A clinical study on the association of Trichomonas vaginalis and Mycoplasma hominis infections in women attending a sexually transmitted disease (STD) outpatient clinic

Swabs from the posterior vaginal fornix were obtained from 804 consecutive female patients visiting a large Dutch sexually transmitted diseases (STD) outpatient clinic. A detailed clinical history was obtained and complaints concerning the lower genital tract, such as vaginal discharge or vulval and vaginal irritation, were recorded. Patients were examined and the presence of non-physiological vaginal secretions was established by speculum examination. The swabs were monitored for bacterial vaginosis (BV) or Candida albicans infection. PCR diagnosis of Chlamydia trachomatis and Trichomonas vaginalis was performed as well. Four groups of patients (n=14-21) with BV or single infections caused by one of these three pathogens and a control group with no pathogens were selected and Mycoplasma hominis PCR was performed additionally. At clinical presentation, controls and single-infected patient groups were comparable with regard to complaints of the lower genital tract and sexual risk behavior defined as having prior STDs and/or admitted prostitution. Only in the T. vaginalis-positive group significantly more women reporting sexual risk behavior were found than in controls. In agreement with former in vitro observations, an in vivo association between the PCR-detected presence of M. hominis and T. vaginalis was established. In 79% of all samples positive for T. vaginalis, M. hominis could be detected, as compared to only 6% in control samples (P=0.0004). However, since single infections by either of the two pathogens were regularly observed, there does not seem to be an exclusive association between the species, as the bacterium is also more frequently found in cases of BV (P=0.026). Co-infection of M. hominis with C. albicans (11%) or C. trachomatis (0%) did not differ significantly from controls (6%). M. hominis did not associate with complaints of the lower genital tract. However, if all groups were combined there appears to be a very significant association between the presence of M. hominis and sexual risk behavior (P=0.0004). M. hominis and sexual risk behavior were more closely associated than M. hominis and T. vaginalis. No indications were found for an enhanced pathogenicity by either of the symbionts.     

Dynamics of vaginal bacterial communities in women developing bacterial vaginosis, candidiasis, or no infection, analyzed by PCR-denaturing gradient gel electrophoresis and real-time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

Genetic and biochemical diversity of Gardnerella vaginalis strains isolated from women with bacterial vaginosis

Gardnerella vaginalis is considered a substantial player in the progression of bacterial vaginosis (BV). We analysed 17 G. vaginalis strains isolated from the genital tract of women diagnosed with BV to establish a potential link between genotypes/biotypes and the expression of virulence factors, vaginolysin (VLY) and sialidase, which are assumed to play a substantial role in the pathogenesis of BV. Amplified ribosomal DNA restriction analysis revealed two G. vaginalis genotypes. Gardnerella vaginalis isolates of genotype 2 appeared more complex than genotype 1 and were subdivided into three subtypes. Biochemical typing allowed us to distinguish four different biotypes. A great diversity of the level of VLY production among the isolates of G. vaginalis may be related to a different cytotoxicity level of the strains. We did not find any correlation between VLY production level and G. vaginalis genotype/biotype. In contrast, a link between G. vaginalis genotype and sialidase production was established. Our findings on the diversity of VLY expression level in different clinical isolates and linking sialidase activity with the genotype of G. vaginalis could help to evaluate the pathogenic potential of different G. vaginalis strains.     

四川地区阴道毛滴虫内人型支原体的PCR检测

从临床上分离获得20株阴道毛滴虫虫株,经纯化培养后,提取基因组DNA.以人型支原体16S rDNA序列设计特异性引物,利用PCR技术检测阴道毛滴虫内的人型支原体,结果有13株为人型支原体阳性,感染率为65%,表明阴道毛滴虫与人型支原体的共生关系在中国四川具有普遍性.     

Frequency of detection of Ureaplasma urealyticum and Mycoplasma hominis in cervical canal and the Douglas pouch of infertile and fertile women

The group of organisms commonly referred to as genital mycoplasmas comprise species most often found in genitourinary tract of sexually active adults as common commensal inhabitants, or pathogens which can possibly cause many different pathologies like: non-gonococcal urethritis, bacterial vaginosis, cervicitis, endometritis or pelvic inflammatory disease. The problem of their morbidity and the possible influence they have on human fertility is still not clear. The aim of this study was to find out whether two investigated species- Ureaplasma urealyticum and Mycoplasma hominis can be detect more often in a group of infertile women. 74 women participated in the study and were assigned to one of 2 groups of patients: infertile women and fertile women without any sign of genital tract infection. Swabs from the cervical canal of the uterus and the fluid from the Douglas pouch were taken during the gynecological examination and laparoscopic procedure. Two diagnostic methods were used: biochemical method- commercial diagnostic kit- Mycoplasma IST 2 and PCR method. The results showed that Ureaplasma urealyticum and Mycoplasma hominis were detected among both fertile and infertile women with nearly the same frequency, much more often in cervical canal than in the Douglas pouch. Ureaplasma urealyticum was more common pathogen than Mycoplasma hominis in both groups and locations. The achieved results point out that the role of genital mycoplasmas in human infertility is still unclear and require further investigations.     

