Introduction

Screening PCRs for enteric bacterial pathogens offer a fast diagnosis or exclusion of bacterial diarrheal disease, except for Salmonella, because they can be isolated using highly sensitive enrichment culture techniques [1]. In this context, the extension of screening stool samples for Shiga toxin genes (stx) in all patients with community-acquired acute diarrhea regardless of bloody stool or suspicion of hemolytic uremic syndrome (HUS) has led to significantly increased numbers of Shiga toxin-producing E. coli (STEC) cases notifiable to public health offices [2, 3]. In Germany, notification criteria by the Infection Protection Act include not only the culture isolation of a stx-positive strain but also the detection of stx genes or Shiga toxin antigen in the stool sample when clinical symptoms occur. However, in cases of uncomplicated diarrhea, a positive stx PCR result does not have a clinical impact in every case because it provides no distinct evidence of the presence of an STEC [4, 5]. In regard to the stx subtype and the association with hemorrhagic colitis and HUS, STEC is classified as low or high virulent, the latter is designated as enterohemorrhagic E. coli (EHEC) [6]. The combination of culture-independent and culture-dependent methods is recommended for the diagnosis of STEC infections and confirmation of the pathological relevance of a positive screening PCR [4, 5, 7]. The isolation of strains in culture is also important to estimate the distribution and frequency of non-O157 serotypes and recognize epidemiological changes [5, 8]. Identification of STEC in culture is challenging because except for O157 which is negative for β-glucuronidase and sorbitol fermentation, most STEC are not biochemically different from nonpathogenic E. coli and their colonies cannot easily be distinguished on differential media [9]. Similar difficulties occur in the diagnosis of Shigella spp. and EIEC. Both pathogens can cause mild diarrhea but also severe dysentery characterized by inflammatory lesions and ulceration of the intestinal epithelium [10]. Whereas Shigella can be easily differentiated by its different biochemical behavior on selective media and by serotyping to identify the 4 species, EIEC can exert different characteristics more similar to Shigella or non-pathogenic E. coli [11]. Serotypes of EIEC can not only overlap with Shigella but also with E. coli serotypes that are not included in standard polyclonal antisera [12]. Now the application of PCR to identify STEC and Shigella spp./EIEC isolates by their virulence factors in culture is the method of choice [13, 14]. An alternative molecular technique of interest is loop-mediated isothermal amplification (LAMP) that uses bst DNA polymerase with strand-displacing activity and offers high-speed amplification under isothermal conditions [15]. In comparison to PCR, LAMP is very fast and involves a low hands-on time, features that are advantageous for using this technique in rapid diagnostic tests [16]. This study was designed to evaluate the performance of the newly introduced eazyplex® EHEC complete LAMP assay (Amplex Diagnostics, Gars-Bahnhof, Germany) as a culture confirmation test for the diagnosis of STEC and Shigella spp./EIEC infections.

Material and methods

Bacterial strains and clinical samples

A total of 12 strains of STEC possessing different stx subtypes and 6 strains of Shigella spp. from the strain collections of the Institute of Medical Microbiology, Jena, and the Institute of Hygiene, Münster, were used as retrospective samples for the evaluation of the eazyplex® EHEC complete assay (Table 1).

Table 1 Performance of the eazyplex® EHEC complete assay for retrospective identification of STEC and Shigella strains
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As prospective samples, 16 isolates and 15 enrichment broths cultured from diarrheal stool specimens as part of the routine patient diagnostics at the Institute of Medical Microbiology, Jena, were analyzed (Tables 2 and 3). The isolates were collected between April 2017 and October 2018 from diarrheal stool samples tested positive for stx or Shigella spp./EIEC using the BD MAX™ Enteric Bacterial Panel (EBP) assay (BD, Heidelberg, Germany) as screening PCR. Formed stools and rectal swabs were excluded.

Table 2 Characteristics of prospectively tested STEC, EIEC, and Shigella isolates
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Table 3 Characteristics of positive GN enrichment broth cultures
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BD MAX™ enteric bacterial panel (EBP) PCR and bacterial culture

Unpreserved stool samples were tested with the BD MAX™ EBP assay within 8 h after receipt according to the manufacturer’s protocol. This automated PCR test uses the following primer sets: spaO for Salmonella spp., ipaH for Shigella spp./EIEC, tuf for Campylobacter jejuni and C. coli, and stx1/stx2 for STEC. Stx1 and stx2 are not differentiated from each other. Samples positive for stx were inoculated onto Brilliance E. coli/coliform chromogenic agar (Oxoid, Thermo Fisher Scientific, Wesel, Germany) and GN broth (BD). For isolation of Shigella Hektoen agar (BD), GN, and selenite broth (BD) were used. Identification of suspect colonies was performed using the RIDA®GENE EHEC/EPEC PCR, agglutination with polyclonal and monoclonal antisera (Sifin Diagnostics, Berlin, Germany) and biochemical differentiation by API20E (bioMerieux, Nürtingen, Germany).

