Real-time PCR detection of the mg219 gene of unknown function of Mycoplasma genitalium in men with and without non-gonococcal urethritis and their female partners in England

Abstract

Real-time PCR was employed to detect a region of the Mycoplasma genitalium mg219 gene, a gene of unknown function, in clinical samples. Amplification of DNA and signal production from 15 other species of human mycoplasmas and 14 other bacteria and viruses did not occur. Using a panel of 208 genital and rectal samples, the sensitivity when compared to the modified mgpa gene (encoding the major surface protein MgPa) real-time PCR assay was found to be 100 % and the specificity of the assay 99.5 % with a positive predictive value of 80 % and a negative predictive value of 100 %. The mg219 gene was found to be in all strains of M. genitalium and was highly conserved. M. genitalium was detected in 3.9 % (11/280, 95 % CI 2.1-6.9) of all male specimens, in 7.7 % (10/130, 95 % CI 4.1-13.7) of patients with non-gonococcal urethritis (NGU) and in 0.7 % (1/150, 95 % CI ,0.01-4.1) of patients without urethritis. The presence of M. genitalium was significantly associated with NGU (P ¡0.01; 95 % Cl 0.88-0.98) and nonchlamydial-non-gonococcal urethritis (P50.0005; 95 % Cl 0.84-0.97). INTRODUCTIONMycoplasma genitalium is a sexually transmitted bacterium that is primarily found in the human urogenital tract. It has also been detected in respiratory (Baseman et al., 1988), rectal (Taylor-Robinson et al., 2003) and synovial (Tully et al., 1995) specimens. Serological detection of M. genitalium is problematic due to cross-reactions with Mycoplasma pneumoniae antibodies (Clausen et al., 2001;Lind et al., 1984). Isolation via cell culture is slow and laborious and real-time PCR is currently used in the diagnosis of infected patients (Jensen, 2006). Originally isolated from men with urethritis (Tully et al., 1981), M. genitalium has been convincingly linked to non-gonococcal urethritis (NGU) and nonchlamydial-non-gonococcal urethritis (NCNGU) (Jensen, 2006). However, the full extent of M. genitalium infection, epidemiology, and role in cervicitis, pelvic inflammatory disease, infertility and other infections requires investigation (Jensen, 2006). M. genitalium is sexually transmitted and a high prevalence has been found (58 %) in individuals with infected sexual partners (Keane et al., 2000). Sequence-based typing has identified identical isolate sequence types from sexual partners, cementing evidence for sexual transmission (Hjorth et al., 2006;Ma et al., 2008).Several real-time PCR assays have been described for the molecular detection of M. genitalium, including those targeting mgpa, the 16S rRNA gene and p115 (for review see Jensen, 2006). The mgpa gene real-time assay has been shown to be of increased sensitivity to the 16S rRNA method (Edberg et al., 2008). We sought to identify a new method for confirmation of PCR results with equal sensitivity to that of the mgpa gene assay. The mg219 gene assay was developed and used to detect the presence of M. genitalium in parallel with the mgpa gene real-time PCR assay in 280 male genitourinary medicine clinic attendees and 23 female partners as part of a case-control doubleblinded study ...

A cross-sectional study ofMycoplasma genitaliuminfection and correlates in women undergoing population-based screening or clinic-based testing forChlamydiainfection in London

“…We introduced a degenerate oligonucleotide (‘wobble’) in the forward primer to account for a frequent detected base substitution that has previously been shown to be successful in another study by Chalker et al 26. As an internal control for PCR inhibition we used murine cytomegalovirus (mCMV) and primers mCMVTAQ1 (forward primer) and mCMVTAQ2 (reverse primer) and mCMVTAQPR probe labelled with JOE (Primers and probe were provided by Eurofins MWG Operon) designed by Garson et al 27.…”

The prevalence of urethral and rectal Mycoplasma genitalium and its associations in men who have sex with men attending a genitourinary medicine clinic

“…Samples were stored at 4°C before testing and all specimens were processed within 6 weeks of collection. MG DNA was detected using a previously published real-time PCR method that targets the MgPa adhesin gene, with a minor modification to the forward primer 7 8. All MG-positive specimens were also examined using a confirmatory real-time PCR that targets the Mg219 gene 8.…”

Gardnerella vaginalis and Mollicute detection in rectal swabs from men who have sex with men

“…Quantification of bacterial loads by real-time quantitative polymerase chain reaction (qPCR) is a useful tool for recording whether synergistic growth between genital Mollicutes and G. vaginalis takes place in male rectal mucosa. Residual anonymous rectal swabs from MSM attending a local Genitourinary Medicine clinic were retained under an approved ethics Bio Archive (REC reference 10/NIR01/20) and tested against five qPCR assays targeting genes from: G. vaginalis 15 ; M. hominis 16 ; M. genitalium 17 ; U. urealyticum and U. parvum . 18 Bacterial prevalence and loads were assessed for evidence of synergistic growth patterns between the respective bacteria.…”