Presence and Concentrations of Select Bacterial Vaginosis-Associated Bacteria Are Associated With Increased Risk of Pelvic Inflammatory Disease.

In a vaginal 16S ribosomal RNA gene quantitative PCR study of 17 pelvic inflammatory disease (PID) cases and 17 controls who tested positive for Chlamydia trachomatis, women who additionally tested positive for Atopobium vaginae, Sneathia spp., Megasphaera spp., Eggerthella-like bacterium or Prevotella amnii were more likely to develop PID.

Identification of novel microbes associated with pelvic inflammatory disease and infertility.

OBJECTIVES: As pelvic inflammatory disease (PID) aetiology is not completely understood, we examined the relationship between select novel bacteria, PID and long-term sequelae. METHODS: Fastidious bacterial vaginosis (BV)-associated bacteria (Sneathia (Leptotrichia) sanguinegens, Sneathia amnionii, Atopobium vaginae and BV-associated bacteria 1 (BVAB1)), as well as Ureaplasma urealyticum and Ureaplasma parvum were identified in cervical and endometrial specimens using organism-specific PCR assays among 545 women enrolled in the PID Evaluation and Clinical Health study. Risk ratios and 95% CIs were constructed to determine associations between bacteria, histologically confirmed endometritis, recurrent PID and infertility, adjusting for age, race, gonorrhoea and chlamydia. Infertility models were additionally adjusted for baseline infertility. RESULTS: Persistent detection of BV-associated bacteria was common (range 58% for A. vaginae to 82% for BVAB1) and elevated the risk for persistent endometritis (RRadj 8.5, 95% CI 1.6 to 44.6) 30 days post-cefoxitin/doxycycline treatment, independent of gonorrhoea and chlamydia. In models adjusted for gonorrhoea and chlamydia, endometrial BV-associated bacteria were associated with recurrent PID (RRadj 4.7, 95% CI 1.7 to 12.8), and women who tested positive in the cervix and/or endometrium were more likely to develop infertility (RRadj 3.4, 95% CI 1.1 to 10.4). Associations between ureaplasmas and PID sequelae were modest. CONCLUSIONS: To our knowledge, this is the first prospective study to demonstrate that S. sanguinegens, S. amnionii, BVAB1 and A. vaginae are associated with PID, failure of the Centers for Disease Control and Prevention-recommended treatment to eliminate short-term endometritis, recurrent PID and infertility. Optimal antibiotic regimens for PID may require coverage of novel BV-associated microbes.

Ureaplasma urealyticum is associated with nongonococcal urethritis among men with fewer lifetime sexual partners: a case-control study.

BACKGROUND: Ureaplasmas have been inconsistently associated with nongonococcal urethritis (NGU). We evaluated the association of the newly differentiated species Ureaplasma urealyticum (UU) and Ureaplasma parvum (UP) with NGU using 2 separate control groups. METHODS: Case patients were men who attended a sexually transmitted disease (STD) clinic in Seattle, Washington, during the period 2007-2009 with NGU (defined as visible urethral discharge and/or ≥5 polymorphonuclear neutrophils per high-powered field; n = 329). Control subjects were STD clinic attendees (n = 191) and emergency department (ED) attendees (n = 193) without NGU. Polymerase chain reaction assays detected UU and UP in ureaplasma culture-positive urine. Multivariable logistic regression was used to assess the associations of UU and UP with NGU. RESULTS: UU was only marginally associated with NGU in aggregate multivariable analyses, irrespective of control group (adjusted odds ratio [aOR](STD-control), 1.6 [95% confidence interval {CI}, 0.9-2.8]; aOR(ED-control), 1.7 [95% CI, 0.97-3.0]). This association was significantly stronger when analyses were restricted to men with fewer lifetime sex partners (<10 vaginal partners: aOR(STD-control), 2.9 [95% CI, 1.2-6.7]; aOR(ED-control), 3.2 [95% CI, 1.3-7.6]; <5 vaginal partners: aOR(STD-control), 6.2 [95% CI, 1.8-21.0]; aOR(ED-control), 5.2 [95% CI, 1.3-20.2]). UP was not positively associated with NGU overall or among subgroups. CONCLUSIONS: The absence of an association of UU with NGU among men with more lifetime sex partners suggests that adaptive immunity may attenuate the clinical manifestation of UU infection. Similar relationships were not observed with UP, which suggests that it is not a urethral pathogen.

