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Nongonococcal urethritis (NGU) is a common genital tract syndrome in men, and up to 50% of cases are considered idiopathic, i.e., no etiological agent is identified. This poses challenges for clinicians in the diagnosis and treatment of NGU and often results in antibiotic misuse and overuse. Therefore, to identify potential infectious causes of urethritis and inform clinical management of urethritis cases, we characterized and compared the urethral microbiota of men with and without idiopathic urethritis. Participants were derived from a case-control study that examined viral and bacterial pathogens and sexual practices associated with NGU. Men with NGU who tested negative for established causes of NGU (Chlamydia trachomatis, Mycoplasma genitalium, Trichomonas vaginalis, adenoviruses, herpes simplex virus [HSV]-1, and/or HSV-2) were classified as idiopathic cases, and the controls were men reporting no current urethral symptoms. Men provided a urine sample that was used to characterize the urethral microbiota using 16S rRNA gene sequencing. Bacterial taxa associated with idiopathic urethritis were identified using analysis of compositions of microbiomes with bias correction. When stratified by sex of sexual partner, we found that the abundance of Haemophilus influenzae was significantly increased in men who have sex with men with idiopathic urethritis, and the abundance of Corynebacterium was significantly increased in men who have sex with women with idiopathic urethritis. Other taxa, including Ureaplasma, Staphylococcus haemolyticus, Streptococcus pyogenes, Escherichia, and Streptococcus pneumoniae

Neisseria meningitidis is a common commensal bacterium found in the respiratory tract, but it can also cause severe, invasive disease. Vaccines have been employed which have been successful in helping to prevent invasive disease caused by encapsulated N. meningitidis from the A, C, W, Y, and B serogroups. Currently, nonencapsulated N. meningitidis groups are more common commensals in the population than in the prevaccine era. One emerging nonencapsulated group of bacteria is the U.S. N. meningitidis urethritis clade (US_NmUC), which can cause meningococcal urethritis in men. US_NmUC has unique genotypic and phenotypic features that may increase its fitness in the male urethra. It is diagnostically challenging to identify and distinguish meningococcal urethritis from Neisseria gonorrhoeae, as the clinical presentation and microbiological findings are overlapping. In this review, the history of meningococcal urethritis, emergence of US_NmUC, laboratory diagnosis, and clinical treatment are all explored.

Mycoplasma genitalium is a common sexually transmitted infection associated with urethritis in men. Little is known about M. genitalium infection in men with urethritis in Uganda. We report that 12% of participants in this study were positive for M. genitalium and that resistance to azithromycin, a macrolide antibiotic, is present. Furthermore, we show that either self-collected penile-meatal swabs or urine can be used for detection of M. genitalium .

Mycoplasma genitalium is a sexually transmitted bacterium associated with nongonococcal urethritis (NGU) in men and cervicitis, endometritis, and pelvic inflammatory disease in women. Effective treatment is challenging due to the inherent, and increasingly acquired, antibiotic resistance in this pathogen. In our treatment trial conducted from 2007 to 2011 in Seattle, WA, we demonstrated poor efficacy of azithromycin (AZM) and doxycycline (DOX) against M. genitalium among men with NGU. In the present study, we cultured M. genitalium from 74 of 80 (92.5%) PCR-positive men at enrollment (V-1) and defined the MICs of AZM (N = 56 isolates) of DOX (N = 62 isolates). Susceptibility to AZM was bimodal; MICs were >8 μg/ml (44.6%) and 8 μg/ml (18/18 men) than those with isolates that had AZM MICs of 8 μg/ml (12/18 men) than with AZM MICs of

Neisseria meningitidis and Neisseria gonorrhoeae are pathogenic bacteria that can cause human infections. While N. meningitidis infections are associated with bacterial meningitis and bacteremia, a strain of N. meningitidis, isolated from the urogenital system, has recently been associated with urethritis. As this strain is becoming prominent as an emerging pathogen, it is essential to assess identification tools for N. meningitidis and N. gonorrhoeae urogenital isolates. Consecutive N. meningitidis isolates recovered from urogenital cultures of symptomatic patients with presumptive diagnoses of gonorrhea and a random selection of N. gonorrhoeae isolates recovered from the same population within the same time frame were characterized with routine identification systems, antimicrobial susceptibility testing, and whole-genome sequencing. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), multilocus sequence typing, 16S rRNA gene sequence, and average nucleotide identity methods accurately identified 95% (18/19) of N. meningitidis and N. gonorrhoeae isolates. With the Aptima Combo 2 CT/NG test, 30% (3/10) of N. meningitidis isolates were misidentified as N. gonorrhoeae, but no misidentifications were found with the Xpert CT/NG nucleic acid amplification test (NAAT). Phylogenetic core genome and single nucleotide polymorphism (SNP)-based grouping analyses showed that urogenital N. meningitidis isolates were highly related and phylogenetically distinct from N. gonorrhoeae and respiratory N. meningitidis isolates but similar to urogenital N. meningitidis isolates from patients with urethritis in the United States. Urogenital N. meningitidis isolates were predominantly azithromycin resistant, while N. gonorrhoeae isolates were azithromycin susceptible. These data indicate that urogenital isolates of N. meningitidis can cause false-positive detections with N. gonorrhoeae diagnostic assays. Misidentification of urogenital N. meningitidis isolates may confound public health-related activities for gonorrhea, and future studies are needed to understand the impact on clinical outcome of N. meningitidis urogenital infection.

The threat of ceftriaxone-resistant Neisseria gonorrhoeae necessitates new gonorrhea treatment regimens. Repurposing older antibiotics not routinely used for N. gonorrhoeae may expeditiously identify new therapies. Ideally, all recommended therapies should eradicate gonorrhea at the pharynx. Between April and September 2019, we enrolled men in an open-label, one-arm clinical trial of single-dose intramuscular aztreonam (2 g). Enrollment criterion included (i) nucleic acid amplification test (NAAT)-positive pharyngeal gonorrhea for ≤14 days or (ii) Gram stain-positive gonococcal urethritis plus report of performing oral sex in ≤2 months. At enrollment, we collected cultures from NAAT-positive or screening sites, and men returned 3 to 8 days following treatment for a test of cure (TOC) by culture. The per-protocol analysis required men to be culture positive at enrollment and to return for TOC. We calculated efficacy as the number of subjects with negative culture at TOC divided by the number culture positive at enrollment by anatomic site. Thirty-two men enrolled in the study; 21 were pharyngeal NAAT positive, and 11 had gonococcal urethritis. The per-protocol analysis included 17 men, 6 with pharyngeal, 9 with urethral, and 4 with rectal gonococcal infections. Aztreonam cured 2 of 6 pharyngeal infections (33%; 95% confidence interval [CI], 4.3% to 78%) and 3 of 4 rectal infections (75%; 95% CI, 19% to 99%). All 11 men with urethritis were cured (100%; 95% CI, 66% to 100%). The aztreonam MIC90 was 0.5 μg/ml (range, 0.06 to 2.0 μg/ml). All treatment failures occurred at a MIC of ≥0.25 μg/ml. Single-dose aztreonam is not a reliable treatment for gonorrhea at the pharynx but may be useful for men with gonococcal urethritis and beta-lactam allergy. (This study has been registered at ClinicalTrials.gov under identifier NCT03867734.)

