LMIC human infant stool whole-genome shotgun metagenomic sequencing to validate qPCR Metagenome

A diagnostic gap exists in the detection of Campylobacter spp. using nucleic acid-based techniques in comparison to standard microbiological culture. A working hypothesis is that uncommon species of Campylobacter play a role in enteric disease among children in low resource settings. C. infans, was recently detected in stool samples from children and hypothesized to play a role in the epidemiology of this pathogen within highly endemic communities. This study aimed to determine the prevalence of C. infans in symptomatic and asymptomatic stool samples from children living in Iquitos, Peru, and to determine if the presence of this pathogen would explain the high discrepancy of the detection of Campylobacter between nucleic acid diagnostics and culture. Stool samples from 215 children with diarrhea and 50 stool samples from children in a diarrhea free interval under the age of two were queried using a Taqman based multiplex assay to detect Campylobacter spp. (16sRNA), Campylobacter jejuni/ Campylobacter coli (cadF gene), C. infans and Shigella spp.. Odds ratios and 95% confidence intervals of diarrhea were calculated based on the presence of these targets using univariate logistic regressions. C. infans was detected in 7.9% (17/215) symptomatic samples and 4.0% (2/50) asymptomatic samples. The presence of C. infans was not associated with higher odds of diarrhea. Twenty nine percent of Campylobacter positive fecal samples are not attributed to C. jejuni, C. coli or C. infans. Whole metagenomic DNA confirmed the presence of C. infans among 13 out 14 positive C. infans positive stool samples. Eight C. infans positive samples were co-infected with at least one additional Campylobacter species. A BLASTn analysis of the qPCR cadF pforward and reverse primers against 3076 C. jejuni and C. coli WGS in pubmlst determined that 41% of the strains (1254/3076) did not have 100% identity at both primer binding sites. Further studies are needed to determine if the targeted section of the cadF gene for the detection of C. jejuni and C. coli can be improved for the detection of strains present in LMICs.