Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study

Abstract

Background: Diarrhoea is the second leading cause of mortality in children worldwide, but establishing the cause can be complicated by diverse diagnostic approaches and varying test characteristics. We used quantitative molecular diagnostic methods to reassess causes of diarrhoea in the Global Enteric Multicenter Study (GEMS).

Methods: GEMS was a study of moderate to severe diarrhoea in children younger than 5 years in Africa and Asia. We used quantitative real-time PCR (qPCR) to test for 32 enteropathogens in stool samples from cases and matched asymptomatic controls from GEMS, and compared pathogen-specific attributable incidences with those found with the original GEMS microbiological methods, including culture, EIA, and reverse-transcriptase PCR. We calculated revised pathogen-specific burdens of disease and assessed causes in individual children.

Findings: We analysed 5304 sample pairs. For most pathogens, incidence was greater with qPCR than with the original methods, particularly for adenovirus 40/41 (around five times), Shigella spp or enteroinvasive Escherichia coli (EIEC) and Campylobactor jejuni o C coli (around two times), and heat-stable enterotoxin-producing E coli ([ST-ETEC] around 1·5 times). The six most attributable pathogens became, in descending order, Shigella spp, rotavirus, adenovirus 40/41, ST-ETEC, Cryptosporidium spp, and Campylobacter spp. Pathogen-attributable diarrhoeal burden was 89·3% (95% CI 83·2-96·0) at the population level, compared with 51·5% (48·0-55·0) in the original GEMS analysis. The top six pathogens accounted for 77·8% (74·6-80·9) of all attributable diarrhoea. With use of model-derived quantitative cutoffs to assess individual diarrhoeal cases, 2254 (42·5%) of 5304 cases had one diarrhoea-associated pathogen detected and 2063 (38·9%) had two or more, with Shigella spp and rotavirus being the pathogens most strongly associated with diarrhoea in children with mixed infections.

Interpretation: A quantitative molecular diagnostic approach improved population-level and case-level characterisation of the causes of diarrhoea and indicated a high burden of disease associated with six pathogens, for which targeted treatment should be prioritised.

Funding: Bill & Melinda Gates Foundation.

Figure 1. Relation between pathogen quantity and diarrhoea

Pathogens are ordered from top to bottom and left to right by prevalence in cases. Data are numbers of dectections (vertical bars) in cases (dark grey) and controls (light grey) with odds ratios (red lines) and 95% CIs (bands). The model-derived cutoffs used for identification of diarrhoea-associated pathogens in individuals (overlaid in blue) are defined as all detections above the point at which the 95% CI no longer includes 1. EAEC=enteroaggregative E coli. EIEC=enteroinvasive E coli. EPEC=enteropathogenic E coli. LT-ETEC=heat-labile enterotoxin-producing E coli. ST-ETEC=STh-producing enterotoxigenic E coli. STEC=Shiga toxin producing E coli.

Figure 2. Attributable incidence of pathogen-specific moderate to severe diarrhoea per 100 child-years, by age stratum, across study sites, in this and the original GEMS

For each age stratum, any pathogens significantly associated with diarrhoea by one or both diagnostic approaches are shown. GEMS=Global Enteric Multicenter Study. EAEC=enteroaggregative E coli. EIEC=enteroinvasive E coli. tEPEC=typical enteropathogenic E coli. ST-ETEC=STh-producing enterotoxigenic E coli. qPCR=quantitative realtime PCR. *Indicates the microbiology in the original GEMS did not test for H pylori or C cayetanensis.

Figure 3. Relative attribution of watery diarrhoea and dysentery to each pathogen

Data are overall adjusted attributable fractions (vertical bars) with 95% CIs. Pathogens are ordered by the proportion of total attributable cases that were watery diarrhoea (dotted line). All pathogens significantly associated with either dysentery or watery diarrhoea are shown. ST-ETEC=STh-producing enterotoxigenic E coli. tEPEC=typical enteropathogenic E coli. EIEC=enteroinvasive E coli.

Figure 4. Detection of co-infections in diarrhoeal cases

(A) Numbers of pathogens at diarrhoea-associated quantities and any quantity in individual cases of diarrhoea. (B) Distribution of pathogens, alone and in co-infections, by quantity and association with diarrhoea. The quantification cycle cutoff used to identify diarrhoea-associated detections is shown in parentheses after each pathogen name. Cq=quantification cycle. EIEC=enteroinvasive E coli. ST-ETEC=STh-producing enterotoxigenic Escherichia coli. tEPEC=typical enterpathogenic E coli.