Design and validation of transcription-mediated amplification nucleic acid amplification tests for Mycoplasma genitalium .

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 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 16s rRNA of M. genitalium Analytical sensitivity, specificity and strain inclusivity of all four TMA tests was determined using nine laboratory strains of M. genitalium and 56 non-target 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 non-target organisms evaluated cross-reacted with any test. A composite comparator reference standard consisting of the 3 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 US clinical sites. Using this reference standard to establish infected specimen status, the sensitivity, specificity, and overall agreement of the AMG IVD assay was 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.