Multipathogen TaqMan array card assay

Since surveillance in Sanjiang was year-round, covering the peak(s) of seasonal diarrhea resulting from bacterial and viral infections, total nucleic acid (TNA) of bacteria and viruses were extracted using MagMAX™ Total Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) on KingFisher Flex (Thermo Fisher Scientific, Waltham, MA, USA). As an extrinsic control, bacteriophage MS2 (ATCC, Manassas, VA, USA) was spiked in lysis buffer to monitor the efficiency or inhibition of extraction and amplification of each sample. While surveillance in Zhengding only focused on viral diarrhea, TNA of viruses were extracted using the Tianlong virus RNA/DNA extraction kit on the NP968 3S system (Tianlong, Xi’an, China). Procedures were performed according to the manufacturer’s instructions unless otherwise stated. All isolated TNA were stored at − 80 °C prior to assays.

The panel of TAC (Applied BiosystemsTM, USA) in US CDC used in this study included 6 viral, 13 bacterial, and 5 parasitic agents as previously described (Additional file 1: Table S1) [6]. Any quantification cycle (Cq) result of < 45 was considered positive.

The Luminex xTAG gastrointestinal pathogen panel assay (GPP) (Luminex Molecular Diagnostics, Austin, TX, USA) as a qualitative multiplex test has been shown to be highly sensitive and specific for a variety of pathogens in clinical settings globally [8–10]. In this study, the xTAG GPP platform in US CDC was used to evaluate the results of rotavirus A, norovirus GI/GII, and adenovirus 40/41 from TAC assays.

We used SAS (SAS Institute Inc., Cary, NC, USA) for statistical analysis. For binary data, the Chi square test was used, or Fisher’s exact test when data were sparse. For comparison of Ct values, the Mann–Whitney U test was used. For validation of the TAC assay, sensitivity, specificity, and kappa coefficient with 95% confidence intervals (95% CI) were calculated using GPP test results as references. The incidence rate of viral diarrhea was calculated as the number of diarrheal illness episodes with stool specimens testing positive for rotaviruses, noroviruses, sapoviruses, adenoviruses, and astroviruses among the surveillance participants per 1,000 child-years. The proportions of cases positive for each agent were calculated. Ct values were presented as the mean of two duplicate wells for specimens yielding positive results. If these two duplicate wells presented discordant results (one was positive via Ct value and one was negative), “indeterminate” comments were assigned to the wells; these wells were not included in subsequent calculations. All P values were two-tailed with < 0.05 considered statistically significant.