This was a cross-sectional study of children aged 6 months to 5 years admitted to Rakai General Hospital, Southern Uganda. Rakai Hospital is a 100-beds capacity hospital that admits approx. 200 sick children every week. The study recruited children with acute diarrhoea (passage of 3 or more loose stool over a 24 hour period lasting less than 2 weeks). The children were consecutively recruited over a period of 3 months (May 2016 to July 2016). HIV-serostatus was performed according to the algorithm of Uganda’s Ministry of Health. The clinical and epidemiological data were collected from the children by using an interviewer-administered questionnaire.
Stool was obtained from each child by using a sterile plastic container labelled with a unique study number. Specimen containers and instructions pertaining specimen collection were given to caretakers of the recruited children. For younger children, mothers collected stool from diapers as soon as the children passed stool while for older children, stool was collected into a disposable plate and immediately transferred into a sterile container. As soon as specimens were obtained, the caretakers were instructed to deliver them to Kakuuto Health Centre IV Microbiology Laboratory, Rakai district where they were processed for growth/identification of E. coli. In the laboratory, each sample was inoculated onto MacConkey agar plate within an hour of reception and incubated at 37 °C for 14 hours. Lactose fermenting colonies suggestive of E. coli were inoculated onto IMViCU (Indole, Methyl red, Voges Proskaeur, Citrate and Urease) media for confirmation as E. coli. Isolates that were positive with indole and methyl red tests but negative with Voges-Proskaeur, citrate and urea were classified as E. coli. To increase chances of detecting DEC, five E.coli colonies were sampled from each plate. Isolates were separately preserved in tryptone soy broth and transported to Makerere University for molecular analysis.
Crude and/or pure chromosomal DNA used in the PCRs was extracted as described previously [28]. Briefly, each sample was inoculated onto Nutrient agar and incubated at 37oC for 24 hours: To extract the DNA, a swipe of colonies was suspended into 300 µl of sterile 0.25X Tris-EDTA (TE) buffer in 1.5 ml microcentrifuge tubes, vortexed for 10 seconds, centrifuged at 13,000 g for 2 minutes, and the supernatant discarded to retain the pellet of clean bacteria. To the cell pellet, we added 100 µl of 0.25X TE buffer, vortexed for 10 seconds, centrifuged at 13,000 g for 5 minutes and discarded the supernatant. Then, cells were suspended in 60 µl of 0.25X TE buffer and heated at 95oC for 10 minutes in a Thermal mixer (Eppendorf, Germany). Then, samples were vortexed for 20 seconds and cooled to room temperature, centrifuged at 13,000 g for 5 minutes and the supernatant which contained the crude DNA was transferred into a sterile 1.5 ml microcentrifuge tube and used as template in PCRs.
Identification of DEC pathotypes
DEC pathotypes can be molecularly identified and/or classified based on the virulence genes specific to each pathotype [11]. To identify the pathotypes, we used previously published PCR primers [29] targeting eleven virulence genes specific to six DEC pathotypes i.e. ETEC, heat stable toxin (ST1 & ST2), heat labile toxin (LT); EHEC, verotoxins (VT1, 2 & 2e); EIEC (Einv); EAEC (Eagg); EPEC, attaching & effacing antigen (eaeA); CDEC (Cell-detaching E. coli), cytotoxic necrotising factors (CNF 1 & 2).
The PCRs were prepared by following the Taq 2X Taq Master Mix protocol (New England BioLabs, Inc.). Briefly, a 25 µL PCR assay was prepared by mixing 12.5 µL Taq 2X master mix, 1 µL each of the forward & reverse primers, 8 µL of nuclease free water, and 2.5 µL of crude DNA extract. Amplification was achieved in a thermocycler by using a programme reported by Todd et al. [30] with minor modifications i.e. Initial denaturation of 94oC for 5 minutes followed by 30 cycles of denaturation at 94oC for 60 seconds, annealing at 55oC for 90 seconds, extension at 72oC for 90 seconds, and a final extension step at 72oC for 10 minutes. Approx. 5 µL each of the PCR product was analysed by agarose gel electrophoresis (2% w/v agarose) in Tris-acetate EDTA (TAE) buffer stained with Ethidium Bromide (5 mg/ml) (Sigma-Aldrich, USA). Gels were run at 120V for 1 hour and visualized with UVP Gel documentation system (Benchtop Trans-illuminator System BioDoc-it, UK). PCRs included negative controls (E. coli ATCC25922 & sterile nuclease free water) and positive controls (PDAS 101, ATCC 35401, 29930, 933W, 35150 & E 2348/69 from the Kenya Medical Research Institute).
Statistical analysis was performed with SPSS v23 and comparisons performed with the Chi square test to assess associations between variables, for which p-values less than 0.05 were considered statistically significant. Outcome variables were presence or absence of DEC pathotypes; predictor variables were age, sex, prior use of antibiotics and HAART, temperature and HIV-serostatus.