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

Abstract

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.IMPORTANCERates of syphilis, including congenital syphilis, are increasing globally, resulting in substantial morbidity and neonatal mortality. A key pillar of syphilis control is timely and accurate diagnosis. Serology is the primary diagnostic test for syphilis. Serological testing for syphilis has been impacted by a withdrawal of the Treponema pallidum particle agglutination (TPPA) assay from several geographical regions, including Australia and Europe. Here, we describe the clinical and laboratory performance of alternative treponemal serological assays, with a focus on alternative agglutination assays (Treponema pallidum hemagglutination assays, TPHAs) as these assays comprise alternative antigens to commercial treponemal immunoassays, require a small volume of sample input and do not require specific instrumentation. This study demonstrates that TPHAs have excellent clinical and analytical performance characteristics and provides confidence that these assays are an acceptable alternative in settings that no longer have access to the TPPA.