Duplex, extraction-free, HiFi-LAMP assay for point-of-care detection of <i>Chlamydia trachomatis</i> and <i>Neisseria gonorrhoeae</i>

ABSTRACT Chlamydia trachomatis ( C. trachomatis ) and Neisseria gonorrhoeae ( N. gonorrhoeae ) are common sexually transmitted pathogens. Although frequently asymptomatic, they can cause severe reproductive complications (e.g., epididymitis, salpingitis, pelvic inflammatory disease, and infertility) if untreated. We developed a duplex high-fidelity loop-mediated isothermal amplification (HiFi-LAMP) assay for simultaneous detection of C. trachomatis and N. gonorrhoeae . The assay demonstrated high sensitivity and specificity, with limits of detection (LODs) of 358 and 30 copies per 25 μL reaction for C. trachomatis and N. gonorrhoeae , respectively. The performance of the assays was evaluated by comparing with the commercial singleplex qPCR assays using 156 paired urine and genital secretion samples. The assays have 100% specificity across all samples and achieved 100% sensitivity for the detection of N. gonorrhoeae in urine and C. trachomatis in genital secretion samples and 98.7% and 88.5% sensitivity for N. gonorrhoeae in genital secretions and C. trachomatis in urines, respectively. Extraction-free HiFi-LAMP assays directly using a small amount of simply treated urine were established and showed 87.9% sensitivity and 100% specificity for the detection of N. gonorrhoeae in 70 urine samples. The new HiFi-LAMP assays can detect all C. trachomatis - and/or N. gonorrhoeae -positive samples within 30 min, implying that the entire HiFi-LAMP assay (including sample process) can be completed within 40 min, significantly shorter than about 90 min by the qPCR assays. The new HiFi-LAMP assays provide a rapid, simple, and cost-effective point-of-care testing (POCT) tool for clinical detection and/or surveillance of C. trachomatis and N. gonorrhoeae in resource-limited settings. IMPORTANCE Asymptomatic C. trachomatis and N. gonorrhoeae infections significantly increase the risk of silent transmission. C. trachomatis and N. gonorrhoeae were cocirculating in sexually active individuals. Of 156 individuals, we detected 7 (4.5%) individuals positive for both C. trachomatis and N. gonorrhoeae , implying an urgent need for rapid accurate and duplex POCT assay for timely detection of C. trachomatis and N. gonorrhoeae . The development of a duplex HiFi-LAMP assay enables the simultaneous detection of C. trachomatis and N. gonorrhoeae with high sensitivity and specificity. The assay offers high diagnostic concordance with commercial qPCR assays and has lower cost and shorter reaction time. Given the availability of various portable fluorescent thermal cyclers and/or isothermal fluorimeters, the development of extraction-free HiFi-LAMP assay directly using simply treated urine will lower the barriers of sexually active individuals to routine screening and facilitate clinical detection and surveillance of C. trachomatis and N. gonorrhoeae in resource-limited settings.