A multiplex polymerase chain reaction (M-PCR) assay that simultaneously detects the three major causes of genital ulcer disease (GUD), Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus, was used to evaluate swab specimens for 38 sequential patients with GUD at a Thai sexually transmitted disease clinic. Subjects received clinical diagnoses and syndromic treatment. Genital ulcer disease (GUD) is an important health problem cultured with methods available to the clinician and is typically diagnosed either serologically or with darkfield microscopy [3]. in many developing country settings. GUD has been implicated in several studies as an important factor in the sexual spread However, the latter is also unavailable in many developing country settings, and the sensitivity of serologic assays in acute of human immunodeficiency virus type 1 (HIV-1) in numerous developing countries, including Thailand [1]. Clinical diagnoinfection can be as low as 30% [4]. ses of 3 of the most common etiologic agents of GUD (HaemoRecently, a multiplex polymerase chain reaction (M-PCR) philus ducreyi, Treponema pallidum, and herpes simplex virus) assay with colorimetric detection has been developed for the in sexually transmitted disease (STD) clinic settings are probsimultaneous amplification of DNA targets of these three lematic, however [2]. H. ducreyi is a fastidious organism; while pathogens using a single swab from genital ulcer secretions culture is the reference standard for diagnosis, sensitivity of [5]. This assay has been used in several studies, including one culture varies considerably [3], and culture is not routinely in Lesotho, where its sensitivity compared with culture for H. done in Thai Ministry of Public Health (MOPH) clinics. Herpes ducreyi and HSV was 95% and 93%, respectively [2]. In a simplex virus (HSV) can be definitively diagnosed by culture, large GUD patient series in New Orleans, the sensitivities of but cost has limited the utility of this assay in most developing M-PCR for HSV, H. ducreyi, and T. pallidum were 100%, country settings, including Thailand, where viral culture is not 98.4%, and 91%, respectively, versus 71.8% for HSV culture, used in government STD clinics. HSV ELISAs are also not 74.2% for H. ducreyi culture, and 81% for darkfield microscopy currently used in Thai MOPH clinics. T. pallidum cannot be for T. pallidum [5]. Chiang Mai City has been one of the epicenters of the Thai HIV epidemic, with HIV prevalence rates among sex workers reaching 40% by 1990 [6]. HIV prevalence among male STD