Composite TMA Reference Standard Enables Clinical Validation of First Mycoplasma genitalium NAAT
Three novel transcription-mediated amplification assays targeting unique M. genitalium rRNA sequences were developed and validated as a composite reference standard, achieving analytical sensitivity down to 0.017 genome equivalents/mL with no cross-reactivity among 56 non-target organisms. Applied to 1,400 clinical specimens, the composite standard confirmed the Aptima M. genitalium IVD assay at 100% sensitivity and 99.9% specificity, establishing a replicable framework for validating diagnostics where no culture-based gold standard exists.
The original study
Design and Validation of Transcription-Mediated-Amplification Nucleic Acid Amplification Tests for Mycoplasma genitalium.
- Authors
- Kirkconnell B, Weinbaum B, Santos K, Le Nguyen T, Vinluan B, Astete S, et al.
- Journal
- Journal of clinical microbiology
- Type
- Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't, Validation Study
- PMID
- 31018983
Original abstract
Mycoplasma genitalium is a sexually transmitted bacterium linked to adverse sexual and reproductive health outcomes in women and men. M. genitalium is difficult to culture, and in the absence of validated amplified molecular methods for diagnosis of infection, there is no reference standard available for use as a comparator for the validation of new M. genitalium diagnostic tests. We evaluated the analytical and clinical performance of three transcription-mediated amplification (TMA) tests for M. genitalium, each targeting unique rRNA sequences, for use as a composite comparator for clinical validation of the Aptima Mycoplasma genitalium (AMG) assay, an in vitro diagnostic (IVD) TMA test that targets 16 s rRNA of M. genitalium Analytical sensitivity, specificity, and strain inclusivity of all four TMA tests were determined using nine laboratory strains of M. genitalium and 56 nontarget bacteria, protozoa, and viruses. Analytical sensitivity of the tests for M. genitalium ranged from 0.017 to 0.040 genome equivalents/ml. None of the nontarget organisms evaluated cross-reacted with any test. A composite comparator reference standard consisting of the 3 alternate (Alt) TMA tests was used to evaluate the clinical performance of the AMG assay by testing residual vaginal swab, female urine, and male urine specimens obtained from 1,400 adult subjects from three U.S. clinical sites. Using this reference standard to establish infected specimen status, the sensitivity, specificity, and overall agreement of the AMG IVD assay were 100%, 99.9%, and 99.9%, respectively. These results demonstrate the utility of molecular composite reference standard methodology for the clinical validation of future IVD tests for this organism.