Hans Verstraelen

Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis

BackgroundThe pathogenesis of bacterial vaginosis remains largely elusive, although some microorganisms, including Gardnerella vaginalis, are suspected of playing a role in the etiology of this disorder. Recently culture-independent analysis of microbial ecosystems has proven its efficacy in characterizing the diversity of bacterial populations. Here, we report on the results obtained by combining culture and PCR-based methods to characterize the normal and disturbed vaginal microflora.ResultsA total of 150 vaginal swab samples from healthy women (115 pregnant and 35 non-pregnant) were categorized on the basis of Gram stain of direct smear as grade I (n = 112), grade II (n = 26), grade III (n = 9) or grade IV (n = 3). The composition of the vaginal microbial community of eight of these vaginal swabs (three grade I, two grade II and three grade III), all from non-pregnant women, were studied by culture and by cloning of the 16S rRNA genes obtained after direct amplification. Forty-six cultured isolates were identified by tDNA-PCR, 854 cloned 16S rRNA gene fragments were analysed of which 156 by sequencing, yielding a total of 38 species, including 9 presumptively novel species with at least five species that have not been isolated previously from vaginal samples. Interestingly, cloning revealed that Atopobium vaginae was abundant in four out of the five non-grade I specimens. Finally, species specific PCR for A. vaginae and Gardnerella vaginalis pointed to a statistically significant co-occurrence of both species in the bacterial vaginosis samples.ConclusionsAlthough historically the literature regarding bacterial vaginosis has largely focused on G. vaginalis in particular, several findings of this study – like the abundance of A. vaginae in disturbed vaginal microflora and the presence of several novel species – indicate that much is to be learned about the composition of the vaginal microflora and its relation to the etiology of BV.

Longitudinal analysis of the vaginal microflora in pregnancy suggests that L. crispatus promotes the stability of the normal vaginal microflora and that L. gasseri and/or L. iners are more conducive to the occurrence of abnormal vaginal microflora

BackgroundDespite their antimicrobial potential, vaginal lactobacilli often fail to retain dominance, resulting in overgrowth of the vagina by other bacteria, as observed with bacterial vaginosis. It remains elusive however to what extent interindividual differences in vaginal Lactobacillus community composition determine the stability of this microflora. In a prospective cohort of pregnant women we studied the stability of the normal vaginal microflora (assessed on Gram stain) as a function of the presence of the vaginal Lactobacillus index species (determined through culture and molecular analysis with tRFLP).ResultsFrom 100 consecutive Caucasian women vaginal swabs were obtained at mean gestational ages of 8.6 (SD 1.4), 21.2 (SD 1.3), and 32.4 (SD 1.7) weeks, respectively. Based on Gram stain, 77 women had normal or Lactobacillus-dominated vaginal microflora (VMF) during the first trimester, of which 18 had grade Ia (L. crispatus cell morphotypes) VMF (23.4%), 16 grade Iab (L. crispatus and other Lactobacillus cell morphotypes) VMF (20.8%), and 43 grade Ib (non-L. crispatus cell morphotypes) VMF (55.8%). Thirteen women with normal VMF at baseline, converted in the second or third trimester (16.9%) to abnormal VMF defined as VMF dominated by non-Lactobacillus bacteria. Compared to grade Ia and grade Iab VMF, grade Ib VMF were 10 times (RR = 9.49, 95% CI 1.30 – 69.40) more likely to convert from normal to abnormal VMF (p = 0.009). This was explained by the observation that normal VMF comprising L. gasseri/iners incurred a ten-fold increased risk of conversion to abnormal VMF relative to non-L. gasseri/iners VMF (RR 10.41, 95% CI 1.39–78.12, p = 0.008), whereas normal VMF comprising L. crispatus had a five-fold decreased risk of conversion to abnormal VMF relative to non-L. crispatus VMF (RR 0.20, 95% CI 0.05–0.89, p = 0.04).ConclusionThe presence of different Lactobacillus species with the normal vaginal microflora is a major determinant to the stability of this microflora in pregnancy: L. crispatus promotes the stability of the normal vaginal microflora while L. gasseri and/or L. iners predispose to some extent to the occurrence of abnormal vaginal microflora.

