Bacterial Vaginosis

Chapter 18

Bacterial Vaginosis Jeanne M. Marrazzo Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA 98104, USA

Sharon L. Hillier University of Pittsburgh Department of Obstetrics, Gynecology and Reproductive Sciences and the Magee-Womens Research Institute, Pittsburgh, PA 15213, USA

Chapter Outline Introduction   Definition   Epidemiology   Role of Sexual Activity   Other Risks for Bv   Microbiology of Bv   Anaerobic Bacteria   Pathogenesis   Role of Host Immunity   Clinical Manifestations   Diagnosis   Treatment   Non-antibiotic Approaches to Managing BV  

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Treatment of Sex Partners   482 Prevention of BV and its Recurrence   483 Complications of Bv   486 Pelvic Inflammatory Disease  487 BV in Pregnancy   488 BV and HIV   489 Conclusions and Future Directions   490 References   491

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INTRODUCTION The microbiota of the human vagina can affect the health of women, their fetuses, and newborns. A vaginal environment that is quantitatively dominated by hydrogen peroxide-producing Lactobacillus species has consistently been associated with healthy pregnancy outcomes, lack of abnormal vaginal symptoms, and reduced risk for several sexually transmitted pathogens, including HIV. Bacterial vaginosis (BV) represents a condition in which the normal protective lactobacilli are replaced by high quantities of commensal anaerobes, resulting in symptomatic vaginitis in many women. BV increases the risk of upper genital tract infection and adverse outcomes of pregnancy. While traditional cultivation has identified numerous BV-associated bacteria involved in these processes, recent Sexually Transmitted Diseases. http://dx.doi.org/10.1016/B978-0-12-391059-2.00018-8 Copyright © 2013 Elsevier Ltd. All rights reserved.

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advances in molecular biology have facilitated the detection and identification of bacteria using cultivation-independent methods. For instance, several novel bacteria in the Clostridiales order are highly specific indicators of BV, and bacteria related to Megasphaera, Leptotrichia, Atopobium, and Dialister species are commonly found in women with BV. These advances have notable implications for research related to the pathogenesis, natural history, diagnosis, and adverse consequences of BV. In addition, largely prompted by investigations into the transmission of HIV, our understanding of innate and acquired immunity as it relates to the genital tract has improved significantly in the last several years. Despite these advances, there are significant gaps in our knowledge concerning the vaginal microbiota. What are the consequences of BV, and what are the gaps in our understanding regarding how these complications arise? What interventions have and have not worked to prevent these consequences, and how do we leverage this knowledge to move forward with appropriate prevention strategies? How might advances in molecular biology contribute to an enhanced approach to diagnosing BV? A more complete understanding of vaginal microbial populations and the host factors that regulate susceptibility to infection and maintenance of healthy microbiota may lead to better strategies to maintain healthy vaginal microbial communities – thus enhancing women’s health – and to create opportunities to explore the role of novel bacteria in reproductive tract diseases. In this chapter, we review the current state of knowledge about BV and discuss ways in which the field can advance towards promoting disease prevention and management.

DEFINITION BV is the most prevalent form of vaginal infection in women of reproductive age, affecting 8–23%, and is the most common etiology of vaginal symptoms prompting women to seek medical care. Symptomatic BV, which accounts for approximately 60% of all cases, typically causes abnormal vaginal discharge that is increased in amount and often malodorous. The discharge results in part from degradation of the normal vaginal mucin gel, which is efficiently performed by mucin-degrading enzymes produced by BV-associated bacteria (particularly Gram-negative anaerobes) (Olmsted et  al., 2003). The odor, usually described as ‘fishy,’ is derived from volatilization of the amines produced by the metabolism of anaerobic bacteria that characterize this disorder. The etiology of BV is not known. At the microbiologic level, BV is a synergistic polymicrobial syndrome characterized by depletion of Lactobacillus species, especially those that produce hydrogen peroxide (H2O2), accompanied by intense overgrowth of commensal vaginal anaerobic bacteria (100to 1000-fold above normal) (Hillier et al., 2008). Conventional cultivation of flora from vaginal fluid of women with BV typically yields a spectrum of primarily anaerobic commensals: Gardnerella vaginalis, Prevotella species, anaerobic Gram-positive cocci, Mobiluncus species, Ureaplasma urealyticum,

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and Mycoplasma hominis. Critically, the initial event leading to this shift is unknown. As discussed in more detail below, BV occurs more frequently among women who report new or higher numbers of male sex partners, is common and highly concordant among female sex partners, and rarely occurs before sexual debut, patterns that invoke the epidemiology of a typical sexually transmitted infection (STI) (Marrazzo, 2011; Fethers et al., 2008). Limited data also suggest that male condoms and circumcision may prevent BV and its recurrence (Marrazzo & Cates, 2011).

EPIDEMIOLOGY Most of our understanding of the prevalence of BV derives from clinical trials of preventive or therapeutic interventions, or from clinic-based populations; relatively few large-scale, representative surveys of vaginal microbiota have been performed. One of the largest studies reporting BV prevalence for women is the National Health and Nutrition Survey (NHANES), which uses a complex, stratified, multistage probability sample design with unequal probabilities of selection to obtain a nationally representative sample of the US civilian non-institutionalized population. Of over 12 000 women in the 2001–2004 NHANES who supplied a self-collected swab of vaginal secretions for Gram stain analysis by Nugent score, BV prevalence was 29.2% (95% confidence interval (CI) 27.2– 31.3, P = 0.003); only 15.7% of women with BV reported symptoms (Koumans et al., 2007). Prevalence was 51.4% among non-Hispanic blacks, 31.9% among Mexican Americans, and 23.2% among non-Hispanic whites (P < 0.01 for each comparison). Detection of BV was associated with increasing number of lifetime sex partners, a previous female sex partner, douching frequency, low educational attainment, poverty, smoking, high body mass index, and having been pregnant. Current use of oral contraceptive pills was inversely associated with BV prevalence. Significant differences by race/ethnicity remained after accounting for other risk factors. A less representative but large cross-sectional study among young women entering the US military reported that BV prevalence was 27% (Yen et al., 2003). The highest prevalence of BV reported in a large population-based sample of women was 51%; this was among 4718 rural Ugandan women, a prevalence that has been confirmed in numerous studies of women in sub-Saharan African settings (Sewankambo et al., 1997; Atashili et al., 2008). Women attending sexually transmitted disease (STD) clinics have had relatively high BV prevalence, typically in the range of 24–37%. In family planning clinics in various countries, BV prevalence has generally been 14–19%. Finally, the prevalence of BV among pregnant women varies widely. In one large study in the USA, 13 747 pregnant women at 23–26 weeks’ gestation underwent evaluation for BV by standardized vaginal Gram stain criteria (Goldenberg et al., 1996a). While 16.3% of women had BV, prevalence varied widely by ethnicity, from 6.1% of Asians to 8.8% of white women, 15.9% of Hispanics, and 22.7% of black women.