Mycoplasma hominis and Trichomonas vaginalis symbiosis: multiplicity of infection and transmissibility of M. hominis to human cells

We recently reported that most Trichomonas vaginalis isolates cultured in vitro are infected by Mycoplasma hominis. In this work, we have characterized some aspects of the relationships between the two microorganisms. PCR, cultivation, and immunological methods revealed that the number of M. hominis organisms carried by T. vaginalis in culture varied from isolate to isolate, suggesting a specific multiplicity of infection. Moreover, infected T. vaginalis isolates were able to pass bacteria not only to M. hominis-free protozoa, but also to human-derived epithelial cells. The in vitro transmission of the bacterium from T. vaginalis to both uninfected parasite isolates and human epithelial cells suggests a role for T. vaginalis as a carrier of the M. hominis infection in vivo.     

Impact of Mycoplasma hominis and Ureaplasma urealyticum on the concentration of proinflammatory cytokines in vaginal fluid

The main aim of this study was to determine impact of Mycoplasma hominis and Ureaplasma urealyticum on the concentrations of selected proinflammatory cytokines in vaginal fluid in pregnant women. The samples were obtained from 120 pregnant women at 22 to 36 weeks gestation. Vaginal fluid were analyzed for the concentrations of IL-1 alpha, IL-1 beta, IL-6 and IL-8 using standard enzyme-linked immunosorbent assay technique (ELISA), and cervical fluid for prevalence of Mycoplasma hominis and Ureaplasma urealyticum. Genital mycoplasmas were diagnosed in 36 of 120 pregnant women (30%), (in 17 of 36 women (47.2%) both M. hominis and U. urealyticum, in 14 women (38.9%) only U. urealyticum, and in 5 cases (13.8%) only M. hominis were diagnosed). Vaginal levels of IL-8 was statistically higher among women with genital mycoplasmas infection, as compared to group without these bacteria (p=0.033), while there was no correlation between IL-1 alpha, IL-1 beta and IL-6 concentrations and genital mycoplasmas infection. Future studies should concentrate on evaluation the impact of other lower genital tract bacteria on concentration of IL-8 and other proinflammatory cytokines.     

生殖道感染与宫颈癌及宫颈癌前病变的相关性研究

目的:探讨生殖道感染与宫颈癌、宫颈癌前病变的相关性及危险因素分析。方法:选取我院收治的185例宫颈癌及宫颈癌前病变患者和同期206例健康体检者分为两组,对人乳头瘤病毒(HPV)、沙眼衣原体(CT)、细菌性阴道病(BV)、阴道滴虫进行检测,观察分析相应病原微生物导致的生殖道感染与宫颈癌及宫颈癌前病变的关系,同时根据HPV不同基因型在宫颈癌及宫颈癌前病变中的致癌作用和程度,判定危险程度。结果:宫颈癌及宫颈癌前病变患者上述指标感染检出率明显高于对照组,差异具有统计学意义(P=0.000、0.001、0.000、0.037),其中高危HPV感染率随宫颈上皮内瘤变级别的升高而呈明显上升趋势,CINⅠ～Ⅲ级感染率分别为57.1%、78.6%和82.9%,宫颈癌感染率最高达91.1%,高危HPV为高危因素。结论:生殖道HPV、CT、BV、滴虫感染与宫颈癌及CIN存在必然或一定相关性,加强对妇女生殖道病原体感染的重视和检测对防治宫颈癌及CIN具有积极意义。     

Performance of swabs, lavage, and diluents to quantify biomarkers of female genital tract soluble mucosal mediators

Background

Measurement of immune mediators and antimicrobial activity in female genital tract secretions may provide biomarkers predictive of risk for HIV-1 acquisition and surrogate markers of microbicide safety. However, optimal methods for sample collection do not exist. This study compared collection methods.

