The incidence of infectious syphilis in men who have sex with men and human immunodeficiency virus-infected patients has increased steadily in Victoria, Australia, since 2002. A TaqMan real-time PCR assay targeting the polA gene of Treponema pallidum (TpPCR) was developed. The analytical sensitivity of the assay was estimated to be 1.75 target copies per reaction. Initially, the assay was used to test a variety of specimens (excluding blood) from 598 patients. Of the 660 tests performed, positive PCR results were obtained for 55 patients. TpPCR results were compared with serology results for 301 patients being investigated for early syphilis. Of these patients, 41 were positive by both TpPCR and serology, 246 were negative by both TpPCR and serology, 4 were TpPCR positive but negative by serology, and 10 were TpPCR negative but showed evidence of recent or active infection by serology. Directly compared with serology, TpPCR showed 95% agreement, with a sensitivity of 80.39% and a specificity of 98.40%. Potential factors leading to the discrepant results are discussed. Concurrent serology on 21 patients with TpPCR-positive primary syphilitic lesions demonstrated that a panel of current syphilis serological tests has high sensitivity for the detection of early syphilis. We found that TpPCR is a useful addition to serology for the diagnosis of infectious syphilis. Direct comparison with other T. pallidum PCR assays will be required to fully assess the limitations of the assay.Since 2002, there has been a marked increase in the number of cases of locally acquired syphilis infection in Victoria, Australia (2). Most cases were detected in men who have sex with men, with a disproportionate number of cases in human immunodeficiency virus (HIV)-infected patients (3).Clinical suspicion, and recognition of the symptoms and signs of syphilis, supported by serology is the mainstay of syphilis diagnosis, as direct detection methods such as dark-field microscopy and direct immunofluorescence are relatively insensitive, require fresh high-quality specimens, and are unsuitable for use on specimens from mucosal sites or if superinfection is present (4). Serology may be problematic in the early stages of primary syphilis, as rapid plasma reagin assay (RPR) responses may take some time to develop (4), particularly in HIV-infected patients, and few laboratories in Australia routinely use immunoglobulin M (IgM) assays.The Victorian Infectious Diseases Reference Laboratory (VIDRL) acts as the state reference laboratory for syphilis serology, confirming positive results from other laboratories and offering less commonly used tests, such as IgM assays. The laboratory also provides primary diagnostic services to a number of Melbourne sexually transmitted infection (STI) clinics and receives large numbers of specimens for culture or PCR for other agents of STI, allowing easy access to specimens for the development and assessment of novel STI detection assays. Our aim was to develop a robust, sensitive, and specific realtime PCR assay to d...