Neisseria meningitidis is a leading cause of bacterial meningitis and sepsis worldwide and an occasional cause of meningococcal urethritis. When isolates are unavailable for surveillance or outbreak investigations, molecular characterization of pathogens needs to be performed directly from clinical specimens, such as cerebrospinal fluid (CSF), blood, or urine. However, genome sequencing of specimens is challenging because of low bacterial and high human DNA abundances. We developed selective whole-genome amplification (SWGA), an isothermal multiple-displacement amplification-based method, to efficiently enrich, sequence, and de novo assemble N. meningitidis DNA from clinical specimens with low bacterial loads. SWGA was validated with 12 CSF specimens from invasive meningococcal disease cases and 12 urine specimens from meningococcal urethritis cases. SWGA increased the mean proportion of N. meningitidis reads by 2 to 3 orders of magnitude, enabling identification of at least 90% of the 1,605 N. meningitidis core genome loci for 50% of the specimens. The validated method was used to investigate two meningitis outbreaks recently reported in Togo and Burkina Faso. Twenty-seven specimens with low bacterial loads were processed by SWGA before sequencing, and 12 of 27 were successfully assembled to obtain the full molecular typing and vaccine antigen profile of the N. meningitidis pathogen, thus enabling thorough characterization of outbreaks. This method is particularly important for enhancing molecular surveillance in regions with low culture rates. SWGA produces enough reads for phylogenetic and allelic analysis at a low cost. More importantly, the procedure can be extended to enrich other important human bacterial pathogens.

Factor H binding protein (FHbp) is an important Neisseria meningitidis virulence factor that binds a negative regulator of the alternative complement pathway, human factor H (FH). Binding of FH increases meningococcal resistance to complement-mediated killing. FHbp also is reported to prevent interaction of the antimicrobial peptide (AMP) LL-37 with the meningococcal surface and meningococcal killing. FHbp is a target of two licensed group B-directed meningococcal (MenB) vaccines. We found a new FHbp variant, peptide allele identification no. 896 (ID 896), was highly expressed by an emerging meningococcal pathotype, the nonencapsulated urethritis clade (US_NmUC). This clade has been responsible for outbreaks of urethritis in multiple U.S. cities since 2015, other mucosal infections, and cases of invasive meningococcal disease. FHbp ID 896 is a member of the variant group 1 (subfamily B), bound protective anti-FHbp monoclonal antibodies, bound high levels of human FH, and enhanced the resistance of the clade to complement-mediated killing in low levels of human complement likely present at human mucosal surfaces. Interestingly, expression of FHbp ID 896 resulted in augmented killing of the clade by LL-37. FHbp ID 896 of the clade was recognized by antibodies elicited by FHbp in MenB vaccines.

Mycoplasma genitalium is an important sexually transmitted pathogen that is commonly associated with nongonococcal urethritis in men and pelvic inflammatory disease in women ([1][1]). Over the past decade, treatment of M. genitalium has become increasingly challenging due to the fact that this

Mycoplasma genitalium is prevalent among attendees in sexually transmitted infection (STI) clinics, and therapy is hampered by rapidly rising levels of resistance to azithromycin and moxifloxacin. In this study, we evaluated, for the first time in Iceland, the prevalence of M. genitalium and azithromycin and moxifloxacin resistance-associated mutations and assessed the diagnostic performance of the CE/in vitro diagnosis (IVD)-marked S-DiaMGTV (Diagenode Diagnostics) versus the U.S. FDA/CE/IVD-approved Aptima MG (AMG; Hologic) for M. genitalium detection. From October 2018 to January 2019, urine and vaginal swabs were provided by male and female attendees at Iceland’s only STI clinic. Specimens were tested with S-DiaMGTV and AMG, and resistance-associated mutations were determined by 23S rRNA gene and parC sequencing. Demographic and clinical data were collected from patient records. M. genitalium prevalence was 9.3% overall; 7.7% (38/491) among male and 10.9% (53/487) among female participants. Azithromycin and moxifloxacin resistance-associated mutations were found in 57.0% (45/79) and 0.0% (0/80) of evaluable specimens, respectively. Sensitivity was 72.5% and 100%, and specificity was 99.9% and 100% for S-DiaMGTV and AMG, respectively. No association was found between M. genitalium and symptoms of urethritis in men. Prevalence rates for M. genitalium and azithromycin resistance-associated genes in Iceland are among the highest reported in Europe. The significantly higher sensitivity of AMG over that of S-DiaMGTV can have important clinical implications. More information is urgently needed to clarify the significance of false-negative results obtained with S-DiaMGTV and other similarly performing widely used real-time PCR methods for diagnosis and management of this sexually transmitted infection.

Mycoplasma genitalium is a sexually transmitted organism that causes nongonococcal urethritis in men and pelvic inflammatory disease in women. In Australia, there is concern over the emergence and spread of antimicrobial resistance (AMR) in M. genitalium. While high levels of AMR have been reported

Data from a large prospective multicenter clinical validation study of a nucleic acid amplification in vitro diagnostic test for Mycoplasma genitalium were analyzed to describe the prevalence of M. genitalium infection, risk factors, and disease associations in female and male patients seeking care in diverse geographic regions of the United States. Among 1,737 female and 1,563 male participants, the overall prevalence of M. genitalium infection was 10., Data from a large prospective multicenter clinical validation study of a nucleic acid amplification in vitro diagnostic test for Mycoplasma genitalium were analyzed to describe the prevalence of M. genitalium infection, risk factors, and disease associations in female and male patients seeking care in diverse geographic regions of the United States. Among 1,737 female and 1,563 male participants, the overall prevalence of M. genitalium infection was 10.3% and was significantly higher in persons ages 15 to 24 years than in persons ages 35 to 39 years (for females, 19.8% versus 4.7% [odds ratio {OR} = 5.05; 95% confidence interval {CI} = 3.01 to 8.46]; for males, 16.5% versus 9.4% [OR = 1.91; 95% CI = 1.20 to 3.02]). The risk for M. genitalium infection was higher in black than in white participants (for females, 12.0% versus 6.8% [OR = 1.88; 95% CI = 1.30 to 2.72]; for males, 12.9% versus 6.9% [OR = 2.02; 95% CI = 1.38 to 2.96]) and higher in non-Hispanic than in Hispanic participants (for females, 11.2% versus 6.0% [OR = 1.97; 95% CI = 1.25 to 3.10]; for males, 11.6% versus 6.8% [OR = 1.80; 95% CI = 1.14 to 2.85]). Participants reporting urogenital symptoms had a significantly elevated risk of M. genitalium infection compared to that for asymptomatic individuals (for females, OR = 1.53 [95% CI = 1.09 to 2.14]; for males, OR = 1.42 [95% CI = 1.02 to 1.99]). Women diagnosed with vaginitis and cervicitis had a higher prevalence of M. genitalium infection than women without those diagnoses, although this was statistically significant only for vaginitis (for vaginitis, OR = 1.88 [95% CI = 1.37 to 2.58]; for cervicitis, OR = 1.42 [95% CI = 0.61 to 2.96]). A diagnosis of urethritis in men was also significantly associated with M. genitalium infection (OR = 2.97; 95% CI = 2.14 to 4.13). Few characteristics distinguished asymptomatic from symptomatic M. genitalium infections. These results from persons seeking care in the United States suggest that M. genitalium infection should be considered in young persons presenting with urogenital symptoms.