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Role of Sexual Activity A persistent conundrum is the extent to which sexual activity precipitates and maintains BV, in part because relatively few prospective studies have defined risks for acquisition of BV. As discussed below, data on a potentially protective effect afforded by male partner condom use support a role for unprotected sex in initiating or maintaining BV. Recent efforts to characterize individual strains of G. vaginalis yielded a high degree of low-level diversity among G. vaginalis sequences, with a total of 46 unique sequence variants (oligotypes), and also found strong correlations of these oligotypes between sexual partners (Eren et al., 2011), a finding that reflects older work in which Piot observed that isolates of G. vaginalis from the vaginas of women with BV and from the urethras of their male sex partners belonged to identical biotypes when the strains were isolated within the same 24-hour period from both partners (Piot, 1984). Among postpubertal females, those reporting no sexual experience have had significantly lower prevalence of BV than have those with sexual experience. One important recent study showed a strong association between BV and penile–vaginal sex with multiple partners, but found no BV in sexually inexperienced women once a history of non-coital sexual practices was elicited, indicating that BV is not present in truly sexually inexperienced women (Fethers et al., 2009). Data on the relationship between BV and numbers of lifetime or recent sex partners are somewhat inconsistent, but generally weigh in favor of a positive association. In heterosexual women, BV generally occurs more frequently among those who report new or higher numbers of male sex partners or more frequent intercourse, consistent with sexual acquisition (Schwebke & Desmond, 2005). Condom use may prevent BV and its recurrence. Women in a large prospective study that evaluated response to treatment had a fivefold reduction in BV persistence and recurrence if their partners consistently used condoms (Sanchez et al., 2004). In another prospective study, women who reported less frequent use of condoms were less likely to have normal vaginal flora sustained over time. Although G. vaginalis, Mobiluncus species, and Mycoplasma hominis have been isolated from the male genital tract, short-course systemic antibiotic treatment of male partners of women with BV does not reduce BV recurrence (Hamrick & Chambliss, 2000). Among heterosexual couples in Kenya, multivariate analysis of factors specific to male partners of women with or without BV showed that crowded living and fewer bathing facilities were associated with higher odds of BV, while poor genital hygiene was associated with fourfold increase in their female partners’ BV risk (Bukusi et al., 2006). Because 97% of men in this study were circumcised, an independent contribution of circumcision status to BV could not be assessed. Subsequently, research from the Rakai area confirmed not only that various BV-associated bacteria (BVAB) could be detected on foreskin that had been removed after circumcision, but that the penile microbiome shifted from a heavily anaerobic community to a less diverse, less anaerobic

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type after circumcision. The finding that male circumcision reduces female partners’ risk of subsequently developing BV (discussed in detail below) (Gray et al., 2009) also strongly suggests that, in some men, the subpreputial area may be a suitable environment for some bacteria associated with BV. BV is considerably more common among women who report sex with other women than among those who do not. In the NHANES study (Koumans et  al., 2007), BV prevalence was 45.2% (95% CI 35.5–57.4) among women who reported having had a female sex partner, compared to overall prevalence of 29.2% (95% CI 27.2–31.3, P = 0.003). Among STD clinic populations, reports of sex with another woman have been a risk for BV in larger, well controlled studies. Moreover, reports of sex with another woman were associated with increased risk of BV recurrence in one prospective study (Bradshaw et al., 2006). The reasons that these women have a high prevalence of BV are unclear, but sexual practices that transmit vaginal fluid increase BV risk, and a high degree of concordance for the presence or absence of BV among lesbian couples has been reported. Among 329 women who were sexually active with women, risks for prevalent BV included higher number of female lifetime partners, shared vaginal use of sex toys, and report of oral–anal sex (Marrazzo et al., 2002). Among 73 couples in this study, 95% were concordant for presence or absence of BV. Both partners had BV in 21 couples, and one woman had BV in only six couples; this degree of concordance differed dramatically from the expected distribution (P < 0.001). The authors concluded that this concordance probably reflected sexual transmission of BV. A more detailed analysis of the Lactobacillus populations in these women revealed that a majority of monogamous female sex partners shared unique Lactobacillus strains, as assessed by DNA homology to type strains and fingerprinting by rep-polymerase chain reaction (PCR) (Marrazzo et al., 2009). In a subsequent prospective cohort study of women who reported sex with women in the previous year, 20% acquired BV in the year of follow-up (Marrazzo et al., 2010b). Risks for incident BV included detection of several BVAB or absence of L. crispatus in vaginal fluid at the enrollment (BV-negative) visit, and evaluation for incident BV in the 2 weeks after menses. Despite collection of extensive sexual behaviors using computerassisted self-interview, only report of a new female sex partner with a history of BV was associated with increased risk of BV acquisition. Intriguingly, a dose– response relationship was observed between risk of BV acquisition and increasing reported number of episodes of receptive vulvovaginal oral sex. The frequently noted association between report of anal sexual behaviors and BV in the few studies that have assessed this practice deserves comment. Perineal transfer of rectal bacteria to the vagina is a well recognized consequence of vaginal intercourse for common bacteria such as E. coli and Group B Streptococcus, and it is possible that BV may be precipitated or promoted if unprotected vaginal intercourse encourages the translocation of key bacteria from the rectum to the vagina. Many of the bacteria that have been described using molecular approaches in the setting of BV are strict anaerobes, and may

