Multiplex PCR testing for Treponema pallidum and herpes simplex virus in primary care to detect primary syphilis in Victoria, Australia: a prospective cohort study

Background Syphilis is caused by the bacteria Treponema pallidum . Primary syphilis might present with multiple, painful lesions that are clinically indistinguishable from herpes. In this prospective cohort study, we aimed to evaluate whether the implementation of routine multiplex PCR testing for T pallidum and herpes simplex virus (HSV) in general practice improves the detection of primary syphilis. Methods From Sept 1, 2022, the PlexPCR VHS (SpeeDx, Eveleigh, NSW, Australia) test, which simultaneously detects T pallidum and HSV-1 and HSV-2 was implemented by Melbourne Pathology (Victoria, Australia). Multiplex testing was done on all samples from patients aged 18 years or older if: (1) the clinician ordered HSV PCR testing only from an anogenital site, or an oral or unspecified site that was also accompanied by a test for another sexually transmitted infection (eg, Chlamydia trachomatis , Neisseria gonorrhoeae , or Mycoplasma genitalium ); or (2) the clinician specifically requested T pallidum PCR. All positive T pallidum PCR results were reported to prompt treatment. The number and proportion of T pallidum PCR tests done and T pallidum PCR-positive cases detected were compared between requested T pallidum PCR and T pallidum PCR added to HSV PCR-only requests due to multiplex testing. Syphilis serology was examined among T pallidum PCR-positive cases in which T pallidum PCR had not been requested, to establish the proportion that might have been missed without multiplex testing. Findings Between Sept 1, 2022, and March 27, 2024, 8873 multiplex tests were done in 5847 (65·9%) female patients and 3026 (34·1%) male patients. In 6667 (75·1%) of 8873 T pallidum PCR tests, and 27 (25·2%) of 107 cases detected, T pallidum PCR was added to HSV-only requests through multiplex testing. Compared with cases in which T pallidum PCR was requested, a higher proportion of the 27 cases identified through added T pallidum PCR testing were from female patients (seven [25·9%] of 27 vs eight [10·0%] of 80; p=0·039), syphilis reinfections (eight [29·6%] of 27 vs nine [11·3%] of 80; p=0·024), clinically atypical presentations (12 [44·4%] of 27 vs 16 [20·0%] of 80; p=0·013), or those co-infected with HSV (three [11·1%] of 27 vs one [1·3%] of 80; p=0·049). In 17 (63·0%) of 27 added cases, serology for syphilis was not done or did not indicate reinfection. Interpretation Multiplex PCR testing for syphilis and herpes among patients for whom HSV PCR alone was ordered improved detection of primary syphilis in primary care. More widespread implementation would reduce misdiagnosis of primary syphilis in primary care, potentially reducing transmission and complications. Funding Australian National Health and Medical Research Council.