Concurrent sexually transmitted infections (STI) can increase the probability of HIV-1 transmission primarily by increasing the viral load present in semen. In this study, we explored the relationship of HIV-1 in blood and seminal plasma in the presence and absence of urethritis and after treatment of the concurrent STI. Primer ID deep sequencing of the V1/V3 region of the HIV-1 env gene was done for paired blood and semen samples from antiretroviral therapy (ART)-naive men living in Malawi with (n = 19) and without (n = 5) STI-associated urethritis; for a subset of samples, full-length env genes were generated for sequence analysis and to test entry phenotype. Cytokine concentrations in the blood and semen were also measured, and a reduction in the levels of proinflammatory cytokines was observed following STI treatment. We observed no difference in the prevalence of diverse compartmentalized semen-derived lineages in men with or without STI-associated urethritis, and these viral populations were largely stable during STI treatment. Clonal amplification of one or a few viral sequences accounted for nearly 50% of the viral population, indicating a recent bottleneck followed by limited viral replication. We conclude that the male genital tract is a site where virus can be brought in from the blood, where localized sustained replication can occur, and where specific genotypes can be amplified, perhaps initially by cellular proliferation but further by limited viral replication. IMPORTANCE HIV-1 infection is a sexually transmitted infection that coexists with other STI. Here, we examined the impact of a concurrent STI resulting in urethritis on the HIV-1 population within the male genital tract. We found that viral populations remain largely stable even with treatment of the STI. These results show that viral populations within the male genital tract are defined by factors beyond transient inflammation associated with a concurrent STI.

Mycoplasma genitalium has been associated with infections of the genitourinary tract, and prevalence is secondary to Chlamydia trachomatis . The clinical observation of increasing treatment failure indicating antibiotic resistance, especially in cases of recurrent urethritis, has been confirmed by molecular testing. Mutations in the 23S rRNA gene can cause macrolide resistance, and topoisomerase/gyrase mutations can cause fluoroquinolone resistance. In this study, 115 M. genitalium DNA-positive samples were analyzed. Eighty-nine (77.4%) samples had a 23S rRNA mutation present, and 26 (22.6%) were wild type (no resistance mutation). Fluoroquinolone mutation screening was performed on 86 (74.8%) of the 115 samples, of which 20 (23.3%) samples had a mutation or mutations associated with increased resistance. This study shows the increasing antibiotic resistance in New Zealand and the need for appropriate guidelines to treat at-risk patients.

Mycoplasma genitalium is an important and emerging agent of sexually transmitted infection in females and males, carrying the potential for postinfection genital tract sequelae. Past efforts to identify this organism on a routine basis, which were problematic due to the fastidious nature of the bacterium and its antigenic intricacies, have recently become supplemented by molecular diagnostics. A number of these assays are available commercially. This minireview describes the format and performance indices of a number of M. genitalium DNA- and RNA-based amplification assays; many of these assays have contributed to an improved clinical and epidemiologic understanding of this organism.

Urethral swabs are the samples of choice for point-of-care Gram stain testing to diagnose Neisseria gonorrhoeae infection and nongonococcal urethritis (NGU) in men. As an alternative to urethral swabs, meatal swabs have been recommended for the collection of urethral discharge to diagnose N. gonorrhoeae and Chlamydia trachomatis infection in certain populations by nucleic acid amplification testing (NAAT), as they involve a less invasive collection method. However, as meatal swabs could be sampling a reduced surface area and result in fewer collected epithelial cells compared to urethral swabs, the adequacy of meatal swab specimens to collect sufficient cellular material for Gram stain testing remains unknown. We enrolled 66 men who underwent either urethral or meatal swabbing and compared the cellular content and Gram stain failure rate. We measured the difference in swab cellular content using the Cepheid Xpert CT/NG sample adequacy control crossing threshold (SAC CT ) and determined the failure rate of Gram stain smears (GSS) due to insufficient cellular material. In the absence of discharge, meatal smears were associated with a significant reduction in cellular content ( P = 0.0118), which corresponded with a GSS failure rate significantly higher than that for urethral swabs (45% versus 3%, respectively; P < 0.0001). When discharge was present, there was no difference among results from urethral and meatal swabs. Therefore, if GSS testing is being considered for point-of-care diagnosis of N. gonorrhoeae infection or NGU in men, meatal swabs should be avoided in the absence of a visible discharge.