RIDA®GENE EHEC/EPEC real-time PCR

RIDA®GENE EHEC/EPEC PCR (R-Biopharm, Darmstadt, Germany) targeting stx1/stx2, eae, and ipaH served as reference method for testing bacterial isolates. A single colony or 5 μl of GN broth were transferred into 1 ml of DEPC water. A total of 20 μl of internal control DNA were added as extraction control. The suspension was vortexed, heated at 95 °C for 10 min, and centrifuged at 12.000 rpm for 1 min. Five microliters of the supernatant was added to 20 μl of PCR mastermix. The test was run on a SmartCycler (Cepheid, purchased from BD) with the following cycling conditions: 95 °C for 1 min, 45 cycles at 95 °C for 10 s and 60 °C for 15 s.

Eazyplex® EHEC complete LAMP assay

The eazyplex® EHEC is a qualitative assay for the detection of different intestinal pathovars of E. coli and their virulence factors. A single eazyplex® test strip contains six oligonucleotide primers in each filled cap and these provide the means for simultaneous, specific amplification of different genes in a single isothermal amplification reaction. A single colony or 5 μl of GN broth were suspended in 500 μl of resuspension and lysis fluid (RALF, Amplex Diagnostics) and boiled for 2 min. After centrifugation at 4000 rpm for 1 min, 25 μl of the supernatant was pipetted into each tube of the eazyplex® test strip containing the lyophilized master mix. The strip was gently knocked to remove air bubbles and loaded into the Genie II Mk2 machine (OptiGene Ltd., West Sussex, UK; purchased from Amplex Diagnostics). Tests were run at 65 °C for 25 min. Amplification was measured by real-time fluorescence detection using a DNA intercalating dye. Data interpretation was automatically performed by the integrated eazyReport™ software.

E. coli subtyping by microarray and sequencing

Because our standard real-time PCR did not determine stx subtypes and ehlyA, the clinical STEC and EIEC isolates were subjected to microarray-based analysis using the E. coli PanType AS2 genotyping kit (Alere Technologies, Abbott, Jena, Germany) to identify the serovar (O:H), the stx subtype, and additional virulence factors. Microarray analysis was performed as previously described [17]. The EIEC isolate was further characterized by whole-genome sequencing (WGS). Genomic DNA was isolated from an overnight culture using the NucleoSpin® Microbial DNA kit (Macherey-Nagel, Düren, Germany). The Nanopore Oxford MinION platform was used for WGS. Briefly, no size selection was performed and the DNA library was generated using the native barcoding expansion kit EXP-NBD103 and the Nanopore sequencing kit SQK-LSK109 (Oxford Nanopore Technologies, Oxford, UK) according to manufacturer’s instructions. The used flow cell FLO-MIN106 (R9-Version) was primed by the flow cell priming kit EXP-FLP001 (Oxford Nanopore, Oxford, UK). The protocol named “1D Native barcoding genomic DNA” was used in version NBE_9065_v109_revB_23May2018 (last update, 03/09/2018). The Albacore basecaller (Oxford Nanopore Technologies, Oxford, UK) translated the minion raw data (FAST5) into short-quality tagged sequence reads (FASTQ). After barcode trimming using Porechop (https://github.com/rrwick/Porechop), canu (https://github.com/marbl/canu) was used to assemble the short reads to nine contigs. All contigs were analyzed by abricate (https://github.com/tseemann/abricate). After nanopolishing (https://github.com/jts/nanopolish), the corrected sequence data were used for a direct comparison to the outbreak strain O96:H19 from Italy in 2007 (LIT) by MUMmer4 software (https://mummer4.github.io/).

Statistical analysis

The qualitative performance of the eazyplex® EHEC complete assay in comparison to the reference method was assessed by calculating the specificity, sensitivity, and accuracy (percent positive agreement).

