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Quantitative PCR Gives New Insights on Large Study of Childhood Diarrhea in Africa, Asia

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NEW YORK (GenomeWeb) – An international team of researchers has used qPCR to re-assess more than 5,300 sample pairs from the Global Enteric Multicenter Study (GEMS) and demonstrated that quantitative methodologies changed estimates of pathogen-attributable diarrhea in children younger than five years.

Specifically, the re-analysis with qPCR suggested that 80 to 90 percent of childhood diarrhea is infectious. Historically most studies have used a combination of detection methods and found infectious causes in less than half of all cases, corresponding author Eric Houpt, a professor of infectious diseases and international health at the University of Virginia School of Medicine, told GenomeWeb. 

The study, which was published last month in Lancet, found that burdens of some pathogens were previously underestimated by up to five times, and that six pathogens — rotavirus, Shigella, cryptosporidium, Enterotoxigenic E. coli (ETEC), adenovirus, and Campylobacter — can explain about 70 percent of diarrhea cases.

Diarrheal disease is the second leading cause of death for children under five worldwide, killing about 760,000 young children per year, according to the World Health Organization.

The original GEMS study, published in Lancet in 2013, was considered the largest and most comprehensive study of childhood diarrhea in developing countries. It used primarily non-molecular methods to assess pathogen-related disease burden in a large sample set from children in seven countries: Gambia, Kenya, Mali, Mozambique, Pakistan, India, and Bangladesh.

That work found that four pathogens — rotavirus, Shigella, cryptosporidium, and ST-ETEC — were responsible for the majority of diarrhea cases, with a burden of disease that equated to about one episode per five children each year.

Led by a group at University of Maryland, the GEMS research also established a network of laboratories in high-burden areas to conduct future studies or roll out diarrheal interventions.

But the original study used culture for bacteria, enzyme immunoassays for rotavirus, adenovirus, and protozoa, and multiplex RT-PCR for norovirus, sapovirus, and astrovirus.

Houpt noted that the original study was "excellent," but the use of multiple different methods to detect pathogens was not ideal. "We wanted to apply the same method to all the pathogens, such that differences in sensitivity were not obscuring the estimates, so we chose qPCR for all 32 pathogens," he said in an email.

The reanalysis deployed a custom Thermo Fisher Scientific TaqMan Array Card designed to detect the 32 enteropathogens.

The group also tried packaging the same assays into several multiplex reactions or into an in-house Luminex-based assay. "All worked similarly well, but the TaqMan Array Card was chosen because it was easier to implement across all the sites and ensure reproducible data," Houpt said.

The global team of researchers then set about assaying approximately 12,000 samples, he said. He likened the logistical challenge to building a space shuttle and then launching it into outer space. "And we finished ahead of schedule," he noted, crediting team members at University of Virginia, University of Maryland, CDC Kenya, as well as at the International Center for Diarrheal Disease Research in Bangladesh, the Medical Research Council of Gambia, and Aga Khan University in Pakistan.

Potentially, focusing on the six pathogens that cause 70 percent of illness could some day save children's lives in lower-resource settings.

There are vaccines for Shigella and ETEC, and new therapies for cryptosporidiosis. Houpt also noted that the qPCR re-analysis found significantly more bacterial causes than previous work. "International guidelines for management of childhood diarrhea are mostly just to re-hydrate with no antibiotics unless [there are] special circumstances — while I don't advocate indiscriminate use of antibiotics, this is hardly indiscriminate, this is a deadly disease in poor countries," he said, adding that studies need to be done first to test if antibiotics improve outcomes.

With the major causes of childhood diarrhea now identified, there may also be more impetus to develop a syndromic panel for this population.

"A panel would be useful for children in resource-limited settings," Houpt said, but noted that the business model and machine requirements might be obstacles. Still, there are trials now underway to see if pathogen-based treatment is beneficial, he said. "If yes, then I think enthusiasm will build."

However, Houpt noted that the quantitative nature of the tests would be critical, as there exist high levels of pathogen carriage in developing countries, so future diagnostics panels would need to use qPCR or other quantitative methods.

But, "We hope this takes a big and historically murky problem and turns it into something manageable — we can devise new strategies to tackle these six [pathogens] and make a huge impact," Houpt said, adding, "Six is doable!"