Demographic, behavioral, and clinical characteristics of men with nongonococcal urethritis differ by etiology: a case-comparison study.

BACKGROUND: Nongonococcal urethritis (NGU) is common, yet up to 50% of cases have no defined etiology. The extent to which risk profiles and clinical presentations of pathogen-associated and idiopathic cases differ is largely unknown. METHODS: Urethral swabs and urine specimens were collected from 370 NGU treatment trial participants who sought care at a sexually transmitted disease clinic in Seattle, WA from 2007 to 2009 and had a visible urethral discharge and/or microscopic evidence of urethral inflammation assessed by Gram-stain (≥5 polymorphonuclear leukocytes per high-powered field [PMNs/HPF]). Neisseria gonorrhoeae, Chlamydia trachomatis (CT), Mycoplasma genitalium (MG), Trichomonas vaginalis (TV), and Ureaplasma urealyticum (UU) were detected in urine, using nucleic acid amplification tests. Cases negative for all assessed pathogens were considered idiopathic. Bivariate and multivariate analyses identified clinical, sociodemographic, and behavioral factors associated with detection of specific pathogens. RESULTS: After excluding 3 participants with gonococcal infection, pathogens were detected in only 50.7% of the 367 eligible cases: CT in 22.3%, MG in 12.5%, TV in 2.5%, and UU in 24.0%, with multiple pathogens detected in 9.5%. In all, 3.5% of cases were negative for CT, MG, and TV but lacked speciated ureaplasma results. The remaining cases (45.8%) were considered idiopathic. Pathogen detection was associated with young age, black race, risky sexual behaviors, cloudy or purulent discharge, and visible discharge plus≥5 PMNs/HPF. In contrast, idiopathic cases were more likely to report prior NGU, were older and less likely to be black, or have an abnormal urethral discharge on examination, compared to all other cases. These cases were not associated with any high risk behaviors. CONCLUSIONS: NGU is a heterogeneous condition. Pathogen detection was associated with a variety of traditional risk factors and clinical features; whereas, idiopathic cases tended to be diagnosed among lower-risk men.

Differential association of ureaplasma species with non-gonococcal urethritis in heterosexual men.

OBJECTIVE: To assess the role of Ureaplasma urealyticum and Ureaplasma parvum in patients with non-gonococcal urethritis (NGU) using specimens from a previously reported study of NGU. METHODS: Species-specific PCR assays for U urealyticum and U parvum were used to detect these organisms in specimens from men enrolled in a case-control study based in a Seattle STD clinic in order to evaluate their association with NGU. Urethritis was defined by clinical examination and the presence of inflammation on Gram stained smear. Controls had normal examination findings and no evidence of inflammation on Gram stain smear or by the leucocyte esterase test. RESULTS: U urealyticum was detected in 26% (31/119) of cases and 16% (19/117) of controls, resulting in an association with NGU (adjusted odds ratio (aOR)=2.3, 95% CI 1.04 to 4.9) after adjusting for age, race, history of prior urethritis and other NGU pathogens (Chlamydia trachomatis, Mycoplasma genitalium). The association of U urealyticum and NGU was strongest in white men <28 years of age (OR=5.4, 95% CI 1.3 to 22.2). U parvum was detected in 14% (17/119) cases and 31% (36/117 controls) and thus was negatively associated with NGU (aOR=0.4, 95% CI 0.2 to 0.8). The prevalence of U urealyticum (16%) in controls was higher than that of C trachomatis (3.4%) or M genitalium (4.3%, p<0.05, each comparison). CONCLUSIONS: Unlike U parvum, U urealyticum was associated with urethritis. The strong effect in younger white men and high rates in controls may suggest variability in virulence among U urealyticum strains or in host innate or acquired immunity.

Clinical presentation of Mycoplasma genitalium Infection versus Neisseria gonorrhoeae infection among women with pelvic inflammatory disease.