Results and discussion

Retrospective evaluation using characterized strains

For a first evaluation of the eazyplex® EHEC assay, a collection of representative strains covering all test targets including stx types of high-virulent (stx 2a, c, and d) and low-virulent (stx 1, 2b, 2e, 2f, and 2g) STEC was used [3]. All strains were correctly identified regarding the stx types 1 and 2, adhesion intimin (eae), and enterohemolysin A (ehlyA) (Table 1). The assay also correctly identified the German outbreak strain O104 from 2011 possessing stx2a and aggregative adherence fimbriae (AAF) which are adhesins of enteroaggregative E. coli (EAEC) [18]. The eazyplex® EHEC assay contains primers targeting aggr encoding a regulatory protein for AAF expression [19].

The stx2f has been included in the assay as a specific target because of its low-sequence homology to other stx2 subtypes [20]. The stx2f gene of strain O128:H2 was detected without cross-reactivity to the common stx2 target detecting only stx2a-e and g (Table 1). It should be noted that stx2f is not covered by all commercial PCRs, e.g., BD MAX™. Although stx2f-positive STEC is rarely isolated, outbreaks have already been reported [21]. Enterohemolysin A (ehlyA) is a characteristic virulence factor especially for classical EHEC strains and the gene was correctly identified in all cases [22]. For identification of Shigella spp./EIEC, the eazyplex® EHEC contains primers for ipaH, a multicopy gene that is encoded chromosomally and on the large invasion virulence plasmid and therefore commonly used as target in diagnostic PCR tests [11, 23]. Several strains of S. dysenteriae, S. flexneri, and S. sonnei were correctly identified (Table 1).

Prospective evaluation of clinical sample isolates

Clinical samples were stool specimens from patients with diarrhea or suspicion of HUS that were screened by BD MAX™ PCR for bacterial pathogens as part of routine diagnostics. A total of 4996 specimens tested between April 2017 and October 2018 yielded 130 Campylobacter jejuni/C. coli, 58 stx, 35 Salmonella spp., and 8 Shigella spp./EIEC-positive results. The overall positive rate was 4.7%, excluding 82 invalid test results. The positive rate for stx was 1.2%. From 27 out of 58 stx-positive samples, a STEC could be isolated in culture or detected in GN enrichment broth, amounting to a culture confirmation rate of 46.6% (Tables 2 and 3). Four out of 8 Shigella spp./EIEC-positive samples were also positive in culture. The discrepancy between PCR and culture may be due to several reasons. BD MAX™ has been shown to be significantly more sensitive than culture [24]. All but one of the isolates were non-O157 E. coli and showed growth characteristics identical to non-pathogenic E. coli on Brilliance E. coli/coliform agar by positive reactions for both β-galactosidase and β-glucuronidase. It should also be noted that a positive stx PCR results does not necessarily implicate the presence of a STEC because the occurrence of free stx-carrying phages in stool specimens from healthy donors has been described [25]. STEC can also lose stx genes during the isolation procedure [26, 27]. This occurred at least in one case of this study in which a patient suffering from HUS was tested positive for stx by BD MAX™ screening PCR but isolates were only positive for eae and ehlyA.

There is an ongoing discussion on the clinical relevance of a positive stx PCR with negative culture in cases without clinical complications [4, 5]. In Germany, as in many other countries, not only is it obligated to report the isolation of STEC in culture to the public health offices but also a culture-independent positive stx test. Introduction of broad screening stx PCR can lead to a significant increase in notifications of STEC infections, resulting in increased workload and costs for hospitals and laboratories [3]. However, clinical significance of diagnostic reports is often questionable when the STEC cannot be classified as high or low virulent, e.g., in the absence of a culture isolate. High-virulent STEC can cause hemorrhagic colitis and HUS and include strains typically possessing stx2a, 2c, 2d, or 1a (the latter included in children < 5 years with bloody diarrhea) [3]. Low-virulent STEC harboring stx1, 2b, 2e, 2f, or 2g can be present in asymptomatic carriers, cause diarrhea, and have also been reported to be associated with long-term gastrointestinal symptoms [28]. In this study, out of a total of 13 STEC isolates 10 were classified as low virulent (Table 2). Most of the low-virulent isolates were of serotype O91:H14 harboring stx1a which belongs to the most common eae-negative STEC types [29, 30]. The three high-virulent isolates were stx2a-positive, as subtyped by microarray, and associated with complicated disease (Table 2). The O157:H7 was isolated from a 5-year-old child with hemorrhagic colitis, the O26:H11 isolate from a 5-year-old child suffering from HUS, and the OR:H2 from a 39-year-old female patient with hemorrhagic colitis and coinfection by Clostridioides difficile. All low-virulent STEC were collected from patients with uncomplicated diarrhea. The stx subtypes and additional virulence factors were determined by microarray analysis. The results of the eazyplex® EHEC assay for identification and characterization of the culture isolates were in accordance with PCR and microarray with only one exception of a false-positive eazyplex® result for eae [isolate O91:H14 (2)] (Table 2). All isolates harbored additional virulence factors such as adhesins and toxins, as shown by microarray analysis (Table 2). From 15 specimens, STEC infection was confirmed by positive GN enrichment broth but no isolate could be recovered from single colonies (Table 3).

