Comparison between culture and a multiplex quantitative real-time polymerase chain reaction assay detecting Ureaplasma urealyticum and U. parvum.

A novel multiplex quantitative real-time polymerase chain reaction (qPCR) for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR

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

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

Development of a Quantitative Real-Time PCR Assay for Detection of Mycoplasma genitalium

Mycoplasma genitalium is known to cause nonchlamydial, nongonococcal urethritis in men and to be associated with pelvic inflammatory disease in women. Specific and sensitive PCR methods are needed for diagnosis of this bacterium because it is very difficult to culture from patient samples. To determine the bacterial load in patients' specimens, a quantitative real-time LightCycler PCR was developed. The housekeeping gene gap encoding glyceraldehyde-3-phosphate dehydrogenase was chosen as the target gene. The assay could consistently detect five genome copies per reaction. To evaluate the PCR, we tested 246 selected urethral swab samples from men attending a clinic for sexually transmitted diseases. Eighty-two of the samples were found positive for M. genitalium by a conventional 16S rRNA gene PCR assay, whereas 164 samples were randomly chosen among those tested negative. Of the positive samples, 78 (95.1%) were found positive, whereas 6 (3.7%) of the negatives were found positive by the LightCycler assay. The patient samples were also tested with a quantitative TaqMan assay, and the bacterial load was compared to the LightCycler results. A good linear correlation between the LightCycler and the TaqMan assays was found with a correlation coefficient of 0.89 and a slope of 0.99. Significantly more M. genitalium-positive men had urethritis, discharge, and dysuria than had M. genitalium-negative men. The M. genitalium DNA load in samples from patients with urethritis was significantly higher than in samples from those without (61 and 2.9 copies/μl, respectively [P = 0.0005]). This assay may prove useful in the monitoring of treatment and for optimizing sample preparation methods

Detection of ureaplasmas in 126 male urethral specimens by culture and qPCR.

<p>Detection of ureaplasmas in 126 male urethral specimens by culture and qPCR.</p

Flow-chart showing the distribution of samples included in the study.

<p>* Four had a matching urethral- or cervical swab sample from the same woman which was also overgrown. One excluded cervical swab sample was not overgrown in the matching urethral swab. <b>**</b> Fifty-five had a paired urethral- or cervical swab sample from the same woman.</p

Comparison of <i>U. urealyticum</i> and <i>U. parvum</i> DNA load by quantitative real-time PCR and culture.

<p>◊ =  female cervical swabs. ♦ =  female urethral swab. * =  male urethral swab. A total of 111 samples negative by both culture and qPCR, and one sample, which was culture positive in CCU group 4 but not re-detected with PCR, are not shown, as the species could not be determined. Co-infected specimens (5 samples) are not shown. Medians are depicted by a horizontal bar.</p

Distribution of <i>U. urealyticum</i> and <i>U. parvum</i> by qPCR in culture positive male and female urethral samples and cervical samples.

<p>M =  man.</p><p>W =  woman.</p><p>* =  Infection with both <i>U. parvum</i> and <i>U. urealyticum</i>.</p

Distribution of ureaplasma positive and negative samples in 60 paired urethral and cervical swabs collected from the same woman as determined by qPCR.

<p><b>*</b>No difference in the distribution (p = 0.25 McNemar's test).</p