Recent emergence of antimicrobial resistance of Neisseria gonorrhoeae worldwide has resulted in limited therapeutic choices for treatment of infections caused by this organism. We performed global transcriptomic analysis of N. gonorrhoeae in subjects with gonorrhea who attended a Nanjing, China, sexually transmitted infection (STI) clinic, where antimicrobial resistance of N. gonorrhoeae is high and increasing. We found that N. gonorrhoeae transcriptional responses to infection differed in genital specimens taken from men and women, particularly antibiotic resistance gene expression, which was increased in men. These sex-specific findings may provide a new approach to guide therapeutic interventions and preventive measures that are also sex specific while providing additional insight to address antimicrobial resistance of N. gonorrhoeae., Neisseria gonorrhoeae is a bacterial pathogen responsible for the sexually transmitted infection gonorrhea. Emergence of antimicrobial resistance (AMR) of N. gonorrhoeae worldwide has resulted in limited therapeutic choices for this infection. Men who seek treatment often have symptomatic urethritis; in contrast, gonococcal cervicitis in women is usually minimally symptomatic, but may progress to pelvic inflammatory disease. Previously, we reported the first analysis of gonococcal transcriptome expression determined in secretions from women with cervical infection. Here, we defined gonococcal global transcriptional responses in urethral specimens from men with symptomatic urethritis and compared these with transcriptional responses in specimens obtained from women with cervical infections and in vitro-grown N. gonorrhoeae isolates. This is the first comprehensive comparison of gonococcal gene expression in infected men and women. RNA sequencing analysis revealed that 9.4% of gonococcal genes showed increased expression exclusively in men and included genes involved in host immune cell interactions, while 4.3% showed increased expression exclusively in women and included phage-associated genes. Infected men and women displayed comparable antibiotic-resistant genotypes and in vitro phenotypes, but a 4-fold higher expression of the Mtr efflux pump-related genes was observed in men. These results suggest that expression of AMR genes is programed genotypically and also driven by sex-specific environments. Collectively, our results indicate that distinct N. gonorrhoeae gene expression signatures are detected during genital infection in men and women. We propose that therapeutic strategies could target sex-specific differences in expression of antibiotic resistance genes. IMPORTANCE Recent emergence of antimicrobial resistance of Neisseria gonorrhoeae worldwide has resulted in limited therapeutic choices for treatment of infections caused by this organism. We performed global transcriptomic analysis of N. gonorrhoeae in subjects with gonorrhea who attended a Nanjing, China, sexually transmitted infection (STI) clinic, where antimicrobial resistance of N. gonorrhoeae is high and increasing. We found that N. gonorrhoeae transcriptional responses to infection differed in genital specimens taken from men and women, particularly antibiotic resistance gene expression, which was increased in men. These sex-specific findings may provide a new approach to guide therapeutic interventions and preventive measures that are also sex specific while providing additional insight to address antimicrobial resistance of N. gonorrhoeae.

The detection of Mycoplasma genitalium was evaluated on 1,080 urine samples by the use of a Panther instrument. Overall sensitivity, specificity, positive predictive values, and negative predictive values were 100%, 99.4%, 93.6%, and 100%, respectively. Detection of M. genitalium by the use of the Panther transcription-mediated amplification assay offers a simple, accurate, and sensitive platform for diagnostic laboratories.

M ycoplasma genitalium has been implicated as a cause of acute and chronic nonchlamydial nongonococcal urethritis (NCNGU) and pelvic inflammatory disease (PID) ([1][1]). In New Zealand, as with many other countries, the preferred treatment for NCNGU is either azithromycin or doxycycline ([2][2]).

We tested the activity of ETX0914 against 187 Neisseria gonorrhoeae isolates from men with urethritis in Nanjing, China, in 2013. The MIC 50 , MIC 90 , and MIC range for ETX0914 were 0.03 μg/ml, 0.06 μg/ml, and ≤0.002 to 0.125 μg/ml, respectively. All isolates were resistant to ciprofloxacin, and 36.9% (69/187) were resistant to azithromycin. Of the isolates, 46.5% were penicillinase-producing N. gonorrhoeae (PPNG), 36% were tetracycline-resistant N. gonorrhoeae (TRNG), and 13% (24 isolates) had an MIC of 0.125 μg/ml for ceftriaxone. ETX0914 may be an effective treatment option for gonorrhea.

金沢大学医薬保健研究域医学系, Liquid-based urine cytology (LB-URC) was evaluated for cytological diagnosis and detection of human papillomavirus (HPV), Mycoplasma, and Ureaplasma. Midstream urine samples were collected from 141 male patients with urethritis and 154 controls without urethritis, and sediment cells were preserved in liquid-based cytology solution. Urethral swabs from urethritis patients were tested for the presence of Neisseria gonorrhoeae and Chlamydia trachomatis. Papanicolaou tests were performed for cytological evaluation. HPV, Mycoplasma, and Ureaplasma genomes were determined by PCR-based methods, and localization of HPV DNA in urothelial cells was examined by in situ hybridization (ISH). The β-globin gene was positive in 97.9% of LB-URC samples from urethritis patients and in 97.4% of control samples, suggesting that high-quality cellular DNA was obtained from the LB-URC samples. HPV DNA was detected in 29 (21.0%) urethritis cases and in five (3.3%) controls (P

Chlamydia trachomatis, a Gram-negative obligate intracellular bacterium with a tropism for mucosal epithelial cells, is the most common cause of bacterial sexually transmitted disease in both developed and developing countries, with more than 90 million new cases occurring each year (113-115). More than 4 million new cases of C. trachomatis infection occur each year in the United States, where costs associated with treating those infections and associated complications are in excess of $2 billion annually (107). In the genital tract, infection with C. trachomatis is propagated within the single-cell columnar layer of the epithelium in the urethra of men and the endocervix of women. Within the epithelial cells, C. trachomatis undergoes a unique biphasic developmental cycle consisting of an infectious, but metabolically inert elementary body (EB) and a noninfectious, but metabolically active reticulate body (RB). After completion of the developmental cycle, the EBs are released and infect neighboring epithelial cells, thereby spreading the infection. Infection can result in acute inflammation characterized by redness, edema, and mucosal discharge and is diagnosed clinically as mucopurulent cervicitis in women and nongonococcal urethritis in men (10, 85). In women, infection can manifest as abnormal vaginal discharge and/or postcoital bleeding, while the infection is limited to the lower genital tract, and irregular uterine bleeding and/or pelvic discomfort once the infection ascends to the upper genital tract (85). Symptoms in males are generally limited to dysuria and moderate clear-to-whitish discharge (85). While these symptoms signify an infection, the absence of such symptoms does not necessarily indicate the absence of infection. It is estimated that >70% of women and 50% of men experience asymptomatic infections (15, 113). Without symptoms providing the impetus, asymptomatic individuals may not seek diagnostic testing and the infection will go untreated. Untreated C. trachomatis infection can wreak havoc on the reproductive organs, profoundly affecting fertility in women. Taken together, the high rate of asymptomatic infections and the severity of the infection-related pathology indicate that, despite the availability of very effective antimicrobial therapy, control of chlamydial infections will most likely require a vaccine.