Out of a total of 8 ipaH PCR- positive stool samples, 2 specimens yielded Shigella isolates, one specimen was culture-positive for EIEC and one specimen was only confirmed by a positive enrichment broth (Tables 2 and 3). Both reference PCR and eazyplex® LAMP correctly identified the isolates by a positive ipaH result (Table 2). The EIEC was further characterized by Alere microarray and tested positive for ipaD as a marker gene for the invasion plasmid pINV and for virF, a regulator of plasmid-encoded virulence genes in Shigella and EIEC (Table 2). Nanopore MinION sequencing of the isolate revealed 99.37% identity with EIEC O96:H19 (NCBI GenBank number CP011416.1), a new emerging strain that has caused two large food-borne outbreaks of diarrhea in Italy (2012) and the UK (2014) with each > 100 cases [31,32,33]. In this study, the strain was isolated from a traveler returning from Tunisia in October 2018 and suffering from diarrhea, abdominal pain, and fever up to 38.5 °C. It is important to note that EIEC O96:H19 grows in lactose-positive colonies in contrast to most EIEC and Shigella and may easily be overlooked.

Performance of the eazyplex® EHEC assay

Analytical performance of eazyplex® LAMP was calculated by combining data from retrospective analysis of representative strains and prospective testing of isolates and GN broths collected from clinical samples. Both standard routine PCR and Alere microarray were defined as reference. Data on the sensitivity, specificity, and accuracy of the eazyplex® parameters are summarized in Table 4. LAMP showed a reliable accuracy compared to PCR. In all cases, stx2 was correctly identified and there was only one false-negative result for stx1 and eae from enrichment broth number 8 (Table 3). Data validation for aggR could not be performed because of a sample size of 1.

Table 4 Overall performance of the eazyplex® EHEC complete assay for identification of STEC in culture
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A great advantage of the eazyplex® real-time LAMP technology is the easy handling and fast time to result. The mean threshold time for fluorescence intensity values of specific targets ranged from 8.5 to 13 min. IC was detected at a mean time of 9.5 min (Table 5).

Table 5 Time to result of the eazyplex® EHEC assay
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Conclusions

The combination of an initial screening PCR for stx and ipaH with subsequent culture has been recommended for identifying STEC and Shigella spp./EIEC infections [34,35,36]. It is suggested to perform Shiga toxin testing from overnight enrichment cultures or primary isolation agars [37]. Both PCR and rapid antigen tests may be suitable for identifying STEC in culture but antigen tests have lower sensitivities [38]. There is a need for rapid, low-tech, and cost-effective assays to enable simple characterization of STEC isolates [7]. This study demonstrates that the eazyplex® EHEC LAMP assay is a suitable alternative molecular test for the characterization of culture isolates as well as testing of enrichment broths. The test can easily be integrated into the daily workflow because there is no need for DNA extraction, the hands-on time is low, and results are available within half an hour including sample preparation.

A limitation of this study is that the assay was not evaluated for direct detection of pathogens in stool samples. Although the consumable costs of eazyplex® LAMP for a single parameter are comparable to those of PCR the costs for a complete test strip covering seven specific targets are relatively high. Therefore, it can be suggested to implement the eazyplex® EHEC assay as a culture confirmation test and to use a sensitive stx PCR as in initial screening test. Nevertheless, it would be of interest to further investigate whether LAMP reaches sufficient sensitivity for its application as a culture-independent rapid diagnostic test. To estimate the pathogenic potential of isolated STEC and to avoid unnecessary follow-up workload it would be attractive if a rapid LAMP assay would allow the differentiation of high- and low-virulent STEC by separate identification of stx 2a, c, and d.