Novel rpsK / rpsD primer-probe assay improves detection of Campylobacter jejuni and Campylobacter coli in human stool

Campylobacter causes bacterial enteritis, dysentery, and growth faltering in children in low- and middle-income countries (LMICs). Campylobacter spp. are fastidious organisms, and their detection often relies on culture independent diagnostic technologies, especially in LMICs. Campylobacter jejuni and Campylobacter coli are most often the infectious agents and in high income settings together account for 95% of Campylobacter infections. Several other Campylobacter species have been detected in LMIC children at an increased prevalence relative to high income settings. After doing extensive whole genome sequencing of isolates of C. jejuni and C. coli in Peru, we observed heterogeneity in the binding sites for the main species-specific PCR assay (cadF) and designed an alternative rpsKD-based qPCR assay to detect both C. jejuni and C. coli. The rpsKD-based qPCR assay identified 23% more C.jejuni/ C.coli samples than the cadF assay among 47 Campylobacter genus positive cadF negative samples verified to have C. jejuni and or C. coli with shotgun metagenomics. This assay can be expected to be useful in diagnostic studies of enteric infectious diseases and be useful in revising the attribution estimates of Campylobacter in LMICs.Author summary: Campylobacter is a leading cause of gastroenteritis among children living in resource poor settings. Infections are predominantly caused by Campylobacter jejuni and Campylobacter coli. To estimate the burden of Campylobacter, nucleic acid diagnostic testing has been utilized in large population based epidemiologic studies. However, there is evidence of heterogeneity in the binding sites for the main C. jejuni and C. coli qPCR assay utilized. This study presents and validates an alternative rpsKD-based qPCR assay to detect both C. jejuni and C. coli. Improved detection of these two species is expected to impact diagnostic studies of enteric infectious disease and improve estimates of the overall burden of Campylobacter.