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

Authors: SCHIAFFINO, FRANCESCA - Universidad Peruana; Parker, Craig; GARCIA-BARDALES, PAUL - Prisma Charitable Association; Huynh, Steven; MANZANARES VILLANEUV, KATIA - Prisma Charitable Association; MOURKAS, EVANGELOS - Oxford University; PASCOE, BEN - Oxford University; PENATARO YORI, PABLO - University Of Virginia; PAREDES-OLORTEGUI, MARIBEL - Prisma Charitable Association; HOUPT, ERIC - University Of Virginia; LIU, JIE - Qingdao University; COOPER, KERRY - University Of Arizona; KOSEK, MARGARET - University Of Virginia

Submitted to: PLOS Neglected Tropical Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/19/2024
Publication Date: 3/1/2024
Citation: Schiaffino, F., Parker, C.T., Garcia-Bardales, P.F., Huynh, S., Manzanares Villaneuva, K., Mourkas, E., Pascoe, B., Penataro Yori, P., Paredes-Olortegui, M., Houpt, E.R., Liu, J., Cooper, K.K., Kosek, M.N. 2024. Novel rpsK / rpsD primer-probe assay improves detection of Campylobacter jejuni and Campylobacter coli in human stool. PLOS Neglected Tropical Diseases. 18(3). Article e0012018. https://doi.org/10.1371/journal.pntd.0012018.
DOI: https://doi.org/10.1371/journal.pntd.0012018

Interpretive Summary: Campylobacter has been classified as a leading cause of bacterial gastroenteritis worldwide. 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 accounting 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.

Technical Abstract: 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 accounting 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.