Safety and Availability of Dapivirine (TMC120) Delivered from an Intravaginal Ring

Vaginal delivery of 200 mg or 25 mg dapivirine from intravaginal rings (IVRs) was evaluated over a 7-day period in two phase 1 safety trials (IPM001 and IPM008, respectively) in a total of 25 healthy women 19 to 46 years of age. The IVR was generally safe and well tolerated with similar adverse events observed in the placebo and dapivirine groups. Across both studies, dapivirine concentrations in vaginal fluids measured at the introitus, cervix, and ring area were within the mean range of 0.7-7.1 microg/ml. Mean dapivirine concentrations in vaginal and cervical tissues on day 7 were 0.3-0.7 microg/g in IPM001 and 1.5-3.5 microg/g in IPM008. Mean plasma concentrations of dapivirine were <50 pg/ml. Dapivirine from both IVRs was successfully distributed throughout the lower genital tract at concentrations >1000x the EC(50) against wild-type HIV-1 (LAI) in MT4 cells suggesting that IVR delivery of microbicides is a viable option meriting further study.

Quantitative determination by real-time PCR of four vaginal Lactobacillus species, Gardnerella vaginalis and Atopobium vaginae indicates an inverse relationship between L. gasseri and L. iners

BackgroundMost studies of the vaginal microflora have been based on culture or on qualitative molecular techniques. Here we applied existing real-time PCR formats for Lactobacillus crispatus, L. gasseri and Gardnerella vaginalis and developed new formats for Atopobium vaginae, L. iners and L. jensenii to obtain a quantitative non culture-based determination of these species in 71 vaginal samples from 32 pregnant and 28 non-pregnant women aged between 18 and 45 years.ResultsThe 71 vaginal microflora samples of these women were categorized, using the Ison and Hay criteria, as refined by Verhelst et al. (2005), as follows: grade Ia: 8 samples, grade Iab: 10, grade Ib: 13, grade I-like: 10, grade II: 11, grade III: 12 and grade IV: 7.L. crispatus was found in all but 5 samples and was the most frequent Lactobacillus species detected. A significantly lower concentration of L. crispatus was found in grades II (p < 0.0001) and III (p = 0.002) compared to grade I. L. jensenii was found in all grades but showed higher concentration in grade Iab than in grade Ia (p = 0.024). A. vaginae and G. vaginalis were present in high concentrations in grade III, with log10 median concentrations (log10 MC), respectively of 9.0 and 9.2 cells/ml. Twenty (38.5%) of the 52 G. vaginalis positive samples were also positive for A. vaginae. In grade II we found almost no L. iners (log10 MC: 0/ml) but a high concentration of L. gasseri ( log10 MC: 8.7/ml). By contrast, in grade III we found a high concentration of L. iners( log10 MC: 8.3/ml) and a low concentration of L. gasseri ( log10 MC: 0/ml). These results show a negative association between L. gasseri and L. iners (r = -0.397, p = 0.001) and between L. gasseri and A. vaginae (r = -0.408, p < 0.0001).ConclusionIn our study we found a clear negative association between L. iners and L. gasseri and between A. vaginae and L. gasseri. Our results do not provide support for the generally held proposition that grade II is an intermediate stage between grades I and III, because L. gasseri, abundant in grade II is not predominant in grade III, whereas L. iners, abundant in grade III is present only in low numbers in grade II samples.

The vaginal microbiota: what have we learned after a decade of molecular characterization?

We conducted a systematic review of the Medline database (U.S. National Library of Medicine, National Institutes of Health, Bethesda, MD, U.S.A) to determine if consistent molecular vaginal microbiota (VMB) composition patterns can be discerned after a decade of molecular testing, and to evaluate demographic, behavioral and clinical determinants of VMB compositions. Studies were eligible when published between 1 January 2008 and 15 November 2013, and if at least one molecular technique (sequencing, PCR, DNA fingerprinting, or DNA hybridization) was used to characterize the VMB. Sixty three eligible studies were identified. These studies have now conclusively shown that lactobacilli-dominated VMB are associated with a healthy vaginal micro-environment and that bacterial vaginosis (BV) is best described as a polybacterial dysbiosis. The extent of dysbiosis correlates well with Nugent score and vaginal pH but not with the other Amsel criteria. Lactobacillus crispatus is more beneficial than L. iners. Longitudinal studies have shown that a L. crispatus-dominated VMB is more likely to shift to a L. iners-dominated or mixed lactobacilli VMB than to full dysbiosis. Data on VMB determinants are scarce and inconsistent, but dysbiosis is consistently associated with HIV, human papillomavirus (HPV), and Trichomonas vaginalis infection. In contrast, vaginal colonization with Candida spp. is more common in women with a lactobacilli-dominated VMB than in women with dysbiosis. Cervicovaginal mucosal immune responses to molecular VMB compositions have not yet been properly characterized. Molecular techniques have now become more affordable, and we make a case for incorporating them into larger epidemiological studies to address knowledge gaps in etiology and pathogenesis of dysbiosis, associations of different dysbiotic states with clinical outcomes, and to evaluate interventions aimed at restoring and maintaining a lactobacilli-dominated VMB.

