Quantitative Molecular Diagnostic Methods Identify Causes of Childhood Diarrhea

The number of cases of childhood diarrhea attributed to pathogens such as bacteria, parasites, viruses or other infections has been substantially underestimated and may be nearly twice as high as previous analysis suggests.

Worldwide, diarrhea remains the second leading cause of death in children under five years old, and is associated with approximately half a million deaths per year. Management of diarrhea usually involves supportive care with rehydration and zinc supplementation. Antibiotics are not usually recommended unless there are signs of dysentery, severe diarrhea with the presence of blood and mucus in the stools or if cholera is suspected.


A large team of scientists collaborating with those at the University of Virginia (Charlottesville, VA, USA) re-analyzed stool samples from 10.608 children with and without diarrhea obtained from regions in seven countries in Asia (Bangladesh, India and Pakistan) and Africa (The Gambia, Kenya, Mali, and Mozambique), which were from the Global Enteric Multicenter Study (GEMS). Previous estimates of the infectious causes of diarrhea were based on a variety of different detection methods, but this study, for the first time, uses a molecular diagnostic testing method called quantitative real-time polymerase chain reaction (qPCR) to test for 32 pathogens.

The scientists formulated a custom TaqMan Array Card (Thermo Fisher, Carlsbad, CA, USA) that compartmentalized probe-based qPCR assays for 32 enteropathogens. This platform has shown high reproducibility within and across sites. All the assays have been described previously and have been extensively validated. Nucleic acid was extracted with the QIAamp Fast DNA Stool mini kit (Qiagen, Hilden, Germany) with pretreatment steps that included bead beating.

The original GEMS study1, published in 2013, estimated that 51.5% of childhood diarrhea cases could be attributed to pathogens, but the new re-analysis finds the proportion is much higher at 89.3%. The original study identified four major pathogens: rotavirus, Shigella spp, Cryptosporidium spp, and heat-stable enterotoxin-producing Escherichia coli (ST-ETEC). This re-analysis reaffirmed these four and added two others: adenovirus 40/41 and Campylobacter jejuni/coli. Together, these six pathogens accounted for 77.8% of all diarrheas. Among the children who had a diarrhea-causing pathogen, about half had more than one infection highlighting the challenges of treating multiple infections.

Eric R Houpt, MD, a professor of Infectious Diseases and a senior author of the study said, “Childhood diarrhea remains an enormous problem, made more confusing by the long list of possible infections, the difficulties in diagnosis, and the large number of undiagnosed cases. We developed sensitive quantitative methods that levelled the diagnostic playing field and closed the diagnostic gap.” The study was published on September 24, 2016, in the journal The Lancet.

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University of Virginia
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