Methods

Secretions were collected from 48 women (24 with bacterial vaginosis [BV]) using vaginal and endocervical Dacron and flocked swabs. Cervicovaginal lavage (CVL) was collected with 10 mL of Normosol-R (n = 20), saline (n = 14), or water (n = 14). The concentration of gluconate in Normosol-R CVL was determined to estimate the dilution factor. Cytokine and antimicrobial mediators were measured by Luminex or ELISA and corrected for protein content. Endogenous anti-HIV-1 and anti-E. coli activity were measured by TZM-bl assay or E. coli growth.

Results

Higher concentrations of protein were recovered by CVL, despite a 10-fold dilution of secretions, as compared to swab eluents. After protein correction, endocervical swabs recovered the highest mediator levels regardless of BV status. Endocervical and vaginal flocked swabs recovered significantly higher levels of anti-HIV-1 and anti-E. coli activity than Dacron swabs (P<0.001). BV had a significant effect on CVL mediator recovery. Normosol-R tended to recover higher levels of most mediators among women with BV, whereas saline or water tended to recover higher levels among women without BV. Saline recovered the highest levels of anti-HIV-1 activity regardless of BV status.

Conclusions

Endocervical swabs and CVL collected with saline provide the best recovery of most mediators and would be the optimal sampling method(s) for clinical trials.     

Limited secretory-IgA response in cervicovaginal secretions from HIV-1 infected, but not high risk seronegative women: lack of correlation to genital viral shedding

The presence and antigen specificity of IgG and secretory-IgA (s-IgA) to HIV-1 were evaluated in cervicovaginal lavages (CVL) from 26 infected and 10 high-risk seronegative women. All the seropositive women had detectable IgG recognizing several viral antigens, while a smaller percentage of women demonstrated s-IgA to the virus. In addition, s-IgA were of limited specificity and provided weak reactivities on Immunoblot bands; an almost constant absence of s-IgA to gp120 was also observed. Neither the presence nor the specificity of either IgG or s-IgA to the virus in CVL prevented the shedding of HIV-1 in this body fluid; in fact, viral RNA was detected in all the women studied and the amounts of viral shedding was unrelated to the genital antibody response. On the other hand, none of the high-risk seronegative women had detectable antibodies to HIV-1 in CVL of either the IgG or s-IgA isotype. Our results a) confirm an impairment of mucosal antibody response during HIV-1 infection and suggest that mucosal immunity is not able to prevent viral shedding in the female genital tract and thus cannot modulate the infectivity of genital secretions; aa) do not provide evidence for a mucosal "memory/protective" antibody response in the genital tract of high-risk seronegative women.     

Mycoplasma hominis in Cuban Trichomonas vaginalis isolates: association with parasite genetic polymorphism

Trichomonas vaginalis can be naturally infected with intracellular Mycoplasma hominis. This bacterial infection may have implications for trichomonal virulence and disease pathogenesis. The objective of the study was to report the presence of M. hominis in Cuban T. vaginalis isolates and to describe the association between the phenotype M. hominis infected with RAPD genetic polymorphism of T. vaginalis. The Random Amplified Polymorphic DNA (RAPD) technique was used to determine genetic differences among 40 isolates of T. vaginalis using a panel of 30 random primers and these genetic data were correlated with the infection of isolates with M. hominis. The trees drawn based on RAPD data showed no relations with metronidazole susceptibility and significantly association with the presence of M. hominis (P=0.043), which demonstrates the existence of concordance between the genetic relatedness and the presence of M. hominis in T. vaginalis isolates. This result could point to a predisposition of T. vaginalis for the bacterial enters and/or survival.     

Trichomonas vaginalis detection using real-time TaqMan PCR

1978 women and 93 men, all suspected of having a Trichomonas vaginalis infection, were tested for the presence of T. vaginalis by real-time PCR using the T. vaginalis-specific 2-kb repeated sequence, and by direct microscopy and culture. 40 samples were positive by T. vaginalis real-time PCR and 27 were positive by wet mount microscopy, either direct or after culture. All samples positive by direct microscopy of culture were also positive by real-time PCR. Of the 13 samples which were real-time PCR positive but negative by direct microscopy and culture 11 were confirmed by another T. vaginalis real-time PCR based on the beta tubulin gene. Only 2 samples (0.1%) showed inhibition in the PCR. The prevalence of T. vaginalis infection in the female patients was 1.8%. The sensitivity, specificity, positive and negative predictive values of the real-time PCR were 100%, 99.9%, 95% and 100%, respectively. The same test characteristics for the combined conventional T. vaginalis detection methods (microscopy+culture) were 71%, 100%, 100% and 99%, respectively. Therefore, real-time PCR is the method of choice for the diagnosis of T. vaginalis infection.     