Biostructure of Fecal Microbiota in Healthy Subjects and Patients With Chronic Idiopathic Diarrhea

BACKGROUND & AIMS Dysbiosis is a key component of intestinal disorders. Our aim was to quantitatively access the biostructure of fecal microbiota in healthy subjects and patients with chronic idiopathic diarrhea and evaluate the responses to Saccharomyces boulardii treatment. METHODS We investigated punched fecal cylinders from 20 patients with chronic idiopathic diarrhea and 20 healthy controls using fluorescence in situ hybridization. Fluctuations in assembly of 11 bacterial groups were monitored weekly for 3 weeks before, during, and after oral S boulardii supplementation. RESULTS The structural organization of fecal microbiota in healthy subjects was stable and unaffected by S boulardii. The assembly of fecal microbiota in idiopathic diarrhea was markedly different, characterized by mucus depositions within feces; mucus septa and striae; marked reduction in concentrations of habitual Eubacterium rectale, Bacteroides, and Faecalibacterium prausnitzii groups; suppression of bacterial fluorescence in the center of the feces; increased concentrations and spatial shift of mucotrop bacteria to the fecal core; and increased concentrations of occasional bacteria. Except for elevated concentrations of some occasional bacterial groups, all parameters typical for diarrhea improved significantly with S boulardii treatment and most changes persisted after cessation of therapy. The improvement of the fecal microbiota was accompanied by partial (40%) and complete normalization (30%) of the diarrheal symptoms. CONCLUSIONS The fecal microbiota is highly structured. Fluorescence in situ hybridization analysis allowed us to quantitatively study the dysbiotic changes. S boulardii significantly improved the fecal biostructure in patients with diarrhea but had no influence on the feces in healthy subjects.

Bacterial vaginosis is associated with uterine cervical human papillomavirus infection: a meta-analysis

BackgroundBacterial vaginosis (BV), an alteration of vaginal flora involving a decrease in Lactobacilli and predominance of anaerobic bacteria, is among the most common cause of vaginal complaints for women of childbearing age. It is well known that BV has an influence in acquisition of certain genital infections. However, association between BV and cervical human papillomavirus (HPV) infection has been inconsistent among studies. The objective of this meta-analysis of published studies is to clarify and summarize published literature on the extent to which BV is associated with cervical HPV infection.MethodsMedline and Web of Science were systematically searched for eligible publications until December 2009. Articles were selected based on inclusion and exclusion criteria. After testing heterogeneity of studies, meta-analysis was performed using random effect model.ResultsTwelve eligible studies were selected to review the association between BV and HPV, including a total of 6,372 women. The pooled prevalence of BV was 32%. The overall estimated odds ratio (OR) showed a positive association between BV and cervical HPV infection (OR, 1.43; 95% confidence interval, 1.11-1.84).ConclusionThis meta-analysis of available literature resulted in a positive association between BV and uterine cervical HPV infection.

