What is the performance of a quantitative, real-time PCR assay for the detection of M. genitalium when used to test known mycoplasma isolates and specimens positive by a conventional 16S rRNA PCR?

A quantitative, M. genitalium real-time PCR assay, which amplified and detected a conserved fragment of the MgPa adhesin gene and included an internal processing control, was developed and validated using urethral swabs and first-void urine samples from M. genitalium positive men. The DNA load was correlated to symptoms and signs.

One hundred first-void urine specimens, collected from men participating in an ongoing study of the clinical and epidemiological aspects of M. genitalium infections at an STD clinic in Sweden, were randomly selected from among those found to be M. genitalium positive by a conventional 16S rRNA gene PCR assay and were used in this study. A matching urethral swab was available for 93 of the 100 urine specimens, of which 13 were M. genitalium negative by 16S rRNA PCR. Twenty-nine men provided more than one urine specimen. Nine men had received antibiotics within the previous 6 months. Among the 62 men included in the study, 56 provided urethral swab specimens and 8 of these were M. genitalium negative by 16S rRNA PCR. The median age of the 62 men was 27 years (range = 18 to 52 years), 14 men did not have urethritis, 9 men were also positive for C. trachomatis. Two hundred randomly selected urogenital specimens from patients found to be negative by 16S rRNA PCR were also tested. 

Smears were prepared from urethral discharge, which was obtained with a plastic loop, stained with methylene blue, and assessed for the number of polymorphonuclear leukocytes per high power microscopic field (PMN/hpf). Patients with smears showing >5 PMN/hpf were considered positive for urethritis. A urethral swab sample, placed into 1.8 ml of SP4 mycoplasma broth medium, and 15 to 20 ml of first-void urine were collected for M. genitalium and C. trachomatis PCRs.  After specimen processing in Chelex 100 resin, approximately 2 μl of the original swab specimen and 36 μl of the urine specimen were added to the M. genitalium PCR assays. The real-time PCR assay primers amplified a 78 bp fragment of the M. genitalium MgPa adhesin gene. The probe was labeled with 6FAM and a minor groove binder and nonfluorescent quencher. An internal process control to detect PCR inhibition was constructed that contained the binding sites of the MgPa primers and internal sequences of the phage lambda genome. Amplification of the control with the MgPa primers produced a 204 bp fragment that was detected using a TAMRA labeled probe. For quantification, standard curves were produced by analyzing 10-fold dilutions of M. genitalium DNA containing 5 to 500,000 genome equivalents. All tests were performed in duplicate.

The specificity of the real-time PCR assay was evaluated by testing DNA extracted from cultures of 15 M. genitalium strains and at least one strain of 19 other Mollicutes and DNA from 200 randomly selected urogenital specimens from patients found to be M. genitalium negative by 16S rRNA PCR. To assess reproducibility, two preparations each of 12 urine and 10 urethral swab specimens were tested in six replicate reactions. To evaluate the applicability of the MgPa real-time PCR primers for amplification of all clinical M. genitalium strains, primers MgPa1 and MgPa3 were used to amplify and sequence the DNA from 42 M. genitalium strains.  

The sensitivity and specificity of the M. genitalium real-time PCR assay was evaluated, and the results compared to those of a conventional 16S rRNA PCR.  

None of the mycoplasma and related species tested, other than M. genitalium, were positive by the real-time PCR assay. The standard curve was linear over a range of 5 log units. The assay was consistently capable of detecting less than 5 genome equivalents (geq) of M. genitalium, corresponding to 6,300 geq/swab specimen and 140 geq/ml of urine. Among the 200 M. genitalium-negative specimens tested, none were inhibited or failed to amplify the internal control. Less than a twofold difference in the M. genitalium DNA load between the mean value for two preparations of urine and urethral swab specimens was found for all specimens containing a mean of >5 geq. Sequencing of the M. genitalium adhesin gene region in which the real-time PCR primers and probe bound indicated that 10 amplicons produced from samples from 9 of 28 patients carried one or two mutations in the forward primer region. For all of the mutation types, the detection limit by the real-time assay was the same or fivefold lower than by the 16S rRNA gene PCR assay.  

All of the 100 urine samples that were M. genitalium positive by a conventional 16S rRNA gene PCR assay were also positive by the real-time assay. Two urethral swabs that were negative by the16S rRNA gene PCR assay were positive by real-time PCR, and one urethral swab specimen positive by 16S rRNA PCR was negative by the real-time PCR. Only 3 geq of M. genitalium was detected in the urine specimen from this patient, and the result of repeat testing by 16S rRNA PCR was negative. The significant associations between M. genitalium DNA load in urine or urethral specimens and clinical signs and symptoms are shown in the table.

Signs and symptoms associated with M. genitalium DNA load in urine and urethral swab samples from M. genitalium positive, C. trachomatis negative men

*5 to 10 PMN/hpf

^>10 PMN/hpf

Sensitivity, specificity, and reproducibility of the quantitative, M. genitalium real-time PCR assay was excellent. There was a significant difference in the M. genitalium DNA load in both urine and urethral swab specimens from patients without urethritis and those from patients with NCNGU. A strong association was observed between a high M. genitalium DNA load and the presence of a discharge for the urethral swab specimens only.  

Source of funding: None given

For correspondence: Jorgen Skov Jensen, Mycoplasma Laboratory, Statens Seruminstitut, Artillerivej 5, DK-2300 Copenhagen S, Denmark. E-mail address: jsj@ssi.dk.