Evaluation of multiplex real-time PCR for detection of H. ducreyi, T. pallidum, HSV-1, and HSV-2 in the diagnosis of genital ulcer disease in Rakai District, Uganda

Abstract

Objective: To develop a real-time PCR assay that reliably and accurately detects the predominant sexually transmitted aetiologic agents of genital ulcer disease (GUD) Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2)) and to assess the utility of real-time PCR diagnostic testing in a rural African field site.

Methods: Two multiplex real-time PCR reactions were used to detect H. ducreyi/T. pallidum and HSV-1/HSV-2 in ulcer swabs from 100 persons with symptomatic genital ulcers in rural Rakai Uganda. Results were compared with syphilis, HSV-1 and HSV-2 serology.

Results: Of 100 GUD samples analyzed from 43 HIV-positive and 57 HIV-negative individuals, 71% were positive for one or more STI pathogens by real-time PCR (61% for HSV-2, 5% for T. pallidum, 3% for HSV-1, 1% for H. ducreyi, and 1% for dual H. ducreyi/HSV-2). The frequency of HSV in genital ulcers was 56% (32/57) in HIV-negative individuals and 77% (33/43) in HIV-positive individuals (p = 0.037). Assay reproducibility was evaluated by repeat PCR testing in the US, with 96% agreement (kappa = 0.85).

Conclusions: STI pathogens were detected in the majority of GUD swab samples from symptomatic patients in Rakai Uganda by real-time PCR. HSV-2 was the predominant cause of genital ulcers. Real-time PCR technology can provide sensitive, rapid and reproducible evaluation of GUD aetiology in a resource limited setting.