Molecular Detection of Enteropathogens in Stool Samples

Field workers collected monthly non-diarrheal stool samples without a fixative, and raw stool aliquots were stored at −80 °C before nucleic acid extraction. All laboratory testing was completed at site-specific laboratories [30 (link),31 (link)]. The QIAmp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) was used to extract total nucleic acid from stool specimens from children who had completed two years of follow-up, as previously described [32 (link)]. The efficacy of extraction and amplification was assessed using extrinsic controls, such as phocine herpesvirus (PhHV) and bacteriophage MS2. Quantitative polymerase chain reaction (PCR) with custom-designed TaqMan Array Cards was used to identify 29 enteropathogens utilizing the AgPath One Step realtime PCR kit (ThermoFisher, Waltham, MA, USA), as described elsewhere [33 (link),34 (link),35 (link)]. Shigella spp. were detected using primer sets specific for the ipaH gene [11 (link),33 (link)], and a cycle of threshold (Ct) value of less than 35 (Ct < 35) was use as the cut-off [11 (link),36 ].