Development of a real-time PCR assay and comparison to CHROMagarTM STEC to screen for Shiga toxin-producing Escherichia coli in stool, Cape Town, South Africa

Introduction Shiga toxin-producing Escherichia coli (STEC) is an emerging infectious pathogen which could lead to haemolytic uremic syndrome. Even though previous studies have compared the performance of CHROMagarTMSTEC to real-time polymerase chain reaction (PCR) in Europe, no study has been done to assess its performance on African isolates. Objectives This project aimed to validate and test an in-house-developed duplex real-time PCR and use it as a reference standard to determine the performance of CHROMagarTMSTEC on African isolates from diarrhoeic stool samples. Methods This study evaluated STEC diagnostic technology on African isolates. An in-house-developed duplex real-time PCR assay for detection of stx1 and stx2 was validated and tested on diarrhoeic stool samples and then used as a reference standard to assess the performance of CHROMagarTMSTEC. Real-time PCR was used to screen for stx in tryptic soy broth and the suspected STEC isolates, while conventional PCR was used to detect the other virulence genes possessed by the isolates. Results The real-time PCR limit of detection was 5.3 target copies/μL of broth. The mean melting temperature on melt-curve analysis for detection of stx1 was 58.2 °C and for stx2 was 65.3 °C. Of 226 specimens screened, real-time PCR detected stx in 14 specimens (6.2%, 95% confidence interval = 3.43% – 10.18%). The sensitivity, specificity, negative predictive value and positive predictive value of the CHROMagarTMSTEC were 33.3%, 77.4%, 95.3% and 11.3%. Conclusions The in-house developed real-time PCR assay is a sensitive and specific option for laboratory detection of STEC as compared to CHROMagarTMSTEC in this setting.


Introduction
Globally, food-and water-borne outbreaks of both O157 and non-O157 Shiga toxin-producing Escherichia coli (STEC) have been successfully detected due to the availability of good baseline data and effective active laboratory-based surveillance systems. 1,2,3,4 Early detection of outbreaks is important to minimise morbidity, mortality and associated economic losses. 5 There is a lack of good baseline data on STEC in Africa, which can be attributed to a lack of laboratory resources and the surveillance strategy employed. STEC has been implicated in outbreaks of bloody diarrhoea in sub-Saharan countries; 6,7,8 however, these have been difficult to track and manage due to laboratory weakness. 9,10 Furthermore, typical haemolytic uremic syndrome, which is overwhelmingly caused by STEC, was reported as the leading contributor to acute renal failure in paediatric patients at a South African academic hospital. 11 Even though several studies have evaluated the performance of CHROMagar TM STEC by comparison to molecular and antigen detection methods in developed countries, 12,13 no study has so far evaluated its performance in Africa. This is necessary, especially given that there are geographical differences in characteristics of STEC that are dependent on the index of suspicion for the different STEC serotypes and on the availability of suitable laboratory methods to detect them. 14 NM), and over 470 non-O157 serotypes have been attributed to clinical disease. 15 Laboratory capacity for molecular detection is increasingly available in African countries and may, in some cases, be simpler than culture-based detection. This project, therefore, aimed to validate and test diarrhoeic stool samples by using an in-house developed duplex real-time polymerase chain reaction (PCR) and use it as a reference standard to determine the performance of CHROMagar TM STEC on African isolates. The duplex assay was used to screen tryptic soy broth (TSB) for stx following overnight stool enrichment, and conventional PCR was used to screen for the other diarrhoeagenic E. coli virulence genes. Diarrhoeagenic E. coli were serotyped, and stx-positive isolates were tested for Shiga toxin production using immunochromatography.

Study design
This study validated an in-house-developed duplex real-time PCR assay for detection of stx 1 and stx 2 . The assay was then tested on diarrhoeic stool samples at a tertiary referral hospital and was used as a reference standard to assess the performance of a commercial chromogenic medium (CHROMagar TM STEC) for STEC screening ( Figure 1).