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prefer the rectal environment; whether and how they may play a pathogenic role in initiating BV is under study. In a large cross-sectional study, Antonio and colleagues reported that rectal colonization with L. crispatus or L. jensenii was significantly associated with lower likelihood of BV (Antonio et al., 2005). In a study of monogamous female sex partners, detection of L. gasseri at rectum or vagina was associated with recent receptive digital–vaginal sex and increased BV risk (odds ratio (OR) 4.2, 95% CI 1.4–13.4) (Marrazzo et al., 2009). Among women without BV who were subsequently followed for an average of 1 year, detection of several BVAB in the vagina at the enrollment (BVnegative) visit was independently associated with subsequent acquisition of BV, and detection of L. crispatus at enrollment was associated with reduced risk of subsequent BV acquisition (Marrazzo et al., 2010b). In extending the analysis to participants’ oral and anal BV-specific microbiology at the enrollment (BV-negative) visit, women who acquired BV over follow-up were more likely to have previous colonization of these extragenital reservoirs with key BVAB (Marrazzo et al., 2012). Detection of G. vaginalis in the oral cavity or anus and of Leptotrichia/Sneathia spp. in the anus was significantly more common at enrollment among women who subsequently acquired BV. In contrast, L. crispatus was detected more frequently in the anus among controls. When these bacteria were present, they were typically detected at higher concentrations of bacterial DNA relative to controls at each site as measured by quantitative PCR assays. These data suggest that some BVAB may be acquired vaginally from pre-existing reservoirs at extravaginal sites, and that changes in the microbiota of these reservoirs may precede the development of BV by a considerable time period.

OTHER RISKS FOR BV For reasons that are not understood, BV prevalence is highest among black women, even after controlling for amount and type of sexual activity, and douching habits (Royce et al., 1999; Allsworth, 2010; Ness et al., 2001). Some investigators have examined the hypothesis that chronic stress associated with residing in urban environments may contribute to the link between African American race and BV. In a cross-sectional study of BV prevalence among 2304 women at first prenatal visit, stress was measured at the individual and community levels with the use of interviews and administrative records (Culhane et al., 2002). Black women had a significantly higher BV prevalence (64%) compared with white women (35%). Almost one-third of black women reported threats to personal safety compared with 13% of white women, and 63% of black women lived in neighborhoods with aggravated assault rates that were above the citywide mean compared with 25% of the white women. After adjustment for sociodemographic, behavioral risk, and perceived stress, the odds of BV associated with the community level stressor of ‘homelessness’ was significant (OR 6.7, 95% CI 1.6–27.8). Inclusion of both individual and community level stressors reduced the black:white BV odds ratio by 27%. The authors concluded that measurement

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of stressors at multiple levels explained a significant proportion of the racial disparity in the rates of occurrence of BV. Stress is known to modulate key cytokines involved in local immune responses, so this hypothesis is of interest. Other risks for BV that have often been reported include douching (which promotes loss of H2O2+ lactobacilli) (Ness et al., 2002), cigarette use (Cherpes et  al., 2008), and the use of non-hormonal intrauterine devices (IUDs) for contraception (Avonts et al., 1990). Hormonal contraception may confer reduced risk, depending on the types and dosages of hormones involved (Riggs et al., 2007; Baeten et al., 2001). Among HIV-uninfected Kenyan female sex workers, increased frequency of vaginal washing was associated with a higher likelihood of BV, as were vaginal lubrication with petroleum jelly, lubrication with saliva, and bathing less than the median for the cohort. The authors concluded that modification of intravaginal and general hygiene practices should be evaluated as potential strategies for reducing the risk of BV (Nelson et al., 2009). Genital hygiene methods for washing after sexual exposure, including vaginal washing and douching, are ineffective in protecting against HIV and STDs, and may increase the risk of BV, some STDs, and HIV (Myer et al., 2005).

MICROBIOLOGY OF BV Gardner and Dukes first isolated the organism that they named Haemophilus vaginalis from 92% of 141 women with ‘bacterial vaginitis,’ and from none of 78 controls (Gardner & Dukes, 1954). In an attempt to fulfill Koch’s postu­ lates, Gardner inoculated 15 women not infected by H. vaginalis with material from the vaginas of infected patients, and reproduced the manifestations of this condition in 11 of 15 women. However, inoculation of pure H. ­vaginalis resulted in these manifestations in only one of 13 women, despite isolation of H. ­vaginalis from the vagina of all of these women 1 or more weeks after ­inoculation (Criswell et al., 1969). Investigations conducted over the past 30 years have substantiated the association of G. vaginalis with BV. However, with use of more sensitive detection methods, including bacterium-specific PCR, G. vaginalis can be detected in women having no signs of vaginal infection, and is estimated to colonize approximately 60% of women whose vaginal fluid is characterized by normal Nugent scores. G. vaginalis may act synergistically with anaerobic bacteria to cause BV (Fredricks et al., 2007). When assessed by specific fluorescent probe, G. vaginalis has been detected in large quantities within adherent biofilms among women with BV; such biofilms appear to persist after treatment failure for BV (Swidsinski et al., 2008).

Anaerobic Bacteria In cultivation of bacteria from vaginal fluid of women with BV, the most common anaerobic Gram-negative rods include Prevotella bivia, the

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black-pigmented species belonging to the genera Prevotella and Porphyromonas, Bacteroides ureolyticus and Fusobacterium nucleatum (Hillier et al., 1993). Members of the Bacteroides fragilis group, although common in the intestinal tract, are less common in the vagina and are not associated with BV. Two species of Mobiluncus, a nutritionally fastidious and strictly anaerobic motile rod, have been strongly associated with BV: M. curtisii and M. mulieris (Schwebke & Lawing, 2001). Mycoplasma hominis and several anaerobic Gram-positive cocci have also been consistently detected in women with BV. New tools in molecular biology have greatly expanded our understanding of microbial diversity in the human vagina by identifying fastidious or cultivation-resistant bacteria (Figure 18.1) (Fredricks et  al., 2005; ZozayaHinchliffe et al., 2008; Verhelst et al., 2004; Burton & Reid, 2002). Molecular methods have been widely adopted to study microbial populations because cultivation-based approaches do not detect or identify a large percentage of microbes present in most niches, including those associated with humans. For example, Escherichia coli and enterococci are not commonly detected in vaginal samples using molecular methods, but are commonly detected using cultivation approaches (Fredricks & Marrazzo, 2005). The vaginal microbiome has been explored using several methodologies. These include

FIGURE 18.1  Molecular methods such as broad-range 16S rRNA gene PCR with cloning and sequencing have demonstrated that the microbiology of BV is complex, and different subjects may have very different vaginal bacterial communities, as evidenced by these two subjects. The eight most abundant phylotypes (species level operational taxonomic units) are displayed for each subject. Please see color plate section at the back of the book.

