Clinical aspects of Mycoplasma genitalium infection

The microorganism Mycoplasma (M.) genitalium was first isolated by culture in 1980. Further attempts to culture this mycoplasma were difficult because of its slow growing nature. The development of polymerase chain reaction methods has made it possible to detect M. genitalium more readily but yet there are no commercial tests available. M. genitalium is sexually transmitted and is an independent cause of non-gonococcal urethritis in men and most likely of cervicitis in women. There is accumulating evidence that M. genitalium might play a role as an important agent causing upper genital tract infections in women with possible sequelae like those found after Chlamydia (C.) trachomatis infections. In this thesis various clinical aspects of M. genitalium infection have been elucidated in patients attending our clinic for sexually transmitted infections (STI-clinic). Paper I: M. genitalium among male patients was found to be associated with nonchlamydial non-gonoccocal urethritis. M. genitalium positive patients also more often had a history of recurrent urethritis compared to controls. Paper II: First void urine (FVU) specimens were found superior to urethral swab specimens in male patients to detect M. genitalium; giving a relative sensitivity of 98 % with the first test regime compared to 89 % with the latter. Among female patients the highest sensitivity was accomplished when a FVU specimen was supplemented with a cervical swab specimen. Paper III: A patient with a unilateral conjunctivitis and a concomitant urethritis was found to be infected with M. genitalium. Identical DNA sequences were found in the eye and the genital tract. After antibiotic treatment the symptoms cleared and tests of cure became negative for M. genitalium at both sites. This is the first publication of M. genitalium associated conjunctivitis. Paper IV: Antibiotic treatment of M. genitalium was studied. Treatment with doxycycline was found much less effective as compared to azithromycin in both men and women. The highest clearance rate was achieved when azithromycin was given in a dose of 500 mg day 1 followed by 250 mg days 2-5. This regimen is today recognized as the treatment of choice for M. genitalium infections. The clinical findings after treatment were poor predictors of successful outcome. Paper V: The clinical findings in women infected with M. genitalium as compared to women with chlamydial infection or women uninfected with these organisms were studied. There were only minor differences in the clinical appearance between these groups regarding symptoms and signs of lower genital tract infection. A substantial number of both M. genitalium and C. trachomatis positive women were symptomless. Thus, in order to detect Mycoplasma genitalium infections in women, as is the case with C. trachomatis infections, tests should be used on wide indications. Conclusions: We recommend a generous testing of men and women for M. genitalium regardless of signs and symptoms - if tests are available! We recommend azithromycin orally 500 mg day 1 followed by 250 mg daily for 4 days as the first line treatment of M. genitalium infection in Sweden

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