Advanced specificity and sensitivity studies relative to a research use only transcription-mediated amplification-based assay for RNA detection.

Unlabelled: Preliminary experimentation has suggested that a research-use-only real-time transcription-mediated amplification assay for (RUO TMA) yields instances of nucleic acid detection that coincide with non-treponemal serology in men who have sex with men (MSM) at increased risk for sexually transmitted infection. To further characterize the specificity of RUO TMA testing, 3,586 rectal swab specimens reported as "not detected" by the assay generated a mean endpoint FAM fluorescence of 637.5 units. Introduction of , , and nucleic acid into matrices generated mean endpoint FAM fluorescence ranging from 620.7 to 633.5 units ( ≥ 0.15 versus control). Introduction of lubricant to a pooled rectal swab matrix did not result in elevated FAM fluorescence (mean endpoint value 628.3 units [95% CI 612.0, 644.6]; = 0.67). Introduction of talcum powder, urine, seminal fluid, and blood also failed to generate increased FAM fluorescence ( ≥ 0.20). Analytic sensitivity assessment was measured by serial 10-fold dilution of whole organism or 23S rRNA transcript in specimen transport medium or pooled rectal swab matrix and interrogation by the assay. Probit analysis estimated sensitivity (95% detection) of RUO TMA at 421-5,707 transcript copies/mL and 9-48 cells/mL, depending on dilution matrix. These results support RUO TMA as a highly sensitive method for detection that is not impacted by potentially cross-reactive organisms or interfering substances and may have adjunctive diagnosis capability for syphilis in MSM.

Importance: Research-use-only transcription-mediated amplification (RUO TMA) has the potential to improve laboratory diagnosis of syphilis, particularly in patients at increased risk for sexually transmitted infection. The high analytic sensitivity and lack of cross-reactivity of the assay can facilitate other laboratories exploring the use of the test in a research setting.