Bacterial vaginosis (BV) is the most common form of vaginitis worldwide and has been linked to multiple reproductive tract complications including preterm birth and increased risk of acquiring STD/HIV.1–3 The pathogenesis of BV is poorly understood and there is lack of consensus on a possible etiologic agent, if any. The majority of epidemiologic data, however, suggests that BV is an STD.4–7 Early on, in the recognition of this syndrome it was accepted that Gardnerella vaginalis was the causative organism; however, this fell out of favor when it was reported that G. vaginalis could be cultured from women who did not meet the Amsel criteria for BV.8 In retrospect, although these women did not meet the clinical criteria for the diagnosis of BV, they also may have not had normal vaginal flora. Although BV flora is characterized by a multitude of organisms, the presence of G. vaginalis in the vaginal flora of women with BV is universal. Studies of G. vaginalis in men have been limited and few studies of concordance among sexual partners have been published and none using polymerase chain reaction (PCR). We determined the presence or absence of this organism from genitourinary specimens of male sexual partners of women with and without normal vaginal flora using G. vaginalis-specific PCR.
Women attending the Jefferson County Department of Health STD Clinic for screening or a new problem were invited to participate in the study. Women were classified as having BV if they met the clinical criteria of Amsel et al.9 or as negative for BV if they did not fulfill this criteria. Women were administered standardized questionnaires regarding sexual behavior and a vaginal smear was obtained for Gram stain using the method of Nugent et al.10 Women were asked to refer sexual partners with whom they had had sexual intercourse within the past 2 weeks to the study for enrollment in the study. Men were also administered a questionnaire and examined for signs of STD. First fraction urine (15–20 mL) was collected, centrifuged, and the pellet resuspended in 5 mL of urine. The pellet mixture was refrigerated on-site then stored at −20 °C before processing. Swabs were obtained from the coronal sulcus and the urethra, placed into a dry sterile screw-capped tube, refrigerated on-site then stored at −20 °C before processing.
Total DNA was extracted from urine sediment, urethral, and coronal sulcus swab specimens by inorganic extraction (Wizard Genomic Purification Kit, Promega, Madison, WI).11 Amplification of a conserved sequence within the G. vaginalis 16 S rRNA gene was carried out using previously reported primers: GV-V6-U2 5′ GACCATGCACCACCTGTGAA 3′ and GV-V2-R1 5′ TCGTGGAGGGTTCGATTCTG 3′.12 Reactions were carried out in 20 μL containing template DNA, 1 × CoralLoad Buffer (Qiagen, Valencia, CA), 200 μmol/L of each dNTP, 0.5 μmol/L of each primer, and 0.025 U/μL of HotStarTaq Plus (Qiagen, Valencia, CA). Amplification conditions on an iCycler (BioRad Laboratories, Hercules, CA) were as follows: 95 °C × 5′/(94 °C × 30′; 60 °C × 30′; 72 °C × 30′) × 30/72 °C × 5′/4°C pause. Two negative controls were run on each amplification batch: water as template for reagent check and Mobiluncus mulieris. The positive control was laboratory cultured G. vaginalis. Amplicons were electrophoresed on 2% agarose/1 × tris acetic acid EDTA (TAE) gels prestained with ethidium bromide, followed by photodigital documentation. G. vaginalis positivity (a band of 1041 base pairs) was defined as a positive PCR from at least 1 sampling of the specimen set.
For comparisons of categorical data, Fisher exact test, and the χ2 test were used. The Wilcoxon rank sum test was used to compare groups about continuous variables because a number of variables were not normally distributed.
A total of 68 men were enrolled into the study, 29 (43%) as partners of women with BV and 39 (57%) as partners of women without BV. For the present analysis we limited inclusion to only those 47 (69.1%) men who had available specimens from all 3 sampling sites (urine, urethral swab, coronal sulcus swab). Women with BV by Amsel criteria had a median baseline Nugent score of 8 versus 2 for the women without BV (P <0.001). The only significant difference other than the baseline Nugent scores of the female partners was race. There was a significantly higher proportion of black subjects who were partners of women with BV than those who were partners to women without BV (Table 1). There were no significant differences between the 47 men who had samples available and the larger cohort (data not shown).