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broad-range or consensus sequence PCR of ribosomal RNA genes, in which primers that anneal with highly conserved regions of the bacterial 16S rRNA gene can be employed in a PCR to amplify segments of the gene from most bacterial species. These amplification products typically contain intervening regions of sequence that are highly variable (or species-/taxon-specific), allowing for the identification of bacteria. Mixed bacterial communities yield mixed PCR products, and the sequences must be sorted in some way to distinguish between individual taxa. There are several approaches for achieving this sorting, including denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) analysis, cloning and sequencing, cloning with amplified ribosomal DNA restriction analysis (ARDRA), and pyrosequencing using the 454 sequencing system. An alternative molecular approach to broad-range PCR is taxon-directed PCR in which one designs primers to PCR-amplify a specific microbe or microbial group (Fredricks et al., 2007), or phyloarrays, which employ microarray platforms to identify sequences generated by broad-range 16S rRNA gene PCR, or rRNA sequences directly isolated from clinical samples without PCR amplification (Yergeau et al., 2009). Fluorescently labeled probes may also be used in a real-time PCR format to increase the specificity of the assay and to generate quantitative data (Fredricks et al., 2009). Finally, fluorescently labeled oligonucleotide or peptide nucleic acid probes targeting bacterial ribosomes can be hybridized with fixed bacteria in tissues or body fluids to detect and identify bacteria by microscopy (Figure 18.2).

FIGURE 18.2  Fluorescence micrograph showing a vaginal epithelial cell coated with bacteria from a subject with BV. Labeled bacteria are shown hybridizing with probes for (BV-associated bacteria) BVAB-1 (green) and BVAB-2 (red). DAPI (4’,6-diamidino-2-phenylindole, a fluorescent stain that binds strongly to A-T rich regions in DNA) stains cell nuclei blue in this image. Please see color plate section at the back of the book.

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PATHOGENESIS BV results from the replacement of the normal vaginal bacteria (Lactobacillus species) with a mixed flora with no single organism being pathognomonic for BV. For this reason, most studies of the pathogenesis of BV have focused on how the microbial ecosystem of the vagina becomes altered (Figure 18.3). The epidemiologic data described above are consistent with the notion that introduction of a particular set of organisms via sexual intercourse is one major factor that initiates the change in vaginal bacteria ­characteristic of BV. Certain vaginal Lactobacillus species may help women to resist vaginal and cervical infection. Vaginal lactobacilli inhibit G. vaginalis, Mobiluncus, and anaerobic Gram-negative rods in vitro. Some strains of Lactobacillus produce hydrogen peroxide (H2O2), and several studies have demonstrated that H2O2producing strains of lactobacilli more frequently colonize the vagina of normal women, compared to women with BV. In several prospective studies, women colonized vaginally with H2O2-positive lactobacilli less often developed BV than did those colonized with H2O2-negative lactobacilli (Bradshaw et al., 2006; Marrazzo et al., 2010b; Cherpes et al., 2008; Hawes et al., 1996). H2O2 may inhibit the growth of anaerobic rods, Gardnerella, Mobiluncus, and Mycoplasma in the vagina, either directly through its toxic activity or by reacting with a halide ion in the presence of cervical peroxidase as part of the H2O2–halide– peroxidase antibacterial system (Klebanoff et al., 1991).

FIGURE 18.3  Factors involved in the pathogenesis of BV. Please see color plate section at the back of the book.

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Amines produced by the microbial community, perhaps through the action of microbial decarboxylases, account for the characteristic abnormal fishy odor that is produced when vaginal fluid is mixed with 10% potassium hydroxide (KOH). This so-called ‘whiff test’ reflects volatilization of aromatic amines, including putrescine, cadaverine, and trimethylamine at alkaline pH. The presence of trimethylamine is thought to be largely responsible for symptoms of malodor experienced by women with BV. The vaginal fluid of women with BV has increased levels of endotoxin, sialidase, and glycosidases that degrade mucin and decrease its viscosity, likely producing the characteristic thin, homogeneous vaginal discharge. Analysis of the vaginal immune response has been limited primarily to measurement of cytokines from cervical or vaginal swabs in women with BV compared to women without BV (Mitchell et al., 2008), although some studies have assessed patterns after treatment (Yudin et al., 2003; Cherpes et al., 2008). While IL-1β is consistently increased in BV (Cauci et al., 2003; Hedges et al., 2006), measurements of other cytokines have yielded variable results. In 81 women, small increases in IL-1β, tumor necrosis factor (TNF), interferon-γ, IL-2, IL-4, IL-10, and granulocyte macrophage colony-stimulating factor (GM-CSF) (not IL-6 or macrophage inflammatory protein (MIP)-1α) were present with BV, and declined with restoration of normal flora (Cherpes et al., 2008). Some studies found that IL-8, a potent neutrophil chemoattractant, was increased with successful treatment (Losikoff et al., 2007). The absence of IL-8 elevation in BV is consistent with a typical (though not universal) paucity of neutrophils in this condition (Cauci et al., 2003), but why it is low in the context of a milieu characterized by elevations in IL-1β and TNF, and the presence of literally billions of anaerobes, is unclear. It may be selectively degraded or inhibited by other unknown factors operating in BV (Cauci et al., 2003).

