Multiplex real-time PCR assay for detecting diarrhoeagenic Escherichia coli
We firstly validated PCR amplification for ETEC, EAEC, EIEC/Shigella, EPEC, and STEC in monoplex using cloned target sequences and then with genomic DNA extracted from the various E. coli pathovars. The sensitivity of the primer and probe sets was determined by generating a series of standard curves using 10-fold dilutions of control plasmid DNA. The limit of detection for all targets was five copies per reaction, with the exception of aggR which could be detected down to 50 copies per reaction. Each primer and probe set were tested against a panel of commonly isolated pathogens found in stool samples, which included Staphylococcus aureus, Klebsiella pneumoniae, Salmonella spp., Campylobacter coli, Campylobacter jejuni, Shigella sonnei, Shigella flexneri, Enterobacter, Proteus, norovirus, and rotavirus. No amplification was observed in any sample other than those containing E.coli.
Ultimately, the PCR assays were multiplexed into four reactions, and the sensitivity, intra-assay and inter-assay CVs across the nine target sequences were calculated for each multiplexed PCR reaction. The Ct values for each target were equivalent between the monoplex and multiplex reactions, confirming that multiplexing did not impact sensitivity. The intra-assay and inter-assay CVs ranged from 0.01 to 1.54% and from 0.01 to 2.12%, respectively (Table 2). The linear regressions of the standard curves were between 0.992-0.999 for all targets tested. The resulting efficiency of the amplification ranged from 90.9 to 105.7%, demonstrating the multiplex real-time PCR assays were well optimized, reproducible, and specific.
The prevalence of diarrhoeagenic Escherichia coli from faecal specimens of children hospitalized with diarrhoea
Between May 2014 and April 2016, we amassed 2,815 MC sweeps (i.e. faecal samples plated on MC media) from 3,166 children hospitalized with bloody and/or mucoid diarrhoea at three tertiary hospitals in HCMC. A single faecal sample was collected from each child within their first two days of hospital admission for diarrhoea. The majority of patients were male (1,731/2,815; 61.5%), with ages ranging from one month to 15 years (median age 10 months, IQR 6.6-17.1 months).
We employed the four multiplex real-time DEC PCRs on all 2,815 MC sweeps to identify DEC targets potentially associated with clinical infection. At least one PCR amplification associated with a DEC variant was positive in 34.7% (978/2,815) of the MC sweeps from paediatric patients hospitalized with diarrhoea. Among the DEC amplification positive samples, EAEC was the most common variant detected, with aggR amplified in 15.7% (443/2,815) of samples (Table 3). Other commonly amplified DEC targets included EIEC/Shigella and EPEC, which were identified in 12.4% (349/2,815) and 12.2% (343/2,815) of the MC sweep samples, respectively.
Within the EPEC pathotype, atypical EPEC positive samples (eae positive, bfpA negative) were more prevalent than typical EPEC positive samples (eae positive, bfpA positive); 93.9% (322/343) vs. 6.1% (21/343), respectively. ETEC was detected in 6% (182/2,815) of samples, with only a limited number of these samples (8.2%; 15/182) producing an amplicon for heat stable toxin (estA). Four diarrhoeal patients harboured samples containing the Shiga toxin-producing genes (stx1/stx2). Among the four cases associated with an STEC positive sample, one was positive for eae and one was positive for both eae and rfbE_O157. Of the two STEC cases that were amplification positive for eae and rfbE_O157, one was additionally positive for eltB (ETEC), the other was positive for aggR (EAEC).
