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

Abstract

Summary 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 or 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.

vAetiology of Diarrhoea in Children Under Five in Zambia Detected Using Luminex xTAG Gastrointestinal Pathogen Panel

“…The high prevalence of single Adenovirus infection, only next to rotavirus, in our study tallies with the GEMS re-analysis study where, when assessed with PCR-based methods, Adenovirus incidence was five times more than previously reported using microbiological methods [17,22], shifting this enteropathogen to become among the top six diarrhoea attributable pathogens [23,24]. However, our study did not find attributable cases of moderate-to-severe diarrhoea due to Adenovirus.…”

“…While the high burden of diarrhoea attributable to rotavirus and Shigella spp. as seen in our study may be due to the high sensitivity of the molecular based diagnosis method we used in this study, other reports such as Liu and colleagues [22] and many other studies [25][26][27][28][29] have noted that Shigella spp. was under detected in cultures compared to PCR.…”

“…For future studies on aetiology of diarrhoea, we propose that the quantitative PCR (qPCR) based assays are used to complement identification efforts by providing burden, species and strain information on enteric pathogens which can help with determining the attributable fraction each detected pathogen contributes to diarrhoea [22].…”

Etiology, epidemiology and clinical characteristics of acute diarrhea in hospitalized children in rural Peru

“…In other studies, the isolation rate and prevalence of each pathogen also varies with the age of the patient and the type of study, i.e. community versus hospital, passive community surveillance versus active surveillance [14]. Although, the rotavirus vaccine was introduced by the Peruvian National Immunization Program in 2009, national reports from 2015 have shown that there is still a low vaccination coverage of 80.9% in children under 12 months [15].…”

Shigella Vaccine Development: Finding the Path of Least Resistance

“…Similarly, a multisite birth cohort study (MAL-ED) conducted in eight countries in South America, Africa, and Asia identified Shigella as an important cause of moderate and severe diarrhea and dysentery in the community during the second year of life (4). Reanalysis of the GEMS data using molecular diagnostic tools suggested that these culture-based burden estimates substantially underrepresent the true burden of Shigella (5,6). Moreover, military personnel and other travelers from high-resource countries who visit these endemic settings are also at risk for acquiring Shigella infections and disseminating them upon returning home.…”