Long-term Assessment of the Physical, Mental, and Sexual Health among Transsexual Women

INTRODUCTION Transsexualism is the most extreme form of gender identity disorder and most transsexuals eventually pursue sex reassignment surgery (SRS). In transsexual women, this comprises removal of the male reproductive organs, creation of a neovagina and clitoris, and often implantation of breast prostheses. Studies have shown good sexual satisfaction after transition. However, long-term follow-up data on physical, mental and sexual functioning are lacking. AIM To gather information on physical, mental, and sexual well-being, health-promoting behavior and satisfaction with gender-related body features of transsexual women who had undergone SRS. METHODS Fifty transsexual women who had undergone SRS >or=6 months earlier were recruited. MAIN OUTCOME MEASURES Self-reported physical and mental health using the Dutch version of the Short-Form-36 (SF-36) Health Survey; sexual functioning using the Dutch version of the Female Sexual Function Index (FSFI). Satisfaction with gender-related bodily features as well as with perceived female appearance; importance of sex, relationship quality, necessity and advisability of gynecological exams, as well as health concerns and feelings of regret concerning transition were scored. RESULTS Compared with reference populations, transsexual women scored good on physical and mental level (SF-36). Gender-related bodily features were shown to be of high value. Appreciation of their appearance as perceived by others, as well as their own satisfaction with their self-image as women obtained a good score (8 and 9, respectively). However, sexual functioning as assessed through FSFI was suboptimal when compared with biological women, especially the sublevels concerning arousal, lubrication, and pain. Superior scores concerning sexual function were obtained in those transsexual women who were in a relationship and in heterosexuals. CONCLUSIONS Transsexual women function well on a physical, emotional, psychological and social level. With respect to sexuality, they suffer from specific difficulties, especially concerning arousal, lubrication, and pain.

Electrospun cellulose acetate phthalate fibers for semen induced anti-HIV vaginal drug delivery

Despite many advances in modern medicine, human immunodeficiency virus (HIV) still affects the health of millions of people world-wide and much effort is put in developing methods to either prevent infection or to eradicate the virus after infection has occurred. Here, we describe the potential use of electrospun cellulose acetate phthalate (CAP) fibers as a tool to prevent HIV transmission. During the electrospinning process, anti-viral drugs can easily be incorporated in CAP fibers. Interestingly, as a result of the pH-dependent solubility of CAP, the fibers are stable in vaginal fluid (the healthy vaginal flora has a pH of below 4.5), whereas the addition of small amounts of human semen (pH between 7.4 and 8.4) immediately dissolves the fibers which results in the release of the encapsulated drugs. The pH-dependent release properties have been carefully studied and we show that the released anti-viral drugs, together with the CAP which has been reported to have intrinsic antimicrobial activity, efficiently neutralize HIV in vitro.

The epidemiology of bacterial vaginosis in relation to sexual behaviour.

BackgroundBacterial vaginosis (BV) has been most consistently linked to sexual behaviour, and the epidemiological profile of BV mirrors that of established sexually transmitted infections (STIs). It remains a matter of debate however whether BV pathogenesis does actually involve sexual transmission of pathogenic micro-organisms from men to women. We therefore made a critical appraisal of the literature on BV in relation to sexual behaviour.DiscussionG. vaginalis carriage and BV occurs rarely with children, but has been observed among adolescent, even sexually non-experienced girls, contradicting that sexual transmission is a necessary prerequisite to disease acquisition. G. vaginalis carriage is enhanced by penetrative sexual contact but also by non-penetrative digito-genital contact and oral sex, again indicating that sex per se, but not necessarily coital transmission is involved. Several observations also point at female-to-male rather than at male-to-female transmission of G. vaginalis, presumably explaining the high concordance rates of G. vaginalis carriage among couples. Male antibiotic treatment has not been found to protect against BV, condom use is slightly protective, whereas male circumcision might protect against BV. BV is also common among women-who-have-sex-with-women and this relates at least in part to non-coital sexual behaviours. Though male-to-female transmission cannot be ruled out, overall there is little evidence that BV acts as an STD. Rather, we suggest BV may be considered a sexually enhanced disease (SED), with frequency of intercourse being a critical factor. This may relate to two distinct pathogenetic mechanisms: (1) in case of unprotected intercourse alkalinisation of the vaginal niche enhances a shift from lactobacilli-dominated microflora to a BV-like type of microflora and (2) in case of unprotected and protected intercourse mechanical transfer of perineal enteric bacteria is enhanced by coitus. A similar mechanism of mechanical transfer may explain the consistent link between non-coital sexual acts and BV. Similar observations supporting the SED pathogenetic model have been made for vaginal candidiasis and for urinary tract infection.SummaryThough male-to-female transmission cannot be ruled out, overall there is incomplete evidence that BV acts as an STI. We believe however that BV may be considered a sexually enhanced disease, with frequency of intercourse being a critical factor.