Treatment and outcome of CPD-associated peritonitis

Backround

Vaginitis is among the most common conditions women are seeking medical care for. Although these infections can easily be treated, the relapse rate is high. This may be due to inadequate use of the diagnostic potential.

Methods

We evaluated the misjudgement rate of the aetiology of vaginal complaints. A total of 220 vaginal samples from women with a vaginal complaint were obtained and analysed for numbers of total lactobacilli, H₂O₂-producing lactobacilli, total aerobic cell counts and total anaerobic cell counts including bifidobacteria, Bacteroides spp., Prevotella spp. Additionally, the presence of Atopobium vaginae, Gardnerella vaginalis, Candida spp. and Trichomonas vaginalis was evaluated by DNA-hybridisation using the PCR and Affirm VPIII Microbial Identification Test, respectively.

Results

The participating physicians diagnosed Bacterial vaginosis (BV) as origin of discomfort in 80 cases, candidiasis in 109 cases and mixed infections in 8 cases. However, a present BV, defined as lack of H₂O₂-lactobacilli, presence of marker organisms, such as G. vaginalis, Bacteroides spp. or Atopobium vaginae, and an elevated pH were identified in only 45 cases of the women examined. Candida spp. were detected in 46 cases. Interestingly, an elevated pH corresponded solely to the presence of Atopobium vaginae, which was detected in 11 cases.

Conclusion

Errors in the diagnosis of BV and candida vulvovaginitis (CV) were high. Interestingly, the cases of misjudgement of CV (77%) were more numerous than that of BV (61%). The use of Amsel criteria or microscopy did not reduce the number of misinterpretations. The study reveals that the misdiagnosis of vaginal complaints is rather high.     

Extraction Parameters Significantly Influence the Quantity and the Profile of PLFAs Extracted from Soils

The female genital tract (FGT) harbors very large numbers of bacterial species that are known to play an important role on vaginal health. Previous studies have focused on bacterial diversity in the vagina, but little is known about the ectocervical microbiota associated with FGT infections. In our study, vaginal swabs and ectocervical swabs were collected from 100 participants in China, including 30 women with bacterial vaginosis (BV; BV group), 22 women with cervicitis (Cer group), 18 women with BV in combination with cervicitis (BC group) and 30 healthy control women (CN group). The diversity and richness of cervicovaginal microbiota were investigated with culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) targeting 11 microorganisms that have been associated with FGT infections. Despite significant interpersonal variations, the PCR-DGGE profiles revealed that vaginal microbiota and ectocervical microbiota were clearly much more complex in the BV group, while the ectocervical microbiota showed no significant difference between healthy and diseased participants. Using species-specific qPCR, BV and cervicitis were significantly associated with a dramatic decrease in Lactobacillus species (p < 0.05), and potential pathogenic species such as Gardnerella, Atopobium, Eggerthella, Leptotrichia/Sneathia, and Prevotella were more common and in higher copy numbers in BV than in CN samples (p values ranged from 0.000 to 0.021). No significant differences were observed between healthy and cervicitis samples (p > 0.05) in ectocervical microbiota. The total numbers of bacteria were significantly lower in the ectocervix as compared in the vagina (p < 0.05). Intriguingly, vaginal microbiota from participants with BV in combination with cervicitis was quite different from that of participants with BV or cervicitis alone. Our study demonstrated that the cervicovaginal microbiota was actively involved in the process of FGT infections. The predominant bacteria of the cervicovaginal communities were clearly associated with BV; however, there was not sufficient evidence that the ectocervical microbiota is directly involved in the development of cervicitis.     

Prevalence of Chlamydia trachomatis and genital mycoplasmas in asymptomatic women.

To establish the prevalence of Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum in women attending a family planning and a prenatal clinic in Halifax, cervical swabs were obtained at the time of the first visit from 491 women who had no symptoms of genital infection. Among the women attending the family planning clinic M. hominis occurred in combination with C. trachomatis more frequently than expected (p less than 0.05). It occurred in the absence of U. urealyticum in only a few cases (13% of the occurrences in the family planning clinic and 6% of those in the prenatal clinic). C. trachomatis was significantly more prevalent in women under 25 years of age (p less than 0.04). However, mycoplasmas were as prevalent in women over 30 years as in those under 30. There were no significant differences in the infection rates of the organisms by trimester among pregnant women. More research is necessary for a proper understanding of the role of M. hominis and U. urealyticum in genitourinary infections and pregnancy outcomes.     