BACKGROUND: Women with pelvic inflammatory disease (PID) often present with a spectrum of symptoms. The characteristics of nongonococcal, nonchlamydial PID have not been well described. Our objective was to examine the characteristics of Mycoplasma genitalium infection among women with clinically suspected PID. METHODS: We evaluated 722 women who were enrolled in the PID Evaluation and Clinical Health study. Women with M. genitalium monoinfection were compared with women with Neisseria gonorrhoeae monoinfection or Chlamydia trachomatis monoinfection. RESULTS: Compared with women with gonococcal PID, women with M. genitalium infection were less likely to have elevated systemic inflammatory markers, including an erythrocyte sedimentation rate >15 mm/h (5 [22.7%] of 22 patients vs. 45 [60.8%] of 74 patients; P = .002), a white blood cell count >10,000 cells/mL (4 [28.6%] of 14 patients vs. 42 [64.6%] of 65 patients; (P = .018), and an oral temperature > or =38.3 degrees C (0 [0.0%] of 22 patients vs. 10 [13.9%] of 72 patients; (P = .001). In addition, they were less likely to present with mucopurulent cervicitis (9 [47.4%] of 19 patients vs. 60 [83.3%] of 72 patients; P = .001), elevated vaginal pH (P = .018), and high pelvic pain score (P = .014). In contrast, women with chlamydial PID had signs and symptoms that were similar to those in women with M. genitalium infection. CONCLUSIONS: Because symptoms might be mild, women with M. genitalium infection might not seek PID treatment. Further studies are needed to assess the potential reproductive tract sequelae of M. genitalium infection of the upper genital tract.

Toll-like receptor variants and cervical Atopobium vaginae infection in women with pelvic inflammatory disease.

PROBLEM: Toll-like (TLR) receptor genetic variants have been implicated in bacterial vaginosis (BV). We determined whether TLR variants are associated with fastidious BV-associated microbes that are linked with infertility following pelvic inflammatory disease (PID). METHOD OF STUDY: Sneathia spp., Atopobium vaginae, BVAB1, and Ureaplasma urealyticum were measured in 250 women from the PID Evaluation and Clinical Health (PEACH) study. Relative risk (RR) and 95% confidence intervals (CI) were calculated adjusting for chlamydia and gonorrhea. Principal component analysis was used to adjust for population stratification. A false discovery rate q-value of 0.05 was significant. RESULTS: TLR2-1733C>A (P = .003) and TLR2-616A>G (P = .004) were associated with cervical A. vaginae. TLR2-1733C>A and TLR6-438C>T were associated with A. vaginae detection in the endometrium, but this was not significant after adjustment for multiple comparisons (FDR q-value = 0.06). CONCLUSION: Host gene variants in TLR2 signaling pathways were modestly associated with cervical A. vaginae in women with clinical PID.

Mycoplasma genitalium among women with nongonococcal, nonchlamydial pelvic inflammatory disease.

Pelvic inflammatory disease (PID) is a frequent condition of young women, often resulting in reproductive morbidity. Although Neisseria gonorrhoeae and/or Chlamydia trachomatis are/is recovered from approximately a third to a half of women with PID, the etiologic agent is often unidentified. We need PCR to test for M genitalium among a pilot sample of 50 women with nongonococcal, nonchlamydial endometritis enrolled in the PID evaluation and clinical health (PEACH) study. All participants had pelvic pain, pelvic organ tenderness, and leukorrhea, mucopurulent cervicitis, or untreated cervicitis. Endometritis was defined as > or =5 surface epithelium neutrophils per x400 field absent of menstrual endometrium and/or > or =2 stromal plasma cells per x120 field. We detected M genitalium in 7 (14%) of the women tested: 6 (12%) in cervical specimens and 4 (8%) in endometrial specimens. We conclude that M genitalium is prevalent in the endometrium of women with nongonococcal, nonchlamydial PID.

High Mycoplasma genitalium organism burden is associated with shedding of HIV-1 DNA from the cervix.