Role of Host Immunity Some in vitro studies have indicated that certain toll-like receptor (TLR) polymorphisms (specifically polymorphisms in TLR2 and TLR4), which modify cellular immune response and production of cytokines, may be involved in BV pathogenesis. Specific cytokine patterns may dampen recruitment of neutrophils in response to abnormal vaginal flora (Goepfert et  al., 2005; Mares et al., 2008; Zariffard et al., 2005). TLRs and Nod-like receptors (NLRs) are families of proteins known as pattern recognition receptors (PRRs) for their detection of microbial ligands and subsequent initiation of inflammatory signaling pathways (Aderem & Ulevitch, 2000; Akira & Takeda, 2004; Beutler et  al., 2006; Janeway Jr. & Medzhitov, 2002). Epithelial cells in vagina and cervix and hematopoietic cells express TLR2. TLR4 is expressed by leukocytes (monocytes, macrophages, and neutrophils) present in the lower female genital tract (Fichorova et al., 2002; Givan et al., 1997). Zariffard assessed the ability of cervicovaginal lavage (CVL) from five women with BV to induce

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proinflammatory pathways resulting in TNF-α secretion relative to CVL from five women without BV. Nine women were also assessed 4 weeks after BV treatment (Zariffard et  al., 2005). Relative to CVL from women without BV, CVL from women with BV induced somewhat higher levels of TNF-α secretion by peripheral blood mononuclear cells (PBMCs) (P = 0.05). Successful treatment of BV resulted in no increase in TNF-α. When assessed as whole bacteria and with use of THP-1 (monocytic transformed) cell lines, G. vaginalis and M. hominis stimulated significant TNF-α secretion relative to controls, but L. crispatus did not. In an effort to characterize the TLR response mediated by the presence of BV, Mares et al. exposed 293 cell types expressing specific TLRs to CVL from women with and without BV; BV-associated CVLs primarily activated cells through TLR2, but not TLR4 (Mares et al., 2008). There is limited evidence for an association of  TLR polymorphisms with BV susceptibility. While Goepfert found no association between BV and the TLR4 299 and TLR4 399 gene polymorphisms among 497 pregnant women with and 388 without BV (Goepfert et al., 2005), other investigators have observed that the TLR4 896A>G polymorphism may modify the vaginal immune response to G. vaginalis and anaerobic Gram-negative rods (Genc et al., 2004). Increases in IL-1β observed in the vaginas of TLR4 896A homozygotes colonized with these bacteria were not observed in carriers of the TLR4 896G allele also colonized, despite the latter group having significantly higher quantities of these bacteria. These investigators hypothesized that a blunted proinflammatory cytokine response in the latter group was permissive for proliferation of these BVAB. Although allelic frequency of genes regulating the immune system differs by self-reported race and ethnicity, whether these patterns influence vaginal microbiota is not known (Nguyen et  al., 2004). In a cross-sectional study of pregnant women, two TLR4 single nucleotide polymorphisms (SNPs) were significantly associated with cervical IL-1 concentrations in European American, but not African American women; this relationship was strengthened in women with BV (Ryckman et al., 2009). The TLR studies offer an intriguing glimpse into how innate immunity, particularly TLR4, may modify risk of acquiring BV, and likelihood of cure and adverse consequences. However, studies performed to date are limited by small size, characterization of subjects and vaginal microbiota, and nature of follow-up after BV treatment.

CLINICAL MANIFESTATIONS In a cross-sectional study of clinic patients, BV defined by Gram stain criteria was significantly associated with symptoms of vaginal malodor (49% of patients with BV versus 20% without BV) and vaginal discharge (50% of patients with BV versus 37% without BV), and with signs of a non-viscous homogeneous, white, uniformly adherent vaginal discharge (69% of women with BV versus 3% without). As noted above, the malodor is attributed to the abnormal presence of amines, particularly trimethylamine. The discharge is

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FIGURE 18.4  Typical vaginal discharge caused by BV. Please see color plate section at the back of the book.

typically greyish and homogeneous, and adheres uniformly to the vaginal walls, often visibly on the labia and fourchette before insertion of a vaginal speculum (Figure 18.4). While most studies have found no significant increase in the mean number of polymorphonuclear neutrophils (PMN) in the vaginal discharge of women with BV, the finding of PMNs in the wet mount should not exclude a diagnosis of BV. This is especially true if cervicitis is detected in the setting of BV. One study showed that leukorrhea (>5–10 white blood cells per high power field on microscopic examination of vaginal fluid) may be a sensitive indicator of cervical inflammation with a high negative predictive value, particularly among women with BV (Geisler et al., 2004). Nearly all women with BV have a vaginal pH above 4.5, although this finding is not specific for BV. A fishy odor was noted when vaginal fluid was mixed with 10% KOH (the ‘whiff test’) in 43% of those with BV versus 1% of those without BV (Amsel et al., 1983).

DIAGNOSIS In clinical practice, BV is typically diagnosed using the Amsel criteria, which include the presence of at least three of four findings: vaginal pH greater than 4.5, homogeneous vaginal discharge on examination, detection of fishy odor on addition of potassium hydroxide to vaginal fluid (positive ‘whiff test’), and presence of significant clue cells (Amsel et al., 1983) (defined as >20% of the total vaginal epithelial cells seen on 100× magnification on saline microscopy). The vaginal fluid pH determination requires pH paper having an appropriate range of pH 4.0–6.0. Commercially available pH paper suitable for this purpose includes a pH range of 3.0–6.5. Vaginal pH is best

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determined by swabbing the lateral or posterior fornices of the vagina, then placing the swab sample directly on the pH paper. Alternatively, the pH paper can be placed on vaginal fluid pooling in the speculum after removal from the vagina. The cervical mucus should be avoided since it has a higher pH (pH 7.0) than the vaginal fluid. Vaginal malodor is the most common symptom of women with BV, and release of the ‘fishy’ amine-odor from vaginal fluid after addition of potassium hydroxide increases detection of malodor. A drop of vaginal fluid should be placed on a glass slide and a drop of 10% KOH added. A coverslip placed over this preparation permits microscopic exam for the pseudohyphal forms associated with candidiasis. Clue cells are squamous vaginal epithelial cells covered with many vaginal bacteria, giving them a stippled or granular appearance. The borders are obscured or stippled owing to adherence of small rods or cocci (Figure 18.5), including Gardnerella, Mobiluncus, and other bacteria. Lactobacilli may also bind to exfoliated vaginal epithelial cells, although seldom in high enough concentrations to mimic clue cells. At least 20% of vaginal epithelial cells resembling clue cells should be present to establish a diagnostic criterion for BV. A sample of vaginal fluid obtained with a swab and mixed on a glass slide with a drop of normal saline is covered by a coverslip, and 10 fields examined under high power (×400) for clue cells. Other point-of-care diagnostic tests take advantage of immediate methods of detecting either high concentrations of G. vaginalis, a variety of the amines that are prominent, including sialidase, trimethylamine, and prolineaminopeptidase, or some combination of amines and abnormal pH (Myziuk et  al., 2003). A specific oligonucleotide probe test adjusted to detect only

FIGURE 18.5  Wet mount of vaginal fluid showing a typical clue cell from a woman with bacterial vaginosis. Note that the cell margins are obscured. (×400 Magnification.) Photograph provided by Lorna K. Rabe.