The pathogenesis of an infection of the male genitourinary tract of mice with a human serovar of Chlamydia trachomatis has not been characterized. To establish a new model, we inoculated C3H/HeN ( H-2 k ) mice in the meatus urethra with C. trachomatis serovar D. To determine the 50% infectious dose (ID 50 ), male mice were inoculated with doses ranging from 10 2 to 10 6 inclusion-forming units (IFU). The mice were euthanized 10 days post infection (p.i.), and the urethra, bladder, epididimydes, and testes were cultured for Chlamydia . Positive cultures were obtained from the urethra, urinary bladder, and epididimydes, and the ID 50 was determined to be 5 × 10 4 IFU/mouse. Subsequently, to characterize the course of the infection, wild-type (WT) and C3H animals with severe combined immunodeficiency (SCID animals) were inoculated with 10 6 IFU/mouse (20 times the ID 50 ). In the WT mice, the infection peaked in the second week, and by 42 days p.i., it was cleared. In contrast, most of the SCID mice continued to have positive cultures at 60 days p.i. C. trachomatis -specific antibodies were first detected in WT animals' sera at 21 days p.i. and increased until 42 days p.i. The immunoglobulin G2a (IgG2a) titers were 32-fold higher than those of IgG1, indicative of a Th1-biased immune response. A lymphoproliferative assay using splenocytes showed a significant cell-mediated immune response in the WT mice. As expected, no humoral or cell-mediated immune responses were observed in the SCID animals. In conclusion, inoculation of WT male mice in the meatus urethra with a human serovar of C. trachomatis resulted in a limited infection mainly localized to the lower genitourinary tract. On the other hand, SCID animals could not clear the infection, suggesting that in male mice, the adaptive immune response is necessary to control an infection with a C. trachomatis human serovar.

The aim of this study was to develop and evaluate a sensitive method for the simultaneous identification of 14 urogenital potential pathogens. A multiplex PCR-based reverse line blot (mPCR/RLB) assay was developed to detect 14 urogenital pathogens or putative pathogens, namely Trichomonas vaginalis , Streptococcus pneumoniae , Neisseria gonorrhoeae , Chlamydia trachomatis , Ureaplasma parvum , U. urealyticum , Gardnerella vaginalis , Haemophilus influenzae , herpes simplex virus type 1 (HSV1) and HSV2, N. meningitidis , Mycoplasma hominis , M. genitalium , and adenovirus, using two species-specific primer pairs and probes for each. The method was validated using a reference strain or a well-characterized clinical isolate of each target organism and was found to be both sensitive and specific. The limits of detection for the mPCR/RLB assay varied among the 14 target organisms from 4.2 × 10 −1 to 7.0 × 10 −11 ng/μl of genomic DNA. There were no cross-reactions among any of the probes. This method was used to test 529 first-voided urine specimens from male patients with and without urethritis attending two Sydney sexual health clinics. One or more target species were detected in 193 (36%) subjects. Of 233 positive results, overall 216 (93%) were concordant between mPCR/RLB and a comparator method (culture and/or species-specific PCR), 9 were positive only by mPCR/RLB, and 8 were positive only by the comparator method. The mPCR/RLB method was an accurate, convenient, and inexpensive method for the detection of multiple potential pathogens in first-voided urine specimens from men.

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.

Mycoplasma genitalium (MG) can cause nongonococcal urethritis and is potentially associated with urethritis, endometritis, and cervicitis. Several assays have been developed to detect MG. Molecular amplification assays for organism detection can be problematic due to the potential for false-positive and false-negative results. Confirmatory testing is often required in these situations, requiring additional time and resources. Use of multigene targets could integrate both detection and verification at lower cost. Utilizing two targets, the MgPa adhesion gene and the 16S rRNA gene, a multitarget real-time (MTRT) PCR for the detection of MG was developed. Samples from patients attending sexually transmitted disease clinics were collected in duplicate. Urine samples from males ( n = 286) and self-collected vaginal swabs from females ( n = 321) were analyzed by MTRT PCR for MG and the Gen-Probe transmission-mediated amplification (TMA) assay, which targets MG rRNA for detection (TMA-MG research use only). Utilizing the criteria of any two targets being positively amplified, the MTRT PCR had a sensitivity and specificity of 91.8% (101 positive samples/110 samples tested) and 99.5% (495/497), respectively, with a positive predictive value (PPV) of 98.1% (101/103) and a negative predictive value (NPV) of 98.2% (495/504). The Gen-Probe TMA-MG assay had a sensitivity, specificity, PPV, and NPV of 98.1% (108/110), 98.1% (488/497), 92.3% (108/117), and 99.5% (488/490), respectively. Comparison between the MTRT PCR and TMA-MG assay by kappa statistic analysis indicated that an overall kappa value was 0.941 (95% confidence interval, 0.907 and 0.976). Both assays demonstrated accuracy in the detection of MG from urine samples from male patients and self-collected vaginal swabs from female patients.

Defensins are key participants in mucosal innate defense. The varied antimicrobial activity and differential distribution of defensins at mucosal sites indicate that peptide repertoires are tailored to site-specific innate defense requirements. Nonetheless, few studies have investigated changes in peptide profiles and function after in vivo pathogen challenge. Here, we determined defensin profiles in urethral secretions of healthy men and men withChlamydia trachomatis-andNeisseria gonorrhoeae-mediated urethritis by immunoblotting for the epithelial defensins HBD1, HBD2, and HD5 and the neutrophil defensins HNP1 to -3 (HNP1-3). HBD1 was not detectable in secretions, and HBD2 was only induced in a small proportion of the urethritis patients; however, HD5 and HNP1-3 were increased inC. trachomatisinfection and significantly elevated inN. gonorrhoeaeinfection. When HNP1-3 levels were low, HD5 appeared mostly as the propeptide; however, when HNP1-3 levels were >10 μg/ml, HD5 was proteolytically processed, suggesting neutrophil proteases might contribute to HD5 processing. HD5 and HNP1-3 were bactericidal againstC. trachomatisandN. gonorrhoeae, but HD5 activity was dependent upon N-terminal processing of the peptide. In vitro proteolysis of proHD5 by neutrophil proteases and analysis of urethral secretions by surface-enhanced laser desorption ionization substantiated that neutrophils contribute the key convertases for proHD5 in the urethra during these infections. This contrasts with the small intestine, where Paneth cells secrete both proHD5 and its processing enzyme, trypsin. In conclusion, we describe a unique defensin expression repertoire in response to inflammatory sexually transmitted infections and a novel host defense mechanism wherein epithelial cells collaborate with neutrophils to establish an antimicrobial barrier during infection.

Two homosexual men were colonized in the urethra with Haemophilus parainfluenzae nonsusceptible to ampicillin (MIC, 8 μg/ml), amoxicillin-clavulanate (MIC, 4 μg/ml), cefotaxime (MIC, 1.5 μg/ml), cefepime (MIC, 3 μg/ml), meropenem (MIC, 0.5 μg/ml), cefuroxime, azithromycin, ciprofloxacin, tetracycline, and chloramphenicol (all MICs, ≥32 μg/ml). Repetitive extragenic palindromic PCR (rep-PCR) showed that the strains were indistinguishable. The isolates had amino acid substitutions in PBP3, L4, GyrA, and ParC and possessed Mef(A), Tet(M), and CatS resistance mechanisms. This is the first report of extensively drug-resistant (XDR) H. parainfluenzae .

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 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.

