The bacterial microbiota in first-void urine from men with and without idiopathic urethritis.

BACKGROUND: Non-gonococcal urethritis (NGU) is a common syndrome in men. NGU may have several causes, but many cases are caused by sexually transmitted infections that may also cause complications in their female partners. Chlamydia trachomatis and Mycoplasma genitalium are the most common causes of NGU, but in up to 35% of the cases, none of the known viral or bacterial causes are found. Traditionally, pathogens have been detected using various culture techniques that may not identify all species present in the urethra. To address this, we used culture-independent methods for analysis of the male urethral microbiota. METHODS: This case-control study analysed first void urine samples, collected at STD clinics in Stockholm, Sweden from men with idiopathic urethritis (IU), i.e. negative for Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Ureaplasma urealyticum, Trichomonas vaginalis, adenovirus, and herpes simplex virus type 1 and -2 together with samples from men without urethritis. Forty-six controls and 39 idiopathic urethritis patients were analysed. RESULTS: The microbiota was highly diverse: None of the 302 operational taxonomic units (OTUs) found in negative controls and IU patients were found in all of the samples or even in all of the samples in one group. More than 50% of the OTUs were only found in one or two of the total of 85 samples. Still the most dominant 1/6 of the genera constituted 79% of the sequences. Hierarchical clustering in a heatmap showed no specific clustering of patients or controls. A number of IU patient samples were dominated by a single genus previously related to urethritis (Gardnerella, Haemophilus, Ureaplasma). CONCLUSION: The male urethra contain a very diverse composition of bacteria, even in healthy controls. NGU may be caused by a number of different bacteria but more studies including a higher number of samples are needed for elucidation of the role of each species.

Urethritis-associated Pathogens in Urine from Men with Non-gonococcal Urethritis: A Case-control Study.

The aetiology of non-gonococcal urethritis (NGU) remains unexplained in 30-40% of patients. Urine samples from men attending Swedish sexually transmitted disease clinics were examined by species-specific quantitative PCRs for Chlamydia trachomatis, Mycoplasma genitalium, Trichomonas vaginalis, Ureaplasma urealyticum, U. parvum, adenovirus, herpes simplex virus, Neisseria meningitidis, Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae. A total of 187 men with acute NGU (symptoms ≤ 30 days) and 24 with chronic NGU (symptoms < 30 days) were cases, and 73 men without NGU were controls. Number of lifetime sexual partners was negatively associated with U. urealyticum bacterial load. C. trachomatis and M. genitalium were associated with NGU, as was U. urealyticum, with bacterial loads ≥ 1.3 × 103 genome equivalents/ml urine. Virus and H. influenzae might explain a few NGU cases, but the aetiology in at least 24% of patients with acute NGU was unexplained. In multivariate analysis, detection of U. urealyticum was significantly more common in acute NGU (20%) compared with controls (11%).

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.

High occurrence of a new variant of Chlamydia trachomatis escaping diagnostic tests among STI clinic patients in Stockholm, Sweden.

BACKGROUND: In 2006, a genetic variant of Chlamydia trachomatis not detectable with the most commonly used diagnostic tests was identified. Initial reports suggested that as many as 10% to 13% of all chlamydia cases would have remained undiagnosed. The aim of the study was to find the occurrence and clinical findings of this genetic variant among a high-risk population in Stockholm, Sweden. METHODS: Samples were analyzed using the Cobas TaqMan CT test (Roche Diagnostics). To detect the new variant, an additional PCR-analysis, artus C. trachomatis LC MOMP PCR Kit (Qiagen) was performed on all negative samples. Positive results in the artus test were confirmed by a mutant specific PCR. Clinical data were retrospectively collected from medical records. RESULTS: Among 1009 samples analyzed, 115 were positive for C. trachomatis and among those, 27 were found to belong to the genetic altered strain. This variant constituted 23% of all chlamydia cases diagnosed, and 29% were found in the age group 20 to 29 years. Women with the new variant were younger and had more often performed another chlamydia test within the previous 6 months compared with those infected with the wild type. CONCLUSION: These results indicate that a large number of sexually active individuals might be infected despite a negative chlamydia test, thus facilitating a rapid transmission of the new variant. Accordingly, it is of great importance to be aware of limitations of the diagnostic methods used.

Sequence-based typing of Mycoplasma genitalium reveals sexual transmission.

