the median turnaround time drastically shortened from 73 to 29 h (P < .0001). Moreover, the detection rate of the targeted pathogens seemed to increase: the positivity rate registered over a twelve month period increased from 4.98% when using bacterial culture, compared to 8.56% when using real-time PCR (P < .0001). For antimicrobial susceptibility testing, samples that are found to be PCR positive are additionally cultured after the PCR result is known. Using this algorithm, we got a positive culture for 71.0% of the PCR positive samples. The samples missed by guided culture had significantly higher quantification cycle (Cq) values compared to the samples picked up by guided culture (P = .0003). Finally; we also tested the effect of extended sample storage on the performance of guided culture. Storage time prior to inoculation did have an effect on the positivity rate of culture; interestingly, these effects were clearly species-dependent.

Introduction

Diarrheal disease poses a worldwide challenge to public health with 1.5 million deaths in 2012 according to World Health Organization estimates (World Health Organization, 2014). Although mortality is low in the developed world, both the incidence and the socioeconomic impact remain high. In Germany the incidence of acute gastroenteritis was estimated to be 0.95 episodes per person per year; whereas in the US an incidence of 0.65 episodes/person per year was reported when excluding episodes of diarrhea due to any chronic illness or condition (Roy et al., 2006, Wilking et al., 2013). The annual cost of gastroenteritis was estimated to be €37-42 per capita in the Netherlands and even up to €207 per capita in Canada (Friesema et al., 2012, Henson et al., 2008).

Although viruses and parasites are frequent causes of acute gastroenteritis, bacteria are often associated with food poisoning, as well as with person-to-person spread of acute gastroenteritis. The Centers for Disease Control and Prevention identified the following bacteria as the most important causes of foodborne disease in the US in 2014, expressed as cases per 100.000 persons: Salmonella (15.5 cases), Campylobacter (13.5 cases) and Shigella (5.8 cases) (CDC – Foodnet, 2014). Moreover, species like Shigella, which need only a low inoculum for infection, have a high potential for person-to-person spread (Wikswo and Hall, 2012).

Conventional culture has for a long time been regarded as the golden standard for diagnosing bacterial gastroenteritis. However, the viability of bacteria may be reduced by extended storage and suboptimal transport conditions of the primary sample, the presence of competing commensals and the prior use of antibiotics (Taylor and Schelhart, 1975). Some bacteria may even attain a viable but non-culturable state (Oliver, 2005). Therefore, many laboratories have been looking into new techniques, such as real-time PCR, as a means for primary screening for bacteria that cause infectious gastroenteritis and many commercial nucleic acid tests have become available on the market (Cunningham et al., 2010, Reddington et al., 2014, Wiemer et al., 2011).

Recently, we have developed and extensively validated a multiplex real-time PCR for the simultaneous detection of Campylobacter jejuni, Salmonella spp., Shigella spp./enteroinvasive Escherichia coli (EIEC) and Yersinia enterocolitica in fecal samples (Van Lint et al., 2015). As this approach offered superior sensitivity over conventional culture and allowed us to perform rapid, sensitive, inexpensive and high-throughput testing of four common bacterial causes of gastroenteritis, we introduced this test in our routine laboratory. A screening algorithm was developed in which initial screening is done by real-time PCR, followed by guided culture of PCR positive samples to allow antimicrobial susceptibility testing and public health surveillance (Cronquist et al., 2012). As is shown in this follow-up study, this screening approach has resulted in a drastic improvement in laboratory workload, turnaround time and detection rates. We have also looked to the performance of guided culture, and studied the effect of extended sample storage.

Section snippets

Clinical specimens

A total of 7008 fresh stool specimens, submitted to our laboratory over two separate twelve month periods (12/2012–11/2013 vs. 2/2014–1/2015) for diagnosis of enteric bacterial pathogens were included in this study. During the first 12 months (n = 3455) the diagnosis was solely made by conventional culture, whereas in the second 12 month period (n = 3553) real-time PCR was used in combination with guided culture for diagnosis. Stool samples were stored at 4 °C upon arrival and were further

Robustness of the PCR method

In order to test the inter-sample variability of the ESwab sampling method, the amount of feces sampled by the ESwab was measured in 12 different fecal samples. On average 170 mg of feces was picked up by the ESwab with a minimum of 110 mg, and a maximum of 280 mg. The coefficient of variation was 28%.

Next, the intra run variation of the assay was determined by testing different samples in triplicate. The resulting Cq values showed an average coefficient of variation of 1.77% (Table 1). All CV%

Discussion

Recently, we have switched to PCR as a screening test for bacterial gastroenteritis. Although several groups have previously developed real-time PCR assays for the diagnosis of bacterial gastroenteritis, this study is one of the first to describe the positive impact of such a test in a routine setting (Cunningham et al., 2010, McAuliffe et al., 2013, Wiemer et al., 2011).

First, the robustness of the assay was confirmed by looking at the inter-sample variation of the ESwab sampling method and

Conflict of interest

The authors declare that they have no conflict of interest.

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