For evaluation of assay performance, genomic materials or reference strains were obtained from American Tissue and Culture Collection (ATCC, Manassas, VA) or BEI resources for adenovirus 1, 5, 40 and 41, human cytomegalovirus, enterovirus 71, Epstein-Barr virus, Aeromonas hydrophila, Bacteroides fragilis, Campylobacter coli, Campylobacter upsalensis, Campylobacter hyointestinalis, Campylobacter jejuni, Helicobacter pylori, Listeria monocytogenes, Mycobacterium tuberculosis, Plesiomonas shigelloides, Salmonella enterica, Vibrio parahaemolyticus, Yersinia enterocolitica, Blastocystis hominis, Cryptosporidium hominis, Cryptosporidium meleagridis, Schistosoma mansoni. Cryptosporidium parvum and Encephalitozoon intestinalis were purchased from Waterborne Inc. (New Orleans, LA). PCR amplicons were generated from the relevant positive clinical samples for Ancyclostoma duodenale, Necator americanus, Strongyloides stercoralis, Cyclospora cayetanensis, Cystoisospora belli, and Enterocytozoon bieneusi. For comparison between stool and swab (FLOQSwabs; Copan Italia, Brescia, Italy), 129 consecutive swab samples were collected from children under five admitted for acute diarrhea in Haydom Lutheran Hospital, Tanzania. A matched stool sample from the same patient was obtained as soon as feasible within the same day. Raw stool samples were transported with a cold chain to the lab within 6 hours and stored at -80°C until testing. Swabs were stored at room temperature until testing. For comparison between different extraction methods and validation of the newly developed qPCR assays on clinical samples, we chose 246 archived stool samples collected in Tanzania, Bangladesh, Nepal, Pakistan, and India through the MAL-ED project (the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development [6 (link)]) in order to obtain specimens positive for 30 diverse enteropathogens. All sites including Haydom Global Health Institute, Tanzania, Aga Khan University, Pakistan, Armed Forces Research Institute of Medical Sciences, Thailand, International Centre for Diarrhoeal Disease Research, Bangladesh, Christian Medical College, India, received ethical approval from their respective governmental, local institutional, and collaborating institutional ethics review boards. Written informed consent was obtained from the parent or guardian of every child.
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