Detection of Mycoplasma genitalium -mediated, chlamydia-negative nongonococcal urethritis and other M. genitalium -linked infectious etiologies has been very challenging. Although M. genitalium is considered a leading cause of genitourinary symptoms in men and women, extreme difficulties in its cultivation due to its highly fastidious nature and the lack of routine and effective diagnostic tests have slowed the generation of clinical data which directly implicate the presence of M. genitalium in disease pathogenesis. In this study, we compared enzyme-linked immunosorbent assays (ELISAs) and immunoblot and PCR assays in M. genitalium culture-positive women over 1 to 3 years of clinical visits to determine the usefulness of independent diagnostic strategies. Furthermore, the value of combinatorial diagnostic assessments is described, which provides insights into the dynamics of M. genitalium -host interactions. Overall, we show that neither ELISA nor PCR, alone or in combination, provides the sensitivity required to confidently predict the existence of viable M. genitalium organisms in cervical and vaginal samples. Additionally, culture-positive women exhibited a range of antibody responsiveness to M. genitalium based upon ELISA and immunoblot assessments, indicating immune diversity among this high-risk population.

We have examined cell-free viral populations in the blood plasma and seminal plasma compartments of men infected with subtype C human immunodeficiency virus type 1 (HIV-1) using the V3-specific heteroduplex tracking assay (V3-HTA). We studied two cohorts of subjects who had visited either a sexually transmitted disease (STD) clinic for genital tract inflammation in the form of urethritis ( n = 43) or a dermatology clinic (controls, n = 14) in Malawi. We have previously shown that the presence of urethritis is associated with an eightfold increase in virus load in the seminal plasma compartment (M. S. Cohen et al., Lancet 349:1868–1873, 1997). The purpose of this study was to determine whether genital tract inflammation and its treatment caused genetic instability in cell-free HIV-1 populations. In a cross-sectional analysis at study entry, three-fourths of the STD and control subjects had multiple V3 populations in their blood while 60% of the STD subjects and 79% of the control subjects had multiple V3 populations in their semen. Overall, one-fourth of all of the subjects showed discordance between results with blood and semen specimens when samples were compared for the presence and absence of subpopulations. When differences in the relative levels of abundance of bands were also taken into account, two-fifths of all of the subjects showed discordance between the compartments. Among the subset of subjects in whom multiple virus populations could be detected, half showed discordance between the compartments. There were no differences between STD and control cohorts for these comparisons of the compartments in this cross-sectional analysis at study entry. Longitudinal analysis of the viral populations from two separate clinic visits over 1 to 4 weeks showed that the complexity of each V3 population as measured by Shannon entropy was different in blood and semen at the two time points, indicating that the blood and semen constitute different compartments for HIV-1. The seminal plasma compartment was more dynamic than the blood plasma compartment for the STD subjects who were treated for urethritis, with changes being noted in the presence or absence of V3-HTA bands in the semen of 29% of these subjects but in the blood of only 9% of these subjects. However, the changes were generally small. Overall, our results suggest that 40% of male subjects show discordance between seminal and blood viral populations and that the complexity of each V3 population was different between the two compartments. Both of these results point to the partial independence of the seminal compartment as a viral niche within the body.

To assess the antimicrobial resistance of Neisseria gonorrhoeae isolated from 1993 through 1998 in Japan, susceptibility testing was conducted on 502 isolates. Selected isolates were characterized by auxotype and analysis for mutations within the quinolone resistance-determining region (QRDR) in the gyrA and parC genes, which confer fluoroquinolone resistance on the organism. Plasmid-mediated penicillin resistance (penicillinase-producing N. gonorrhoeae ) decreased significantly from 1993–1994 (7.9%) to 1997–1998 (2.0%). Chromosomally mediated penicillin resistance decreased from 1993–1994 (12.6%) to 1995–1996 (1.9%) and then increased in 1997–1998 (10.7%). Chromosomally mediated tetracycline resistance decreased from 1993–1994 (3.3%) to 1997–1998 (2.0%), and no plasmid-mediated high-level tetracycline resistance was found. Isolates with ciprofloxacin resistance (MIC ≥ 1 μg/ml) increased significantly from 1993–1994 (6.6%) to 1997–1998 (24.4%). The proline-requiring isolates were less susceptible to ciprofloxacin than the prototrophic or arginine-requiring isolates. Ciprofloxacin-resistant isolates contained three or four amino acid substitutions within the QRDR in the GyrA and ParC proteins.

Adenoviruses (Ads) are an important cause of respiratory illness, conjunctivitis, and gastroenteritis, but they are seldom recognized as a potential cause of sexually transmitted disease. We performed virus cultures on approximately 7,000 patients attending a sexually transmitted disease clinic or other health department clinics for the evaluation of genital ulcers, urethritis, or conjunctivitis. Ads were isolated from genital or conjunctival specimens obtained from 23 (0.33%) patients. Among the 20 Ad-positive men, 15 (75%) had urethritis, 12 (60%) had conjunctivitis, and 10 (50%) had both. All three Ad-positive women had vaginal discharge and genital ulcers or fissures. Ad isolates from 17 patients were available for serotyping. Ad type 37 was isolated from 14 patients, Ad type 8 was isolated from 2 patients, and Ad type 2 was isolated from 1 patient. In three of the Ad type 37 cases, Ad was recovered from both urethral and conjunctival specimens. One of the Ad type 8 cases had conjunctivitis, but the Ad type 2 case did not. Ads, particularly type 37, may be a sexually transmissible cause of genital ulcers, urethritis, and conjunctivitis.

TThe Syva MicroTrak Chlamydia enzyme immunoassay (EIA; Syva Company, San Jose, Calif.) with cytospin and direct fluorescent-antibody assay (DFA) confirmation was evaluated on 43,630 urogenital specimens over a 1-year period in the Provincial Laboratory in Regina, Saskatchewan, Canada. This was a two-phase study intended to define a testing algorithm for Chlamydia trachomatis that would be both highly accurate and cost-effective in our high-volume (> 3,000 tests per month) laboratory. The prevalence of C. trachomatis infection in our population is moderate (8 to 9%). In phase 1, we tested 6,022 male and female urogenital specimens by EIA. All specimens with optical densities above the cutoff value and those within 30% below the cutoff value were retested by DFA. This was 648 specimens (10.8% of the total). A total of 100% (211 of 211) of the specimens with optical densities equal to or greater than 1.00 absorbance unit (AU) above the cutoff value, 98.2% (175 of 178) of the specimens with optical densities of between 0.500 and 0.999 AU above the cutoff value, and 83% (167 of 201) of the specimens with optical densities within 0.499 AU above the cutoff value were confirmed to be positive. A total of 12% (7 of 58) of the specimens with optical densities within 30% below the cutoff value were positive by DFA. In phase 2, we tested 37,608 specimens (32,495 from females; 5,113 from males) by EIA. Only those specimens with optical densities of between 0.499 AU above and 30% below the cutoff value required confirmation on the basis of data from phase 1 of the study. This was 4.5% of all specimens tested. This decrease in the proportion of specimens requiring confirmation provides a significant cost savings to the laboratory. The testing algorithm gives us a 1-day turnaround time to the final confirmed test results. The MicroTrak EIA performed very well in both phases of the study, with a sensitivity, specificity, positive predictive value, and negative predictive value of 96.1, 99.1, 90.3, and 99.7%, respectively, in phase 2. We suggest that for laboratories that use EIA for Chlamydia testing, a study such as this one will identify an appropriate optical density range for confirmatory testing for samples from that particular population.

