Development and Performance of a Microwell-Plate-Based Polymerase Chain Reaction Assay for Mycoplasma genitalium

In Brief Background Mycoplasma genitalium is associated with, and could be the cause of, idiopathic cases of urethritis, endometritis, and cervicitis. Further epidemiologic studies on this organism are needed, but currently used polymerase chain reaction (PCR) assays are labor-intensive and culture is insensitive. Goal The goal was to develop and evaluate a microwell-plate-based PCR assay for M. genitalium. Study Design We adapted an M. genitalium PCR assay targeting the MgPa gene to a 96-microwell plate format with colorimetric detection of PCR products and incorporation of an internal inhibition control to determine the limit of detection of this assay (termed MgPa-IMW) for M. genitalium DNA and evaluate its performance on cervical and male urine specimens. Results The MgPa-IMW PCR assay detected 1 and 17 genome copies of M. genitalium (with 27% and 95% confidence) and was able to detect specimens inhibited for amplification. This assay was 100% concordant (50 positive and 50 negative) with the Southern-blot-based PCR assay with cervical specimens. Similarly, this test was 89% concordant with the Southern-blot-based assay for 64 male urine specimens (25 positive, 32 negative, 7 discordant), 97% concordant after correcting for specimens no longer positive by the Southern blot-based assay after freezer storage. Conclusion The MgPa-IMW assay is sensitive and specific for the detection of M. genitalium in patient specimens and should facilitate large-scale screening for this organism. An M. genitalium PCR assay incorporating an internal inhibition control and using 96-well microtiter plate EIA assay to score positive and negative tests has been developed and evaluated on cervical and male urine specimens. This test should facilitate high-throughput screening of patient specimens for this organism.