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high concentrations (>107/mL vaginal fluid) of G. vaginalis was 87.7% sensitive and 96% specific for the diagnosis of BV in 115 women (Affirm VP III, Becton-Dickinson, Cockeysville, MD) (Gazi et al., 2006). This test simultaneously detects Candida species as well as Trichomonas vaginalis, and provides a laboratory-based option for detection of all three vaginal pathogens. The advantage of this system is that it is objective, detects mixed vaginal infections, and can be used in any setting approved for moderately complex laboratory tests. Despite the ease of use of point-of-care tests, clinicians unfortunately do not regularly pursue a specific diagnosis of vulvovaginal complaints, and rely (usually inappropriately) on syndromic management to direct treatment. Moreover, accuracy of the Amsel criteria in clinical practice is likely limited by clinicians’ lack of, or limited, skill in using microscopy to detect clue cells and rule out other important findings, including trichomonads and yeast forms. BV may also be diagnosed using a score applied to Gram stains of vaginal fluid, the Nugent criteria, which quantifies the number of lactobacilli relative to BV-associated bacterial morphotypes to create a 10-point scale of flora abnormality ranging from normal (score 0–3) through intermediate (score 4–6) to frank BV (score 7–10) (Nugent et  al., 1991). This method, based upon the shift in bacterial morphotypes, from predominance of lactobacilli (Figure 18.6) to a predominance of G ­ ardnerella and anaerobic bacterial morphotypes including Mobiluncus (Figure 18.7), has had 89% sensitivity and 83% specificity for diagnosis of BV in comparison with clinical criteria,

FIGURE 18.6  Gram stain of normal vaginal fluid, showing Gram-positive rods with blunt ends consistent with lactobacilli. (×1000 Magnification.) Photograph provided by Lorna K. Rabe. Please see color plate section at the back of the book.

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FIGURE 18.7  Gram stain of vaginal fluid from a woman with bacterial vaginosis showing absence of lactobacilli and large numbers of Gram-negative or Gram-variable coccobacilli. Curved Gram-variable rods are consistent with Mobiluncus. (×1000 Magnification.) Photograph provided by Lorna K. Rabe. Please see color plate section at the back of the book.

and has had excellent inter-center reproducibility (Joesoef et al., 1991). The vaginal smear for Gram stain can be prepared at the same time that the wet mount is prepared by rolling (not streaking) the swab across the surface of a glass slide. After air-drying, the slide can be stored for months or years prior to staining, with no appreciable loss in quality. Self-obtained swabs have also been proven to be acceptable for preparation of Gram-stained vaginal smears (Brotman et al., 2010). The slide should be heat-fixed and stained as usual in the clinical lab. The advantages of Gram stain for diagnosis include interpretation by standardized objective criteria by a microbiologist, suitability for quick screening, and storage for batch reading or later confirmation if desired. Most recently, targeted qualitative and quantitative PCR assays for detection of various BV-associated bacteria have been studied; this approach may offer some utility in the future, but has not been widely validated for diagnosis of BV in large, diverse populations of women and is costly (Fredricks et al., 2007; Menard et al., 2008). Moreover, quantitative PCR has not yet been well studied for its ability to differentiate between women who have intermediate flora versus frank BV as determined by the Nugent score; women with intermediate flora by Nugent score may have relatively high quantities of the BV-associated bacteria G. vaginalis and Atopobium vaginae as determined by quantitative PCR (qPCR) (Menard et al., 2008). The currently commercially available panel using PCR offers detection of G. vaginalis, Bacteroides fragilis, Mobiluncus mulieris, and Mobiluncus curtisii, and thus detects only a small proportion of the bacterial species that characterize BV. The optimal molecular methodology

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or criteria, both qualitatively and quantitatively, to serve as the next generation diagnostic modality has not been determined. Thus the current commercial tests require additional validation. Cultures for G. vaginalis or other individual microbes have little utility for the diagnosis of BV. G. vaginalis can be recovered from nearly all women with BV, but also from up to 70% of those without BV (Fredricks et al., 2007). A positive vaginal culture for G. vaginalis in the absence of the clinical signs of BV does not warrant therapy. Likewise, G. vaginalis culture does not constitute ‘test of cure’ since many women without clinical signs of BV have positive cultures for this organism following effective treatment.

TREATMENT Interpreting the results of treatment trials for BV requires an understanding of the current guidance from the US Food and Drug Administration, which recommends that clinical cure be defined as the resolution of all four clinical signs (Amsel criteria) of BV (U.S. Department of Health and Human Services, 2009). If this definition of cure is used, cure rates for all of the available therapies, whether administered orally or vaginally, are usually about 50%. Using criteria that are more likely to reflect improvement in women’s vaginal symptoms (absence of BV by Amsel or Nugent criteria), most trials indicate that over 85% of women respond initially to currently recommended regimens. More perplexing is the high rate of early recurrence (30% at 3 months, 50% at 6 months), reflecting early relapse and more likely late reinfection, for which successful management has not been forthcoming. Although each symptomatic episode usually responds rapidly to conventional antibiotic treatment, rapid recurrence is frequently inevitable. Antimicrobial compounds with broad activity against most anaerobic bacteria are effective at relieving symptoms of BV (Koumans et al., 2002). Metronidazole and clindamycin are the mainstays of therapy (Workowski & Berman, 2010). Tinidazole, a nitroimidazole with antiprotozoal and antibacterial activity, is also approved for the treatment of BV in two oral dosing regimens (2 g daily for 2 days or 1 g given for 5 days). The published studies have consistently reported resolution of BV in 71–89% or more of women 1 month after administration of these regimens (Hillier et al., 2008). Intravaginal therapies have had efficacy similar to that of oral metronidazole regimens with fewer side effects. In a meta-analysis of metronidazole treatment of BV, Lugo-Miro reported initial cure in 87% of 280 women receiving oral metronidazole (400–500 mg) two to three times daily for 7 days, and an 86% response in 317 women receiving 5 days of oral therapy (Lugo-Miro et  al., 1992). From analysis of over 1200 women, over 85% of women responded initially to metronidazole therapy. However, these authors reported that the 2 g stat dose was equivalent to 7 days of oral therapy, a finding refuted in subsequent studies. An analysis of six studies which compared the single 2 g dose of metronidazole versus 800–1200 mg

