Viability assessment of Chlamydia trachomatis in men who have sex with men using molecular and culture methods

Abstract

Objectives: Chlamydia trachomatis (CT) is the most commonly reported bacterial sexually transmitted infection worldwide. Diagnosis relies on nucleic acid amplification techniques, such as PCR, which does not distinguish between viable pathogens and residual bacterial DNA, leading to potential overdiagnosis and overtreatment. PCR with confirmation of pathogen viability has not been widely explored in the sexually transmitted infection field. We aimed to establish a CT viability PCR (V-PCR) and to apply it to anorectal swabs from men who have sex with men (MSM).

Methods: We validated a published V-PCR protocol by preparing artificial samples with known ratios of viable and non-viable CT. Mock samples were treated with propidium monoazide (PMAxx) before DNA extraction and quantitative PCR (qPCR) to detect CT. The V-PCR was then applied to CT PCR-positive anorectal swabs from MSM. Viability was expressed as the difference in CT copies between PMAxx untreated and treated samples (ΔLog10 CT/mL). The anorectal samples were inoculated in cell culture for isolation. Genotyping was performed by examining the ompA gene sequence.

Results: Of 236 anorectal swabs, 69 (29.2%) were CT PCR positive, and we obtained V-PCR data from 54. There were 7 of 54 (12.9%), samples with <1% viable CT (>2.52 ΔLog10 CT/mL) 4 of 54 (7.4%) samples with 1% to 10% viable CT (1.59-2.52), 16 of 54 (29.6%) with 10.01% to 50% viable CT (0.86-1.59) and 27 of 54 (50.0%) with 50.01% to 100% viable CT (<0.35-0.86). CT was isolated successfully from 39 of 69 (56.5%) samples in cell culture. Genotypes based on ompA were obtained for 62 of 69 (89.9%) samples: G (n = 15/62), D/Da (n = 15/62), J (n = 15/62), E (n = 11/62), L1 (n = 4/62), and L2 (n = 2).

Discussion: We successfully implemented a viability test based on PCR, which can distinguish, detect and quantify viable CT in anorectal swabs from MSM. Rapid, reliable assessment of CT viability could help to improve antimicrobial stewardship.

Keywords: Cell culture; Chlamydia; Men who have sex with men; Pre-exposure prophylaxis; Viability.