Alternative treponemal serology assays for diagnosis and confirmation of syphilis in a diagnostic laboratory: a retrospective evaluation of four agglutination assays and one ELISA

The Treponema pallidum particle agglutination (TPPA) assay is no longer available in some settings. Here, we report the results of a clinical laboratory evaluation of alternative treponemal assays, including three T. pallidum hemagglutination assays (TPHAs), a T. pallidum IgG enzyme-linked immunosorbent assay (ELISA), and a detailed laboratory evaluation of the only TPHA with Australian regulatory approval. The clinical laboratory evaluation comprised 300 sera (120 TPPA reactive and 180 TPPA non-reactive) collected from individuals ≥18 years between 1 June 2021 and 1 June 2023. Median age was 33, 92% were from a sexually transmitted infection clinic, 71% were male, 15.3% were living with HIV and 5% were pregnant. Twenty-four percent had active untreated syphilis, 25% had prior treated syphilis, and 50.7% had no syphilis. Compared to TPPA, positive percent agreement (PPA) was 99.2% (95% confidence interval [CI], 95.4-99.9%), 100% (95% CI, 96.9-100%), 99.2% (95% CI, 95.4-99.9%), and 93.3% (95% CI, 87.4-96.6%), respectively, for the Arlington, Fortress, and Randox TPHAs, and Euroimmun IgG ELISA. Negative percent agreement (NPA) was 97.8% (95% CI, 94.4-99.1%) for each TPHA and 98.9% (95% CI, 96.0-99.8%) for the Euroimmun IgG ELISA. Clinical sensitivity of the Arlington and Fortress TPHAs for active untreated syphilis infection was equivalent to TPPA at 97.2% (95% CI, 90.4-99.5%) and clinical specificity of these assays was 99.3% (95% CI, 96.3-100%). In the evaluation of the NewBio TPHA, comprising 322 clinical serum and plasma samples and 22 quality assurance panel samples; overall PPA compared to TPPA was 100% (95% CI, 97.5-100%) and NPA 98.9% (95% CI, 94.1-98.9%). TPHAs are an acceptable alternative for confirmatory treponemal serology.