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per day for 7 days and followed patients for at least 1 month after treatment reported that five of these six studies showed lower cure rates with the single dose (Hillier & Holmes, 1999). When results from the six studies are combined, a 2 g single dose of metronidazole was significantly less effective than 7-day regimens (82% versus 73%, P = 0.03) for treatment of BV. These data led the CDC to remove this regimen from its list of recommended therapies for BV in 2006. Side effects of oral metronidazole include nausea, metallic taste, headaches, and gastrointestinal distress. Oral metronidazole may also lead to a disulfiramlike reaction after alcohol consumption, which may decrease patient compliance. To reduce systemic absorption of metronidazole and decrease side effects, intravaginal therapies were developed (Box 18.1) (Koumans et  al., 2002; Livengood 3rd et al., 1999). Use of intravaginal metronidazole resulted in fewer side effects than were observed after use of oral metronidazole. A direct randomized comparison of metronidazole vaginal gel twice daily for 5 days versus oral metronidazole 500 mg twice daily for 7 days showed that, 5 weeks after therapy, BV was cured in 71% (29 of 41) of the intravaginal metronidazole group, and in 71% (32 of 45) of the oral metronidazole group (Hanson et al., 2000). In a randomized trial of 7 days of oral versus five daily doses of a topical 0.75% metronidazole gel for treatment of BV during pregnancy, efficacy was similar for both groups (Yudin et al., 2003). Finally, limited data suggest that increasing the dose of vaginal metronidazole may effect higher cure rates for BV. Sanchez compared metronidazole vaginal gel (Metrogel™) to Flagystatin™ (ovules with 500 mg metronidazole and 100 000 U nystatin) given nightly for 5 nights (Sanchez et al., 2004). Of 138 women evaluable for follow-up at mean 42 days, BV persisted in 38% in the gel and 17% in the ovule group (P = 0.01). While the higher dose of metronidazole was presumed to be the reason for the superior efficacy of the combination product, a potential contribution of the antifungal component could not be ruled out in this study design. Moreover, the study was limited by relatively low rates of follow-up and assessment of cure at variable intervals. Nonetheless, the data are intriguing and support a potential route of investigation towards more effective regimens for BV treatment. Efficacy of oral clindamycin has generally been equivalent to that of oral metronidazole, while vaginal clindamycin has sometimes produced somewhat lower cure rates than those of vaginal metronidazole regimens (48% in a recent multicenter study). However, not all studies have measured cure at similar times with the same criteria for cure. Intravaginal clindamycin for the treatment of BV in non-pregnant women has been evaluated in numerous studies. Several double-blinded, placebo-controlled trials have been published comparing 7 days of intravaginal clindamycin cream (and twice daily oral placebo) to 7 days of oral metronidazole administered twice daily (and once-daily placebo vaginal cream) (Koumans et al., 2002). In all of these trials, intravaginal clindamycin vaginal cream was as efficacious as 7 days of oral metronidazole therapy for the treatment of BV after both 1 week and 1 month of follow-up. Another study

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compared topical clindamycin, in the form of ovules used intravaginally for these days, with metronidazole gel applied daily for 5 days. Cure rates were not statistically different for the two groups, although clindamycin-resistant microorganisms were reported frequently among women in the clindamycin treatment group (Beigi et al., 2004). In summary, intravaginal therapies with metronidazole gel or clindamycin cream, as described above, are considered effective treatments for vaginal infections. Intravaginal therapies have been well tolerated. However, the costs of such therapies currently exceed the cost of a comparable course of oral metronidazole. Tinidazole, like metronidazole, is a nitroimidazole with antiprotozoal and antibacterial activity, and is approved in the USA for the treatment of BV, as well as trichomoniasis. Outside of the USA, tinidazole has been used widely for the treatment of BV in various oral dosing regimens. In recent studies, cure rates using a composite of both Amsel and Nugent criteria were 88% for the 2 g oral dose and 73% for a 2 g single dose of oral metronidazole, with recurrent BV seen at 12 weeks in 3% of the group given tinidazole and 30% of those given metronidazole (P = 0.04). In a multicenter, placebo-controlled randomized trial of oral tinidazole given at either 2 g daily for 2 days or 1 g given for 5 days, resolution of BV (as defined by absence of at least three Amsel criteria) was seen in 46% of subjects who received the 2-day regimen and in 64% of those who received the 5-day regimen (Livengood 3rd et al., 2007). Factors that determine whether a woman with BV will respond to these standard regimens are not clear. One prospective study indicated that detection of any of several BVAB at the onset of treatment with intravaginal metronidazole predicted treatment failure at 30 days (Marrazzo et al., 2008). Clindamycin-resistant bacteria have been reported among women treated with vaginal clindamycin, although this was not associated with reduced cure rates (Beigi et  al., 2004). Although metronidazole is active against Gram-negative anaerobes and Mobiluncus mulieris, it is less active against G. vaginalis, anaerobic Gram-positive cocci and M. curtisii, and inactive against M. hominis and Atopobium vaginae (Austin et al., 2005). M. curtisii is not susceptible to metronidazole in vitro, but is usually eradicated following metronidazole therapy. It is possible that inhibition or elimination of metronidazole-susceptible members of the vaginal bacteria in BV results in a decline in certain non-susceptible members as well.

Non-antibiotic Approaches to Managing BV Because BV results from an ecological shift in the vaginal microflora, a number of researchers have evaluated therapies that either act as vaginal disinfectants or are aimed at restoring the vaginal ecosystem. The efficacy of vaginal acidifiers in the form of gels, suppositories, and acidsoaked tampons has varied widely, from 18% to 80% in several small studies.