Target plasmid preparation
The real-time PCR previously described by Grys et al. 16 was used to amplify stx 1 and stx 2 gene targets from a STEC O157:H7 NCTC control strain (C4193-1) with both stx 1 (subtype 1a) and stx 2 (subtype 2a). PCR amplicon size was confirmed visually by agarose gel detection (~208 bp for stx 1 and ~204 bp for stx 2 ) before confirmation by sequencing using the Big Dye® Terminator v3.1 Cycle Sequencing Kit (Life Technologies Corporation, Carlsbad, California, United States). We used primers 1a and 2a (Table 1) for unidirectional Sanger sequencing of the amplicons. Resultant sequences were then trimmed and submitted for BLAST analysis against the NCBI database and confirming stx 1 or stx 2 target sequences in comparison to O157:H7 EDL933 (NCBI Reference: NC_002655.2). 17 Purified amplicons (Mini Elute Gel extraction kit, Qiagen, Madrid, Spain) were cloned using CloneJet PCR cloning kit (Thermofisher Scientific, Austin, Texas, United States) into a pJet 1.2/blunt vector using the sticky end cloning protocol and transfected into the JM109 competent cells by calcium chloride transformation. Plasmids containing stx 1 and stx 2 were separately extracted using a Genopure plasmid Maxi kit (Roche Life Sciences, Rotkreuz, Switzerland) and quantified by spectrophotometry. To verify successful preparation purified plasmids were subjected to PCR amplification using primers 1a and 1b for stx 1 and 2a and 2b for stx 2 with amplicon size visually confirmed by agarose gel detection and subsequent sequence analysis. Plasmid quantification was determined spectrophotometrically employing the BioDrop-µLite (Isogen Life Science, B.V, Veldzigt, Netherlands). The A 260 was used to calculate the plasmid concentration expressed as the number of molecules/µL.

Polymerase chain reaction assay validation
To assess the potential for PCR cross-reactivity and assess the analytical specificity of the hybridisation probe-based realtime PCR described by Grys et al., 16    Sciences, Rotkreuz, Switzerland). The resulting amplicon size was visualised using agarose gel electrophoresis and subjected to DNA sequencing and BLAST alignment to reference stx 1 a and stx 2 a sequences (NC_002655.2). To mimic the sample matrix for sensitivity determination, TSB was inoculated with a pea-size amount of stool (from a single donor) shown to be stx-negative by PCR. To this inoculated broth 1 mL of plasmid stock (5.3*10 6 copies/µL) containing both stx 1 and stx 2 was added and serially diluted eight times in 9 mL of TSB, to a lowest dilution of 1:10 8 (53 plasmid copies/ml). Nucleic acid extraction was performed on 200 µL broth employing the MagNApure LC instrument (Roche Diagnostics, Rotkreuz, Switzerland) to yield 100 µL of extract. Initially, real-time PCR was performed in triplicate using a template from each of the eight dilutions to estimate a limit of detection (LOD). Subsequently, real-time PCR was performed in eight replicates on the dilution with the estimated LOD, as well as one dilution above and one dilution below the estimate. The LOD was defined as the lowest plasmid concentration spiked into TSB, before nucleic acid extraction, yielding a positive signal, as described above in all eight replicates. Nucleic acid extractions from STEC subtypes 1d (Reference strain MH1813, GenBank accession No. AY170851), 2b (Reference strain EH250, GenBank accession No. AF043627), 2c (Reference strain 031, GenBank accession No. L11079), 2d (Reference strain C165-02, GenBank accession No. DQ059012), 2e (Reference strain S1191, GenBank accession No. M21534), 2f (Reference strain T4/97, GenBank accession No. AJ010730) and 2g (Reference strain 7V, GenBank accession No. AY286000) were also subjected to PCR amplification to assess impact of strain variation on detection. The reproducibility of melting temperature assessment for stx 1 and stx 2 differentiation was determined by testing 24 replicates of TSB spiked with cloned stx 1 and stx 2 plasmids. To further assess the reproducibility of melting temperature across the subtypes, three stx 1 subtypes and seven stx 2 subtypes were tested similarly.

Isolate characterisation
Isolates yielding mauve colonies on CHROMagar TM STEC and presumptively identified as E. coli were subjected to stx characterisation employing the real-time PCR assay characterised herein. Other diarrhoeic E. coli virulence genes, including the fimbrial adhesion gene for diffusely adherent E. coli, the anti-aggregation protein transporter gene for enteroaggregative E. coli, heat-stable and heat-labile enterotoxin genes of enterotoxigenic E. coli, the intimin coding gene eae for enteropathogenic E. coli (EPEC) and the bundle-forming pili gene for the typical EPEC, were determined using standard gel-based PCR as previously described using primers as shown in Table 2. 18 To confirm Shiga toxin production among stx-positive isolates, the Immunocard STAT! ® EHEC (Meridian Biosciences, Inc., Cincinnati, Ohio, United States) was used to detect Shiga toxin 1 and 2 (by employing immunochromatography with toxindirected monoclonal antibodies labelled with red-coloured gold particles). All mauve isolates found to carry virulence genes were serotyped at the Centre for Enteric Diseases, National Institute of Communicable Disease, Johannesburg, by employing antisera (Statens Serum Institut, Copenhagen, Denmark) and the detection of somatic O-antigens as previously described. 19,20 H-antigen serotyping was not undertaken.

