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

June L Chan; Stacey Cerón; Stephanie M Horiuchi; Jewell P Yap; Erika G Chihuahua; Allison T Tsan; Edwin Kamau; Shangxin Yang

doi:10.1016/j.jmoldx.2023.07.004

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

J Mol Diagn. 2023 Sep 7:S1525-1578(23)00195-2. doi: 10.1016/j.jmoldx.2023.07.004. Online ahead of print.

Authors

June L Chan¹, Stacey Cerón¹, Stephanie M Horiuchi¹, Jewell P Yap¹, Erika G Chihuahua¹, Allison T Tsan¹, Edwin Kamau¹, Shangxin Yang²

Affiliations

¹ Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California.
² Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California. Electronic address: shangxinyang@mednet.ucla.edu.

PMID: 37683891
DOI: 10.1016/j.jmoldx.2023.07.004

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 nongonococcal urethritis, pelvic inflammatory disease, and infertility. Mycoplasma hominis and Ureaplasma species 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 and 2 weeks. We developed a high-throughput, real-time multiplex PCR assay for the rapid detection of M. hominis and Ureaplasma species in urine, genital swab, body fluid, and tissue. In total, 282 specimens were tested by PCR and compared with historic culture results; a molecular reference method was used to moderate discrepancies. Overall result agreement was 99% for M. hominis (97% positive percentage agreement and 100% negative percentage agreement) and 96% for Ureaplasma species (96% positive percentage agreement and 97% negative percentage agreement). 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 laboratory. With this method, >90 samples can be tested in one run, with a turnaround time of 4 to 5 hours from specimen extraction to reporting of results. This PCR test is also more labor effective and cost-effective than the conventional culture-based test, thus improving laboratory efficiency and alleviating strains because of labor shortages.