French Prospective Clinical Evaluation of the Aptima Mycoplasma genitalium CE-IVD Assay and Macrolide Resistance Detection Using Three Distinct Assays

Abstract

The aim of this study was to evaluate the clinical performance of the Aptima Mycoplasma genitalium transcription-mediated amplification (MG-TMA) CE-marked for in vitro diagnosis (CE-IVD) assay for the detection of Mycoplasma genitalium in male and female clinical samples in comparison with the in-house real-time PCR (in-house PCR) assay routinely used in our laboratory. A total of 1,431 clinical specimens obtained from 1,235 patients were prospectively collected at the Bacteriology Department of Bordeaux University Hospital (France). Additional research-use-only Aptima M. genitalium transcription-mediated amplification (TMA) assays, Alt1-TMA and Alt2-TMA, were performed on discordant specimens to determine M. genitalium infection status. All confirmed M. genitalium-positive specimens were tested for macrolide resistance using three assays: the in-house 23S rRNA FRET PCR assay, the SpeeDx ResistancePlus MG assay and the nested reverse transcription-PCR (RT-PCR) sequencing assay. The comparison of the MG-TMA assay with the in-house PCR results showed a moderate correlation (kappa value, 0.69). The MG-TMA assay had higher clinical sensitivity compared to that of the in-house PCR assay (100% versus 59.74%, respectively) and similar specificity (99.10% versus 100%, respectively) for M. genitalium detection. In this study, the prevalence of M. genitalium infection was 5.90% (72/1,220 patients). The nested RT-PCR sequencing assay was the most sensitive but the most laborious assay for detecting macrolide-resistance-associated mutations. The prevalence of resistance was 8.33% (6/72). To our knowledge, this is the first clinical evaluation of the MG-TMA CE-IVD assay. The MG-TMA assay performed on the automated Panther system is a very sensitive and specific method for the detection of M. genitalium in clinical specimens.

Evaluation of the Mycoplasma genitalium Resistance Plus kit for the detection of M. genitalium and mutations associated with macrolide resistance

“…Overall, the results reported in this evaluation were in agreement with previously reported sensitivity and specificity rates for this assay10; however, we found the assay to be more sensitive for macrolide-genotype detection than Le Roy et al (2017) 11…”

Design and Validation of Transcription-Mediated-Amplification Nucleic Acid Amplification Tests for Mycoplasma genitalium

“…In this study, we validated the performance characteristics of four TMA NAAT assays for the detection of ribosomal RNAs from M. genitalium. We found that these assays are sensitive and specific for the detection of multiple stains of M. genitalium and have Similarly to NAATs designed to detect other sexually transmitted infections such as Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis, the detection of rRNA of M. genitalium affords a particularly sensitive method for detecting the organism in vivo, leading to improved clinical diagnosis of M. genitalium infection compared to that with NAATs that target genomic DNA of the organism (20)(21)(22). This difference in relative test clinical sensitivity may be due to the disparity between genomic DNA and rRNA content in the cell (i.e., a single DNA genome versus an estimated hundreds to thousands of rRNA copies per cell) (29), enabling the detection of low titers of M. genitalium in patient specimens (30,31).…”

“…This is the first study to use only rRNA-specific NAATs for the components of a composite comparator reference standard for the evaluation of an rRNA-based molecular test for M. genitalium detection. Previous studies investigating the clinical perfor- mance of rRNA-based NAAT assays for M. genitalium used a reference standard consisting of a combination of tests that detected rRNA and genomic DNA of M. genitalium (20,21). This mixed reference standard approach has the potential to introduce bias into the analyses, since the lower sensitivity of DNA-based tests can decrease the rate of DNA-positive/RNA-positive consensus results obtained in the analysis, potentially resulting in artificially lowered specificity estimates for the RNA-based diagnostic test under evaluation.…”

“…Finally, we did not compare test clinical performance to an absolute reference standard such as PCR Sanger sequencing for the identification of M. genitalium infection. This concern is mitigated by previously published data showing the AMG IVD assay has 100% agreement with a validated reverse transcription-PCR/Sanger sequencing assay for M. genitalium 23S rRNA (19,21).…”

“…The TMA-based Aptima Mycoplasma genitalium assay (AMG) is an FDA-cleared (510k number DEN180047) in vitro diagnostic (IVD) test for the detection of 16S rRNA from M. genitalium. Application of the AMG IVD assay outside the United States has been described (20)(21)(22). In the present study, we developed and optimized three additional alternate (Alt) TMA assays, Alt TMA-1, Alt TMA-2, and Alt TMA-3, to capture, amplify, and detect unique regions of 16S rRNA (Alt TMA-1) or 23S rRNA (Alt TMA-2 and Alt TMA-3) of M. genitalium.…”