Development of a Rapid and High-Throughput Multiplex Real-Time PCR Assay for Mycoplasma hominis and Ureaplasma Species

Abstract

Bacterial commensals of the human genitourinary tract, Mycoplasma hominis and Ureaplasma species (parvum and urealyticum) can be sexually transmitted, with the potential to cause non-gonococcal urethritis, pelvic inflammatory disease, and infertility. M. hominis and Ureaplasma spp. may also cause severe invasive infections in immunocompromised patients. Current culture-based methods for Mycoplasma/Ureaplasma identification are costly and laborious, with a turnaround time between 1-2 weeks. We developed a high-throughput, real-time multiplex PCR assay for the rapid detection of M. hominis and Ureaplasma spp. in urine, genital swab, body fluid, and tissue. In total, 282 specimens were tested by PCR and compared with historical culture results; a molecular reference method was used to moderate discrepancies. Overall result agreement was 99% for M. hominis (97% positive percent agreement (PPA), 100% negative percent agreement (NPA)) and 96% for Ureaplasma spp. (96% PPA, 97% NPA). Specimen stability was validated for up to 7 days at room temperature. This multiplex molecular assay was designed for implementation in a high-complexity clinical microbiology lab. With this method, >90 samples can be tested in one run, with a turnaround time of 4-5 hours from specimen extraction to reporting of results. This PCR test is also more labor- and cost-effective than the conventional culture-based test, thus improving laboratory efficiency and alleviating strains due to labor shortages.