Clinical manifestations of diarrhoeagenic Escherichia coli mono-infection
To investigate clinical syndromes associated with the various DEC in Vietnam, clinical data associated with the patients were accessed and compared between pathotype groups (Table S2). Patient samples from which multiple DEC pathogens were amplified were excluded. Notably, ~70% of those with an ETEC, EAEC, EPEC, or STEC O157 positive sample were associated with mucoid, non-bloody diarrhoea, whereas EIEC/Shigella was significantly associated with visible bloody diarrhoea (39.7%, 46/116, p<0.001, χ2 test). EAEC was the most commonly identified DEC in mono-infection. This pathotype was more commonly associated with children that had wasting or severe wasting (13.5%, 23/170; p=0.013, Fisher’s exact test) than the other DEC variants. Whilst EHEC_O157 was identified less frequently than other pathotypes, it was significantly associated with moderate and severe dehydration (40%, 8/20; p=0.010, Fisher’s exact test), which commonly required intravenous rehydration therapy.
Generally, we found that infections associated with DEC positive samples were uncomplicated; >90% of patients had improved or recovered after three days and their median hospital stay was five days [IQR 3-7 days]. The use of antimicrobials within this study population was high, with 81.3% (1,513/1,861) of patients receiving empirical antimicrobial treatment prior to any diagnostic testing. Fluoroquinolones, specifically ciprofloxacin, were the most commonly used class of antimicrobials in those with a DEC in their stool (957/1,512, 63.3%).
Diarrhoeagenic Escherichia coli from faecal specimens of diarrhoeal hospitalized children vs. healthy non-diarrhoeal children
Between March 2016 and August 2016, 498 MC sweeps were additionally collected from faecal samples taken from healthy children residing in HCMC and participating in a cohort study. The majority of healthy children were male (269/498; 54.0%), with their age when sampled ranging from 24 months to 5 years (median age 46.4 months, IQR 35.6-52.5 months). In a comparable manner to the diarrhoeal samples, we screened the MC extractions from these healthy children with the multiplex real-time PCRs to detect DEC. At least one pathotype of DEC was detected in 41.2% (205/498) of samples associated with non-diarrhoeal children (Table 4).
To determine the prevalence and distribution of the various DEC in healthy and diarrhoeal children, we compared the data from the healthy children with a subset of the data from matched children in the diarrhoeal study which were between the ages of 2 and 5 years old (319 children; median age 31.5 months, IQR 26.7-38.9 months). The prevalence of ETEC, EAEC, and EHEC_O157 in faecal samples was not significantly different between children with or without diarrhoea (Table 4, Figure 2). Furthermore, EPEC was detected significantly more frequently in the non-diarrhoeal samples (18.7%, 93/498) than the diarrhoeal samples (11.4%, 33/289) (p=0.019, c2 test) (Table 4). The only DEC that was significantly associated with the diarrhoeal samples was EIEC/Shigella, which was detected in 34.3% (99/289) of diarrhoeal samples vs. 0.8% (4/498) non-diarrhoeal samples (p<0.001, Fisher’s exact test).
The distribution of DEC co-infection among the cases and the controls was complex and highly variable (Figure 2). The most common co-infections in the diarrhoeal group were EAEC + EIEC/Shigella (3.8%, 12/319) and EAEC + EIEC/Shigella + ETEC (2.2%, 7/319); whereas EPEC + EAEC (3.4%, 17/498) was more common in the healthy control group. Co-infection with more than one DEC was more likely to be associated with diarrhoeal disease than with healthy controls (16.3%, 52/319 vs. 9.6%, 48/498, p=0.005, c2 test). However, due to the predominant presence of EIEC/Shigella in the diarrhoeal group, EIEC/Shigella infection was a potential confounder.
To disaggregate the potential confounding effect of EIEC/Shigella, we performed binary univariate and multivariate logistic regression to identify variables and DEC that were associated with diarrhoeal disease in children aged 24-60 months (Table 5). In the univariate model, co-infection with ETEC, mono-infection with EIEC/Shigella, co-infection with EIEC/Shigella, and co-infection without EPEC, EHEC_O157, and STEC were significantly associated with diarrhoea. However, after controlling for confounders, only mono or co-infection with EIEC/Shigella and wasting were determined to be significantly associated with diarrhoea. Conversely, mono-infection with ETEC, EAEC, and obesity were significantly more common in the non-diarrhoeal children.