In a study of 1,486 men attending two sexually transmitted disease clinics, of whom 891 had no symptoms of urethritis, we compared an enzyme immunoassay (EIA) (Baxter-Bartels, formerly Northumbria AntigEnz) of urine sediment to urethral culture for the detection of Chlamydia trachomatis. C. trachomatis prevalence by culture alone was 7.7% in asymptomatic men and 10.9% in symptomatic men. Discrepant results between EIA of urine and urethral culture were evaluated by direct fluorescent-antibody staining (DFA) for elementary bodies in urine sediment or in culture transport media. When chlamydial infection was defined as either a positive urethral culture or positive EIA confirmed by DFA, chlamydia prevalence increased to 8.9% in asymptomatic men and 11.6% in symptomatic men. The urine EIA sensitivity, specificity, and positive and negative predictive values for chlamydia detection in asymptomatic men were 84.8, 99.3, 91.8, and 98.5%, respectively, with nearly identical results for symptomatic men. The sensitivities of urethral culture alone compared with the combination of urethral culture and urine EIA (with DFA confirmation) were 87.3 and 94.3% for asymptomatic and symptomatic men, respectively. The present EIA of urine sediment is both highly sensitive and specific for the detection of C. trachomatis in asymptomatic men, thus providing a noninvasive screening method for chlamydia infection in asymptomatic men attending sexually transmitted disease clinics.

SUMMARY The history, replication, genetics, characteristics (both biological and physical), and factors involved in the pathogenesis of Mycoplasma genitalium are presented. The latter factors include adhesion, the influence of hormones, motility, possible toxin production, and immunological responses. The preferred site of colonization, together with current detection procedures, mainly by PCR technology, is discussed. The relationships between M. genitalium and various diseases are highlighted. These diseases include acute and chronic nongonococcal urethritis, balanoposthitis, chronic prostatitis, and acute epididymitis in men and urethritis, bacterial vaginosis, vaginitis, cervicitis, pelvic inflammatory disease, and reproductive disease in women. A causative relationship, or otherwise strong association, between several of these diseases and M. genitalium is apparent, and the extent of this, on a subjective basis, is presented; also provided is a comparison between M. genitalium and two other genital tract-orientated mollicutes, namely, Mycoplasma hominis , the first mycoplasma of human origin to be discovered, and Ureaplasma species. Also discussed is the relationship between M. genitalium and infertility and also arthritis in both men and women, as is infection in homosexual and immunodeficient patients. Decreased immunity, as in HIV infections, may enhance mycoplasmal detection and increase disease severity. Finally, aspects of the antimicrobial susceptibility and resistance of M. genitalium , together with the treatment and possible prevention of mycoplasmal disease, are discussed.

Mycoplasma genitalium accounts for as much as 15 to 20% of cases of male nongonococcal urethritis (NGU) (7). It is also implicated in female cervicitis and increasingly so in pelvic inflammatory disease (3). Since it is a fastidious microorganism that is very difficult to culture, nucleic acid amplification tests (NAAT) have become invaluable in its detection and microbiological evaluation. Currently recommended treatments of NGU include multidoses of doxycycline (100 mg twice daily for 7 days) or a single dose of azithromycin (1 g). With regard to M. genitalium-positive NGU, a recent randomized study has shown multidose treatment with doxycycline to be inferior to single-dose azithromycin (cure rates of 45% versus 87%, respectively) (8). However, even single-dose azithromycin can be associated with treatment failure rates of up to 28% (2). An extended 5-day course of azithromycin (500 mg on day 1, followed by 250 mg on days 2 to 5) has a much higher cure rate of up to 96% (1) but becomes less efficacious when used after initial treatment failure with single-dose azithromycin (6). Jensen et al. (4) have shown that macrolide resistance in M. genitalium can be induced by insufficient therapy with single-dose azithromycin and found the resistance to be strongly associated with three single-base mutations (A2058C, A2058G, A2059G) in region V of the 23S rRNA gene (4). They developed a conventional PCR to detect all of these mutations. In September 2009, we started M. genitalium NAAT testing of genital samples from selected patients with recurrent NGU at the Christchurch Sexual Health Centre, Christchurch, New Zealand. The PCR was performed as described by Jensen et al. (5) with the 2× Universal TaqMan gene expression master mix (Applied Biosystems, Life Technologies, Foster City, CA). Between September 2009 and October 2010, 52 samples were collected and M. genitalium was detected in 11 (21%) samples. The nucleic acid extracts were stored at −80°C until October 2010, when we retrospectively performed PCR and DNA sequencing on these extracts to look for the single-base mutations mentioned above, using the method described by Jensen et al. (4). Of the 11 nucleic acid extracts, we were able to amplify and sequence the 23S rRNA genes of 9 extracts. PCR of the other 2 nucleic acid extracts did not yield an amplicon upon repeat testing, probably due to nucleic acid degradation during storage. Of the 9 sequenced nucleic acid extracts, 4 extracts contained the A2059G mutation, whereas none contained mutations at position 2058. We retrospectively reviewed the notes for the relevant patients (those with extracts containing the mutation) and found that all four patients had previously been treated multiple times for sexually transmitted infections. We were able to determine that three of these patients had received single-dose azithromycin between 34 and 58 days preceding the positive test for M. genitalium; the fourth patient had also received single-dose azithromycin in the few weeks preceding a positive test, but we were unable to determine the exact time frame. All four patients had received an extended course of azithromycin following the diagnosis of M. genitalium urethritis: two experienced treatment failure, whereas the other two were subsequently cured, based on the fact that M. genitalium DNA was no longer detectable. Of the 5 patients whose M. genitalium extracts did not contain any of the macrolide resistance mutations (of the 9 extracts whose rRNA genes we were able to amplify and sequence), 3 had experienced successful empirical therapy with single-dose azithromycin (2 patients) or extended-dose azithromycin (1 patient); 1 patient was lost to follow up. The remaining 1 patient had received treatment with single-dose azithromycin, but upon subsequent testing, M. genitalium DNA was detectable and in this case contained the A2059G mutation, and the patient was included in our group above. This nicely demonstrates the induction of macrolide resistance by single-dose azithromycin treatment. To our knowledge, this is the first report from New Zealand describing macrolide resistance in M. genitalium. Despite a very limited number of samples, we have evidence that the A2059G mutation conferring induced macrolide resistance is present at a high frequency in M. genitalium extracts from patients with recurrent NGU at the Christchurch Sexual Health Centre. This brings into question whether or not single-dose azithromycin should be the first-line treatment of choice for M. genitalium-positive urethritis. At the Christchurch Sexual Health Centre, the current practice is to prescribe an extended course of azithromycin for all cases of M. genitalium-positive urethritis (primarily because moxifloxacin is currently not readily accessible in New Zealand). We also note with interest that in two patients with the resistant isolate, infection was still cleared with an extended course of azithromycin. This would suggest that macrolide resistance conferred by the A2059G mutation was not absolute. Clearly, the subject of macrolide resistance in M. genitalium requires more study.