We assessed the relationship between infection with Mycoplasma genitalium, an emerging sexually transmitted pathogen, and cervical shedding of human immunodeficiency virus (HIV)-1 DNA among 303 HIV-1-positive Kenyan women. HIV-1 shedding was detected by qualitative polymerase chain reaction (PCR) in 154 women (51%); M. genitalium was detected by qualitative PCR in 52 (17%), and organism burden was determined by quantitative PCR. Women with high M. genitalium organism burdens (more than the median of 3195 genomes/mL) were 3-fold more likely to shed HIV-1 DNA than were M. genitalium-negative women (adjusted OR, 2.9 [95% confidence interval, 1.1-7.6]), yet this did not appear to be mediated by traditional measures of cervical inflammation (elevated polymorphonuclear leukocyte count).

mgpB and mgpC sequence diversity in Mycoplasma genitalium is generated by segmental reciprocal recombination with repetitive chromosomal sequences.

Mycoplasma genitalium is associated with sexually transmitted infections in men and women that, if untreated, can persist, suggesting that mechanism(s) exist to facilitate immune evasion. Approximately 4% of the limited M. genitalium genome contains repeat sequences termed MgPar regions that have homology to mgpB and mgpC, which encode antigenic proteins associated with attachment. We have previously shown that mgpB sequences vary within a single strain of M. genitalium in a pattern consistent with recombination between mgpB and MgPar sequences (Iverson-Cabral et al.). In the current study, we show that mgpC heterogeneity similarly occurs within the type strain, G-37(T), cultured in vitro and among cervical specimens collected from a persistently infected woman. In all cases, alternative mgpC sequences are indicative of recombination with MgPar regions. Additionally, the isolation of single-colony M. genitalium clonal variants containing alternative mgpB or mgpC sequences allowed us to demonstrate that mgpB and mgpC heterogeneity is associated with corresponding changes within donor MgPar regions, consistent with reciprocal recombination. Better-defined systems of antigenic variation are typically mediated by unidirectional gene conversion, so the generation of genetic diversity observed in M. genitalium by the mutual exchange of sequences makes this organism unique among bacterial pathogens.

Mycoplasma genitalium infection and persistence in a cohort of female sex workers in Nairobi, Kenya.

OBJECTIVE: The objective of this study was to assess the risk factors for and persistence of Mycoplasma genitalium (MG) in a highly exposed female population in Kenya. STUDY DESIGN: Two hundred fifty-eight sex workers in Nairobi, Kenya, 18 to 35 years of age, were enrolled. Every 2 months, cervical samples were collected for MG, Chlamydia trachomatis (CT), and Neisseria gonorrhoeae (GC) testing by polymerase chain reaction. RESULTS: At enrollment, 16% were infected with MG. Seventy-seven subjects acquired 107 MG infections, giving an incidence of 22.7 per 100 women-years. Incident CT (adjusted hazard ratio [HR] = 2.4; 95% confidence interval [CI] = 1.5-4.0), GC (HR = 2.0; 95% CI = 1.2-3.5), and HIV infection (adjusted HR = 2.2; 95% CI = 1.3-3.7) were associated with an increased risk of MG. Seventeen percent, 9%, and 21% of MG infections persisted 3, 5, and >or=7 months, respectively. CONCLUSION: The high incidence of MG, greater than that for both CT (14.0%) and GC (8%), association with common sexually transmitted infection risk factors, and persistence in the female genital tract supports its role as a common sexually transmitted infection in Kenyan women.

Sentinel surveillance of sexually transmitted infections/HIV and risk behaviors in vulnerable populations in 5 Central American countries.

