Use of TaqMan 5' nuclease real-time PCR for quantitative detection of Mycoplasma genitalium DNA in males with and without urethritis who were attendees at a sexually transmitted disease clinic

Abstract

Mycoplasma genitalium is a cause of nongonococcal urethritis, particularly in patients not infected with Chlamydia trachomatis. A quantitative 5' nuclease assay (TaqMan PCR) was developed and validated. The assay detected a fragment of the MgPa adhesin gene by use of a TaqMan MGB (minor groove binder) probe and included an internal processing control to detect PCR inhibition. Urethral swab specimens and first-void urine samples from M. genitalium-positive men were examined, and the M. genitalium DNA load was correlated to symptoms and signs. The assay consistently detected <5 genome copies without cross-reactions with other mycoplasmas. Urine and urethral swab specimens from men with urethritis had higher M. genitalium DNA loads than specimens from men without urethritis. However, a very broad overlap of DNA loads between patients with and without urethritis was observed. Urethral swab specimens from patients with urethral discharge had a significantly higher DNA load than specimens from patients without discharge. This correlation was not found in first-void urine specimens.

FIG. 1.

Flowchart illustrating selection and elimination of FVU specimens from men attending an STD clinic. A random sample of 100 FVU specimens found to be positive by the conventional PCR for detection of the M. genitalium 16S rRNA gene were blindly tested by the TaqMan quantitative PCR. After the code regarding the identities of the patients and recent antibiotic treatment was broken, specimens were excluded from the subsequent analysis.

FIG. 2.

Alignment of MgPa sequences from positions 1271 to 1501 of the MgPa operon sequence (GenBank accession number M31431) from cultured M. genitalium strains and PCR products from 30 specimens from 28 patients. The sequences of the primers used in the TaqMan assay are underlined, and the sequence of the TaqMan MGB probe is shaded.

FIG. 3.

M. genitalium DNA load in FVU specimens from patients without urethritis (<5 PMNLs/hpf), low-grade NCNGU (5 to 10 PMNLs/hpf), and high-grade NCNGU (>10 PMNLs/hpf) and patients with a concomitant C. trachomatis infection (C.tr. pos). The median DNA load for each group is marked by a horizontal line.

FIG. 4.

M. genitalium DNA loads in urethral swab specimens from patients without urethritis (<5 PMNLs/hpf), low-grade NCNGU (5 to 10 PMNLs/hpf), and high-grade NCNGU (>10 PMNLs/hpf) and patients with a concomitant C. trachomatis infection (C.tr. pos). Specimens without detectable M. genitalium DNA are marked as not detectable (ND). The median DNA load for each group marked by a horizontal line.

FIG. 5.

M. genitalium DNA loads in 71 FVU specimens from men who attended the STD clinic for the first time during the study and who were grouped by the intensity of the 16S rRNA gene amplicon detected in an ethidium bromide-stained agarose gel. Column 1, a faint band; column 2, a clear band but the IPC is still visible; column 3, a strong band with no IPC visible. Data were recorded during the screening of the specimens. The median DNA load for each group is marked by a horizontal line.