Of the 331 Neisseria gonorrhoeae strains isolated in Greece from 1996 to 1999, 39 (11.8%) exhibited decreased susceptibility to quinolones due to gyrA andparC mutations. Conventional typing and pulsed-field gel electrophoresis showed that 34 of these isolates were clonally related. Epidemiological data indicated that the epidemic clone was sustained in a group of high-frequency transmitters.

Trichomonas vaginalis infection in men is an important cause of nongonococcal urethritis. Effective detection of the parasite in men using culture requires examination of multiple specimens. We compared culture and PCR-enzyme-linked immunosorbent assay in urethral swabs, urine, and semen for T. vaginalis detection in male sexual partners of women with trichomoniasis identified by wet mount and culture. Trichomonads were detected by at least one positive test in 205/280 men (73.2%) who submitted at least one specimen for culture and PCR. Whereas InPouch TV culture detected only 46/205 cases (22.5%), PCR detected 201/205 (98.0%). Urethral swab cultures from men with urethritis were more likely to be positive with shorter incubation than specimens from men without urethritis. T. vaginalis was detected more often in men with wet-mount-positive partners. Even with a sensitive PCR assay, reliable detection of T. vaginalis in male partners required multiple specimens. The majority of male sexual partners in this study were infected, emphasizing the importance of partner evaluation and treatment.

The lack of an experimental model has significantly limited the understanding of the pathogenesis of Chlamydia trachomatis infections in males. In an attempt to establish a model using the natural route of infection, we inoculated male mice in the meatus urethra. To establish the 50% infectious dose (ID 50 ), C3H/HeN ( H-2 k ) male mice were inoculated in the meatus urethra with doses ranging from 10 1 to 10 7 inclusion-forming units (IFU) of C. trachomatis mouse pneumonitis biovar (MoPn) and were euthanized at 10 days postinfection (p.i.). Approximately 50% of the animals inoculated with 5 × 10 4 IFU had positive cultures of the urethra, urinary bladder, epididymides, and/or testes. Subsequently, to characterize the course of the infection, a group of animals was inoculated with 10 6 IFU/mouse (20 times the ID 50 ). Positive cultures from the urethra, urinary bladder, epididymides, and testes were obtained from the animals. The infection peaked in the first 2 weeks p.i. and subsequently declined over the 7 weeks of observation. C. trachomatis -specific antibodies were first detected in serum by 2 weeks p.i. and rose over the period of observation. The titers of immunoglobulin G2a (IgG2a) were 16-fold higher than those of IgG1. A lymphoproliferative assay using splenocytes and local lymph nodes showed a strong cell-mediated immune response. Levels of gamma interferon were significantly higher than those of interleukin-4 in the supernatants from stimulated lymphocytes. An acute inflammatory infiltrate consisting of polymorphonuclear leukocytes was detected in the urethra at 1 week p.i. At 3 weeks p.i., a mixed acute and chronic inflammatory infiltrate was observed in the urethra that by 5 to 6 weeks was mainly composed of mononuclear cells. Similar findings were also observed in the urinary bladder, although the inflammatory infiltrate was delayed by approximately a week relative to that in the urethra. Sections of the epididymides showed a focal acute inflammatory infiltrate at 2 weeks p.i. Immunohistochemical staining demonstrated multiple chlamydial inclusions in the epithelium of the urethra and urinary bladder. No chlamydial inclusions were observed in the epididymides or testes. In conclusion, inoculation of male mice in the meatus urethra with C. trachomati s MoPn results in an infection of the genitourinary tract that closely parallels that described in humans. This model should help to characterize the pathogenesis of chlamydial infections in males and to test therapeutic and preventive measures.

Mycoplasma genitalium is a leading cause of chlamydia-negative, nongonoccocal urethritis and has been directly implicated in numerous other genitourinary as well as extragenitourinary tract pathologies. Detection of M. genitalium has relied almost entirely on PCR amplification of clinical specimens and evidence of seroconversion since these mycoplasmas are highly fastidious and culture isolation by microbiological techniques is very rare. We have established a combinatorial strategy using confocal immunoanalysis (CIA) and real-time PCR to qualitatively and quantitatively assess patterns of M. genitalium infection in women attending a sexually transmitted disease-related health clinic in San Antonio, Tex. CIA allows spatial examination of mycoplasmas on surfaces and inside human target cells, plus the ability to evaluate cell-to-cell patterns and variances within samples. Real-time PCR permits determination of genome copy numbers of mycoplasmas and human cells by multiplex amplification using mycoplasma gyrA and human RNase P gene sequences, which indicates overall levels of mycoplasma infection and degree of parasitism. These assays are strongly correlated and, in combination, permit detection and elucidation of heretofore-unrecognized patterns of M. genitalium infections in clinical and experimental samples.

Trichomonas vaginalis infection is highly prevalent worldwide and is associated with urethritis, prostatitis, and urethral strictures in men. However, the natural history and importance of T. vaginalis in men are poorly understood, in part because of difficulties in diagnosing infection. Traditional detection methods rely on culture and wet-mount microscopy, which can be insensitive and time consuming. Urethral swabs are commonly used to detect T. vaginalis in men, but discomfort from specimen collection is a barrier to large studies. One thousand two hundred twenty-five Malawian men attending sexually transmitted disease and dermatology clinics were enrolled in this cross-sectional study to validate detection by urine-based PCR-enzyme-linked immunosorbent assay (ELISA) with urine and urethral swab culture as the reference standard. This assay for detection of amplified T. vaginalis DNA in first-catch urine (≤30 ml) performed with a sensitivity of 92.7%, a specificity of 88.6%, and an adjusted specificity of 95.2% compared to culture of urethral swabs or urine sediment. For clinical research settings in which urethral swabs are not available and culture is not feasible, the urine-based PCR-ELISA may be useful for detection of trichomoniasis in men.