Targeting surface cell antigen 2 increases sensitivity of Rickettsia typhi detection

Background Murine typhus is a flea-borne disease caused by Rickettsia typhi that typically presents as an acute febrile illness. The diagnosis is often missed, leading to delays in appropriate treatment. A qPCR targeting a single gene ( ompB) for R. typhi is widely used for diagnosis; however, it has low sensitivity for detecting bacterial DNA in patients’ blood. We aimed to increase sensitivity of detection of R. typhi using qPCR by targeting a gene containing repetitive sequences, sca2 (surface cell antigen 2) . Methodology We compared diagnostic accuracy with the standard assay targeting single sequence ompB (outer membrane protein B). Specificity, sensitivity, and bacterial load measurement of both assays were compared using stored EDTA-anticoagulated buffy coat samples from 88 patients with febrile illness at Mahosot Hospital or provincial hospitals in Laos. Among these, 55 cases were confirmed as positive or negative by first culturing Rickettsia spp. from patients’ EDTA blood, followed by assessment with IFA and ompB PCR, and buffy coat from 6 additional cases was confirmed by ompB PCR. Ten further positive cases were confirmed by IFA using paired sera, and 17 cases classified as negative for scrub typhus and murine typhus based on Rapid Diagnostic Test (RDT) results were included in the evaluation. Results The sca2 qPCR assay showed 59.09% sensitivity (95% CI, 38.73-76.74%) and 100% specificity (93.98-100%) for detection of R. typhi . In comparison, ompB assay demonstrated 36.36% sensitivity (95% CI, 19.73-57.05%) and 100% specificity (95% CI 93.98-100%). DNA copy number determined using the sca2 gene was approximately 2.933 log unit higher than that determined using ompB gene (median, 16,500 copies/μL; IQR, 13,045–40,000 versus median, 19.25 copies/μL; IQR, 11.11-56.62, P < 0.0001). Conclusion This study suggests qPCR targeting sca2 increases frequency of detection of R. typhi in patients with low bacterial DNA concentrations.