G. vaginalis was detected in 12 patients [25%, 95% confidence interval (CI) from 14% to 40%]. Urethral specimens were positive in 8/12 (66.6%) of the infected subjects. One patient had a positive result only from the coronal sulcus and 3 patients were positive only in the urine (Table 2). Twenty-six percent of partners of women with BV tested positive for Gardnerella versus 25% of partners of women without BV. There was no significant difference in the rates of G. vaginalis between men who were sexual partners of women with and without BV by Amsel criteria, women with and without normal Nugent scores of 0 to 3, or women with and without bacterial morphotypes on vaginal Gram stain that were consistent with G. vaginalis.
None of the 11 participants who stated that they had used a condom at the last sexual encounter tested positive for G. vaginalis in contrast to 12/36 (33%) of those who did not use a condom at the last sexual encounter (P = 0.04). None of the 5 participants who stated that they always used a condom in the previous 3 months were positive for G. vaginalis. In comparison, 12/42 (29%) of those who did not always use a condom in the past 3 months were positive for G. vaginalis (P = 0.31) (Table 3).
The significance of G. vaginalis in the pathogenesis of BV remains controversial; however, nearly all women with BV have high concentrations of this organism.13 Like Neisseria gonorrhoeae, it is a fastidious bacterium that is recovered primarily from genitourinary sites in both sexes and is unstable when removed from its natural environment.14 The human vagina provides an especially rich culture medium, the presence of glycogen being an in vivo growth requirement.15 In males, G. vaginalis has been repeatedly recovered from the urethra and from seminal fluid.16–20
Sexual contact studies point to the sexual transmissibility of G. vaginalis. In 1955, Gardner and Dukes reported that they had isolated Gardnerella from the urethras of 86% of husbands of women with BV.16 Similarly, in 1978, Pheiffer et al. demonstrated the concordance of Gardnerella among 79% of couples in which the woman had BV versus 0% of couples in which the woman did not have BV.21 Biotyping of G. vaginalis has yielded compelling results supporting the sexual transmission of this bacterium. Piot et al. found that biotypes isolated from women with BV and from the urethras of their sexual partners were identical 90% of the time.22 In a longitudinal study of G. vaginalis biotypes, Briselden and Hillier found that lipase-producing strains were associated with BV and that although not statistically significant there was an association between acquisition of a new biotype and new sex partners.23 Ninety percent of women who developed BV acquired a new biotype suggesting a new infection rather than relapse.23 Holst cultured the urethra, coronal sulcus, and seminal fluid of male partners of women with and without BV. They found that 17% of male partners of women with BV were infected versus 2% of partners of women without BV. Further, among men who had specimens taken within 20 hours of unprotected sex with women with BV and then 2 weeks later after interval condom usage, G. vaginalis was only isolated from specimens taken after recent unprotected intercourse.24
In our study, using PCR, we found an overall prevalence of G. vaginalis of 25% but found no difference in the prevalence rates between male sexual partners of women with and without BV. This unexpected finding may be the result of the fact that both groups of men were recruited from the same high risk cohort of patients attending an STD clinic. In fact, the overall prevalence of BV among women attending the clinic is between 50% and 60% (unpublished data). It is therefore possible that the male partners of women without BV were either exposed to abnormal vaginal flora in their referring partners at an earlier time or that they were exposed via a second partner. Although a few men had G. vaginalis detected externally from the coronal sulcus, most cases were detected using either urethral swab specimens or urine. Condom use with the last sexual encounter was significantly associated with a reduced rate of G. vaginalis detection and there was a similar trend for those men who reported always using condoms in the previous 3 months. Condom use has been shown in several recent studies to be protective for acquisition of BV in women.25,26 Additional studies examining concordance of BV-related pathogens in couples, especially couples at lower risk of STD, may be beneficial in contributing to our knowledge of the pathogenesis of BV.
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