Mycoplasma genitalium causes male nonchlamydial, nongonococcal urethritis and is associated with cervicitis and pelvic inflammatory disease in women. Epidemiological studies indicate that M. genitalium is sexually transmitted, and the aim of the present study was to further substantiate this by means of a DNA typing system. A typing assay based on a diagnostic mgpB gene PCR was developed, evaluated, and applied directly to urogenital specimens. The assay had a low limit of detection and hence a high typeability. Sequences of isolates from 52 unrelated patients were divided into 29 different sequence types, giving a discriminatory index of 0.95. Two to six M. genitalium-positive specimens were collected from each of 44 patients over a median interval of 56 days (range, 11 to 1,395). Forty had the same sequence type in consecutive specimens. Specimens collected from two men were repeatedly positive at intervals of 472 and 1,395 days, respectively, but the sequence types had changed. A new strain was introduced in one sexual dyad, and the sequence types changed subsequently. Seventy-nine M. genitalium-positive specimens from 19 couples were investigated, and all partners initially had concordant sequence types, but one couple had discordant types at one time point before a newly introduced strain took over. The present typing system is simple and reproducible and has an excellent discriminatory capacity which might prove useful in studies of sexual networks and for evaluation of treatment failures. In the laboratory, this system may document the uniqueness of newly isolated M. genitalium strains.

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/microl, respectively [P = 0.0005]). This assay may prove useful in the monitoring of treatment and for optimizing sample preparation methods.

Conjunctivitis associated with Mycoplasma genitalium infection.

Conjunctival and first-void urine specimens from a patient with unilateral conjunctivitis and urethritis were found to be positive for Mycoplasma genitalium but negative for Chlamydia trachomatis, by polymerase chain reaction analysis. Identical M. genitalium DNA sequences were found in both types of specimens.

Comparison of first void urine and urogenital swab specimens for detection of Mycoplasma genitalium and Chlamydia trachomatis by polymerase chain reaction in patients attending a sexually transmitted disease clinic.

OBJECTIVE: The objective of this study was to compare urogenital swab specimens and first void urine (FVU) specimens from male and female patients at a sexually transmitted disease clinic for the detection of Mycoplasma genitalium and Chlamydia trachomatis infections using in-house, inhibitor-controlled polymerase chain reaction (PCR). STUDY DESIGN: Urethral swabs and FVU were collected from 1856 men and 753 women who also had a cervical swab collected. A positive diagnosis of infection was made if any 1 of the specimens tested positive and were confirmed in a second PCR assay targeting independent genes. RESULTS: M. genitalium DNA and C. trachomatis DNA were detected in 126 (6.8%) and 246 (13.3%) of the male sample sets and in 51 (6.8%) and 73 (9.7%) of the female specimen sets, respectively. Using our in-house PCR and sample preparation methods, FVU was found to be the most sensitive diagnostic specimen for both pathogens, but for optimal sensitivity, it should be supplemented with a cervical specimen in women. In a small subset of female FVUs, storage at -20 degrees C led to false-negative M. genitalium PCR results in 27% of specimens found positive when a sample preparation was performed before freezing. The age-specific prevalence of M. genitalium in men was almost constant between 18 and 45 years of age in contrast to C. trachomatis infections, which were more common in younger men. CONCLUSION: Urine appeared to be a better diagnostic specimen than the urethral swab for M. genitalium and C. trachomatis detection by PCR in this cohort of sexually transmitted disease clinic attendees but should be supplemented with a cervical specimen in women.

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.

Imiquimod to treat different cancers of the epidermis.

BACKGROUND: Topical immunomodulatory therapy with imiquimod has been recently used for the treatment of actinic keratoses, intraepithelial carcinoma, and small basal cell carcinoma (BCC) besides the licensed indication of extragenital warts (condyloma). METHODS: We treated several patients with particular epidermal neoplasias such as squamous cell cancer (SCC) and basal cell cancer of sclerodermiform type three times per week for 4 to 12 weeks. RESULTS: We report several novel aspects of the treatment of epidermal cancers with self-applied, nonpainful, immunomodulatory therapy. First, we treated-for the first time-two immunosuppressed renal transplant patients for invasive SCC with imiquimod. Interestingly, systemic immunosuppression did not adversely affect the response to therapy. Second, one patient with the high-risk and aggressive growth pattern of basal cell cancer (sclerodermiform histology) was cured from his disease at a particular location in the face, suggesting sufficient penetration despite scarring. No recurrence was detected in another patient who suffered from 29 BCCs until almost 2-years follow-up. Third, the treatment of actinic keratoses in the face is substantially shorter (in the order of 4 to 6 weeks) as opposed to other skin cancers. Immunomodulatory treatment with imiquimod led to the demarcation of in situ actinic keratosis lesions that could not be identified using the dermatologist's experience, probably because of the existence of exclusive alterations on the molecular level. CONCLUSION: Several novel aspects of immunomodulatory treatment with imiquimod and new indications such as selected cases of sclerodermiform BCC and SCC have been described. The texture of the skin at various different body locations may explain the varying sensitivities to imiquimod when facial skin is compared with skin on the extremities.