Real-time polymerase chain reaction validation
The BLAST analysis of the primers and probe sequence specificity yielded no significant homology to non-stx targets (data not shown). Real-time PCR amplicons generated were confirmed as 208 bp for stx 1 and 204 bp for stx 2 (Figure 2). Sequencing and BLAST analysis confirmed the identity of both stx 1 and stx 2 amplicons. The serially diluted plasmidstool-TSB was successfully amplified in 8/8 replicates in the sixth dilution, whereas the seventh dilution yielded an amplification signal in 3/8 replicates, yielding a LOD of 5.3 target copies/µL of broth. All other stx subtypes investigated (stx 1 a, stx 1 b, stx 2 a, stx 2 b, stx 2 c, stx 2 d, stx 2 e, stx 2 f, and stx 2 g) were successfully amplified by this assay (data not shown). stx 1 and stx 2 were successfully distinguished by a melting temperature of 58.2 °C (SD = 0.033) and 65.3 °C (SD = 0.037) (Figures 3-6). The T m for stx 2 subtypes 2a, 2b, 2c, 2d, 2e, 2f and 2g were the same at 65.3 °C (SD = 0.037, 0.041, 0.035, 0.039, 0.034, 0.033 and 0.032, respectively), whereas that of 1d was 44.7 °C (SD = 0.042). The efficiency of the assay was 1.99 as calculated from the amplification curves generated using the Light Cycler ® 480 software. The duplex assay detected both targets in the same run, and these could be differentiated by the melt curve with two distinct peaks at 58.2 °C for stx 1 and 65.3 °C for stx 2 (Figure 7).

Discussion
We validated the use of a previously described duplex realtime PCR assay with modification able to detect and differentiate stx 1 (melting temperature = 58.2 °C) and stx 2 (melting temperature = 65.3 °C) from overnight broth enrichment with a LOD of 5.3 target copies/µL broth. This assay was able to detect both stx 1 and stx 2 in the same run, thus potentially reducing process turn-around time in a busy laboratory setting. Timely reporting of STEC infections is important, because use of certain antibiotics is contraindicated in STEC infections.
Compared to the validated duplex real-time PCR, CHROMagar TM STEC showed a sensitivity of 33%, specificity of 77.4%, negative predictive value of 9.4% and positive predictive value of 95% for detection of STEC in stool following TSB enrichment. Of the 53 mauve isolates, 48 were negative for stx genes on use of the validated real-time PCR, whereas nine of the 14 PCR-positive broths did not yield any mauve colonies when cultured on CHROMagar TM STEC. Reasons for the poor performance of this medium in relation to the in-house developed duplex real-time PCR include the following: (1) delays in reporting of diarrhoea cases to a tertiary hospital (where samples were collected) may have led to loss of stx genes; STEC numbers are sharply reduced in stool after one week of illness, and the Shiga toxin genes might be lost by the bacteria. 21 (2) CHROMagar TM STEC selects for tellurite resistant strains but misses the tellurite susceptible STEC whose prevalence in this setting is not known.
For a chromogenic medium to be considered for routine screening purposes, it must have high specificity so as not to waste scarce laboratory resources on false positives. The false positivity rate in this setting (48/53 [90.6%]) is higher than has previously been reported in Europe (16.3% reported by Gouali et al., 2013 and 18.3% by Wylie et al., 2013). 12

Conclusions
The in-house developed real-time PCR assay is a sensitive and specific alternative to the currently used diagnostic strategy.   Due to the high false positivity rate, CHROMagar TM STEC can only be used as an adjunt to a more sensitive and specific assay such as real-time PCR.

Trustworthiness
To the best of our knowledge, the findings of this study can be used as per the scope of the study and in light of the study limitations as clearly pointed out.

Reliability
We confirm that the experiments conducted in this study will yield the same results during repeated trials using the same reagents and detection platforms.

Validity
To the best of our knowledge, the findings of this study, as obtained using the methods we employed, are valid for the study area and season. The in-house developed real-time PCR may, however, be adopted in other laboratories in developing countries.