In El Salvador, Guatemala, Honduras, Nicaragua, and Panama, we recruited 2466 female sex workers (FSWs) by probabilistic or comprehensive sampling and 1418 men who have sex with men (MSM) by convenience sampling to measure sociobehavioral risk and sexually transmitted infections. For MSM, HIV seroprevalence ranged from 7.6% in Nicaragua to 15.3% in El Salvador, and estimated HIV seroincidence per 100 person-years ranged from 2.7 in Panama to 14.4 in Nicaragua; 61% reported using condoms consistently with casual male partners, 29% reported exposure to behavioral interventions, and 22% reported recent sex with male and female partners. For FSWs, HIV seroprevalence ranged from 0.2% in Nicaragua and Panama to 9.6% in Honduras, where estimated HIV seroincidence was also highest (3.2 per 100 person-years); 77% and 72% of FSWs reported using condoms consistently with new and regular clients. Herpes simplex virus (HSV)-2 seroprevalence averaged 85.3% in FSWs and 48.2% in MSM, and syphilis seropositivity averaged 9.6% in FSWs and 8.3% in MSM. Chlamydia trachomatis and Neisseria gonorrhoeae prevalences in FSWs averaged 20.1% and 8.1%, and Trichomonas vaginalis and bacterial vaginosis prevalences averaged 11.0% and 54.8%. An ongoing HIV epidemic involves Central American MSM with potential bridging to women. In FSWs, HSV-2 infection was associated with HIV infection (odds ratio = 11.0, 95% confidence interval: 2.9 to 7.9). For these vulnerable populations, prevention must incorporate acceptable and effective sexual health services, including improved condom access and promotion.

Intrastrain heterogeneity of the mgpB gene in Mycoplasma genitalium is extensive in vitro and in vivo and suggests that variation is generated via recombination with repetitive chromosomal sequences.

Mycoplasma genitalium is associated with reproductive tract disease in women and may persist in the lower genital tract for months, potentially increasing the risk of upper tract infection and transmission to uninfected partners. Despite its exceptionally small genome (580 kb), approximately 4% is composed of repeated elements known as MgPar sequences (MgPa repeats) based on their homology to the mgpB gene that encodes the immunodominant MgPa adhesin protein. The presence of these MgPar sequences, as well as mgpB variability between M. genitalium strains, suggests that mgpB and MgPar sequences recombine to produce variant MgPa proteins. To examine the extent and generation of diversity within single strains of the organism, we examined mgpB variation within M. genitalium strain G-37 and observed sequence heterogeneity that could be explained by recombination between the mgpB expression site and putative donor MgPar sequences. Similarly, we analyzed mgpB sequences from cervical specimens from a persistently infected woman (21 months) and identified 17 different mgpB variants within a single infecting M. genitalium strain, confirming that mgpB heterogeneity occurs over the course of a natural infection. These observations support the hypothesis that recombination occurs between the mgpB gene and MgPar sequences and that the resulting antigenically distinct MgPa variants may contribute to immune evasion and persistence of infection.

Detection of novel organisms associated with salpingitis, by use of 16S rDNA polymerase chain reaction.

Although Chlamydia trachomatis and Neisseria gonorrhoeae are established causes of salpingitis, the majority of cases have no known etiology. We used broad-range 16S rDNA polymerase chain reaction to identify novel, possibly uncultivable, bacteria associated with salpingitis and identified bacterial 16S sequences in Fallopian-tube specimens from 11 (24%) of 45 consecutive women with laparoscopically confirmed acute salpingitis (the case patients) and from 0 of 44 women seeking tubal ligations (the control subjects) at Kenyatta National Hospital, Nairobi, Kenya. Bacterial phylotypes most closely related to Leptotrichia spp. were detected as the sole phylotypes in 1, and mixed with other bacterial phylotypes in 2, specimens. Novel bacterial phylotypes and those associated with bacterial vaginosis, including Atopobium vaginae, were identified in 3 specimens. N. gonorrhoeae and Streptococcus pyogenes were identified in 2 and 1 specimens, respectively. The finding of novel phylotypes associated with salpingitis has important implications for the etiology, pathogenesis, and treatment of this important reproductive-tract disease syndrome.

Pilot study of COBAS PCR and ligase chain reaction for detection of rectal infections due to Chlamydia trachomatis.

We tested rectal specimens from men who have sex with men for Chlamydia trachomatis by using COBAS PCR (Roche Diagnostics) and ligase chain reaction LCR (Abbott laboratories) and compared three PCR specimen-processing procedures. Chlamydiae were detected by one or more procedures in 22 of 186 specimens. All three PCR tests were positive for 17 specimens, all of which also tested positive by LCR.