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

Area of Science:

• Microbiology
• Infectious Diseases
• Molecular Diagnostics

Background:

• Campylobacter species cause significant enteritis, dysentery, and growth faltering in children, particularly in low- and middle-income countries (LMICs).
• Current diagnostic methods, often culture-independent, face challenges in accurately detecting diverse Campylobacter species prevalent in LMICs.
• Campylobacter jejuni and Campylobacter coli are the primary agents in high-income settings, but other species are more common in LMICs.

Purpose of the Study:

• To address limitations in existing PCR assays for Campylobacter detection, specifically the cadF assay, due to observed heterogeneity in binding sites.
• To develop and validate a novel, more inclusive quantitative PCR (qPCR) assay for simultaneous detection of Campylobacter jejuni and Campylobacter coli.

Main Methods:

• Whole genome sequencing of Campylobacter jejuni and Campylobacter coli isolates from Peru.
• Design and implementation of a new species-specific qPCR assay targeting the rpsKD gene.
• Comparative analysis of the novel rpsKD assay against the established cadF assay using shotgun metagenomics for verification.

Main Results:

• Heterogeneity in cadF binding sites was identified, impacting assay accuracy.
• The novel rpsKD-based qPCR assay detected 23% more Campylobacter jejuni/coli samples compared to the cadF assay among verified positive samples.
• Shotgun metagenomics confirmed the presence of Campylobacter jejuni and/or coli in samples missed by the cadF assay.

Conclusions:

• The rpsKD-based qPCR assay offers improved sensitivity and inclusivity for detecting Campylobacter jejuni and coli.
• This assay is a valuable tool for diagnostic studies of enteric infectious diseases in LMICs.
• Findings can help refine attribution estimates for Campylobacter infections in LMICs.