Evidence for Gardnerella vaginalis uptake and internalization by squamous vaginal epithelial cells: implications for the pathogenesis of bacterial vaginosis

Bacterial vaginosis (BV), a common condition seen in premenopausal women, is associated with preterm labor, pelvic inflammatory disease, and delivery of low birth weight infants. Gardnerella vaginalis is the predominant bacterial species associated with BV, although its exact role in the pathology of BV is unknown. Using immunofluorescence, confocal and transmission electron microscopy, we found that VK2 vaginal epithelial cells take up G. vaginalis after exposure to the bacteria. Confocal microscopy also indicated the presence of internalized G. vaginalis within vaginal epithelial cells obtained from a subject with BV. Using VK2 cells and (35)S labeled bacteria in an invasion assay, we found that a 1 h uptake of G. vaginalis was 21.8-fold higher than heat-killed G. vaginalis, 84-fold compared to Lactobacillus acidophilus and 6.6-fold compared to Lactobacillus crispatus. Internalization was inhibited by pre-exposure of cells to cytochalasin-D. In addition, the cytoskeletal protein vimentin was upregulated in VK2 cells exposed to G. vaginalis, but there was no change in actin cytoskeletal polymerization/rearrangements or vimentin subcellular relocalization post exposure. Cytoskeletal protein modifications could represent a potential mechanism for G. vaginalis mediated internalization by vaginal epithelial cells. Finally, understanding vaginal bacteria/host interactions will allow us to better understand the underlying mechanisms of BV pathogenesis.     

Comparison of polymerase chain reaction assay and Mycoplasma IST 2 test with culture for detection of infections caused by Ureaplasma urealyticum and Mycoplasma hominis

The polymerase chain reaction (PCR) technique and commercial Mycoplasma IST 2 test were compared with culture for the detection of U. urealyticum and M. hominis in 173 clinical samples obtained from patients without clinical symptoms from genito-urinary tract. The presence of U. urealyticum was diagnosed by culture in 24 samples, by PCR in 33 samples and by Mycoplasma IST 2 test in 39 samples. The presence of M. hominis was diagnosed in 26 samples only by Mycoplasma IST 2 test--culture and PCR were negative. The study showed the excellent sensitivity (100%) and good specificity (appropriately 94.0% and 90.0%) for U. urealyticum in PCR and Mycoplasma IST 2 test. The discrepancy of results obtained in Mycoplasma IST 2 test and culture as well as in PCR may suggest the over sensitivity of the commercial test for detection of M. hominis.     

联合法对5种常见下生殖道感染的鉴别诊断

目的摘要：目的对比分析白带常规镜检法、细菌预成酶谱分析法及联合法对5种常见下生殖道感染鉴别诊断的有效率。方法分别采用白带常规镜检法、细菌预成酶谱分析法对来我院就诊的3420例患者的阴道分泌物进行检测。结果采用白带常规镜检检出阴道感染患者1636例,其中BV患者543例、VVC患者820例、TV患者225例、CV患者48例;采用细菌预成酶谱分析法检出阴道感染患者1078例,其中AV患者461例、BV患者617例;采用联合法检出阴道感染患者2171例,其中AV患者461例、BV患者617例、CV患者48例、TV患者225例、VVC患者820例。结论联合法集白带常规镜检法和细菌预成酶谱分析法的优点与一体,能够鉴别诊断5种常见下生殖道感染。     

Prevalence of cervical pathogens in women with and without inflammatory changes on smear testing.

OBJECTIVE--To assess correlation between nonspecific cervicitis, inflammation, or exudate on cervical smears tests and confirmed presence of known cervical pathogens. DESIGN--Investigation of women attending a family practice clinic for smear test by microbiological screening for Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, Trichomonas vaginalis, Candida species, group B streptococcus, Gardnerella vaginalis, and Neisseria gonorrhoeae. SETTING--Family practice teaching clinic in a university hospital. PATIENTS--411 women presenting for a smear test. MAIN OUTCOME MEASURES--Prevalence of genital infections associated with presence or absence of inflammatory changes on cervical smear. RESULTS--Of the 132 women with inflammatory changes on cervical smear, 64 (48%) had positive cultures. Of the 248 without inflammatory changes, 117 (47%) had positive cultures. Subgroup analysis on individual organisms also showed no significant difference between the two groups. CONCLUSION--Reports of inflammatory changes on cervical smear testing are a poor indicator of infection.