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However, intermittent use over weeks is usually required. Vaginal acidifiers will suppress, but not kill, vaginal anaerobes, so may suppress without effecting a cure. Hydrogen peroxide douches have been advocated for BV treatment as an alternative to antimicrobial therapy. In one study of 23 women with recurrent BV, a 3-minute vaginal treatment with 3% H2O2 was reportedly effective in most women. Concerns with this approach include that it involves douching (a procedure most experts feel should be generally discouraged), that simple application of H2O2 may produce short-term disinfection but will not sustain resolution of abnormal flora over time, and that even H2O2-producing lactobacilli may be killed by high concentrations of H2O2. Even though certain lactobacilli may play an important role in maintaining the normal vaginal flora, it remains to be determined whether the application of such lactobacilli is sufficient to restore the vaginal microflora (Senok et  al., 2009). At least one human-derived strain of Lactobacillus crispatus (CTV-05) is currently under study as treatment for BV, but is not yet commercially available (Antonio & Hillier, 2003; Patton et al., 2003). In a randomized placebo-controlled trial of this product administered with oral metronidazole as treatment for BV, women randomized to the CTV-05 group were less likely to be colonized by endogenous strains of lactobacilli 1 month after treatment, indicating that the L. crispatus contained in the CTV-05 disrupted vaginal colonization by endogenous lactobacilli, suggesting that probiotic strains of lactobacilli may disrupt colonization by endogenous strains of lactobacilli through competitive inhibition (Antonio et al., 2009). Further support for this hypothesis comes from the same product’s success in reducing the rate of recurrent urinary tract infections when given in conjunction with an antibiotic treatment regimen (Stapleton et al., 2011). Another study of orally administered lactobacilli reported a high level of effectiveness among African women (Anukam et al., 2006).

Treatment of Sex Partners Several placebo-controlled trials have demonstrated that treatment of male partners does not improve the clinical outcome of treatment of BV, or reduce recurrence. For example, Moi reported no differences in initial response of BV to therapy or in recurrence at 4–12 weeks after therapy for women whose partners were or were not treated with oral metronidazole (Moi et al., 1989). In a double-blind randomized trial, Vejtorp observed no effect of treatment of the male partner on symptoms, clinical signs of BV, or vaginal isolation of G. vaginalis 1 and 5 weeks after BV treatment (Vejtorp et al., 1988). Similarly, Vutyavanich reported that tinidazole treatment of male partners in Thailand had no effect on clinical response of BV, but was associated with a significant increase in side effects in the men who received tinidazole compared to placebo-treated men (22% versus 7%, P = 0.006) (Vutyavanich et  al., 1993). In a study in which women were treated for BV with clindamycin vaginal cream and their male

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partners were randomly treated with oral clindamycin or placebo, recurrences of BV were unrelated to treatment of partners (Colli et al., 1997). Because controlled trials of male partner treatment demonstrated no benefit to their female partners, current guidelines do not recommend routine treatment of male partners (Workowski & Berman, 2010). The discrepancy between data suggesting sexual acquisition of BV and the lack of benefit of treating the male partner remains puzzling. Part of the issue may be that the selection or dosing of the antibiotics used in the trials done to date were not appropriate or adequate for eradicating a potential reservoir for BVAB in men. Wives of men who participated in a randomized trial of circumcision to prevent HIV acquisition in Uganda were followed to assess effects on vaginal infections. Among women without BV at enrollment, BV at follow-up was significantly less common in wives of men who had been circumcised compared to wives of men who had not (prevalence risk ratio (PRR) 0.80, 95% CI 0.65–0.97). In women with BV at enrollment, persistent BV at 1 year was significantly lower in the circumcision arm than control arm women (PRR 0.83, 95% CI 0.72–0.96). Assessment of local bacteria pre- and post-circumcision among 12 participants revealed not only that several anaerobic bacterial families were detected in the subpreputial space, but also that they were significantly decreased in quantity and species diversity after circumcision (Gray et al., 2009).

Prevention of BV and its Recurrence While short-term response to standard treatment regimens is acceptable, symptomatic BV persists or recurs in 11–29% of women at 1 month and in 50–70% by 3 months. Long-term recurrence rates may approach 80% in certain populations. Possible reasons for this include failure to suppress the growth of BVAB continuously; reinoculation with these organisms from an exogenous source (for example, sexually); persistence of host risk factors (for example, douching or use of an intrauterine device); failure to recolonize the vagina with H2O2producing lactobacilli; and infection with a Lactobacillus phage that destroys vaginal lactobacilli. None of these mechanisms has been conclusively shown to explain the high rates of BV recurrence, or to identify women at increased risk for BV incidence, recurrence, or sequelae. To date, the only proven interventions that prevent development or recurrence of BV are circumcision of male partners (Gray et al., 2009) and chronic suppressive antibiotic therapy (Sobel et al., 2006). Sobel and colleagues performed a prospective multicenter study in which women with current BV and at least two prior episodes of BV in the previous year were initially treated with 10 days of vaginal metronidazole gel then, if cured, randomly assigned to receive twice-weekly metronidazole vaginal gel or placebo for 16 weeks (Sobel et al., 2006). They were subsequently followed off therapy for 12 weeks. Of 157 eligible women, 112 of 127 returning evaluable women (88.2%) responded clinically and were randomly assigned. During suppressive therapy, recurrent BV occurred in 13 women (25.5%)

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receiving metronidazole and 26 (59.1%) receiving placebo (modified intentto-treat analysis, relative risk (RR) 0.43, CI 0.25–0.73, P = 0.001). During the entire 28-week follow-up, recurrence occurred in 26 (51.0%) on treatment compared with 33 (75%) on placebo (RR 0.68, CI 0.49–0.93, P = 0.02). Probability for remaining cured was 70% for metronidazole compared with 39% on placebo, which declined to 34% and 18%, respectively, by 28 weeks’ followup. Adverse effects were uncommon; however, secondary vaginal candidiasis occurred significantly more often in metronidazole-treated women (P = 0.02). The authors concluded that suppressive therapy with twice-weekly metronidazole gel achieves a significant reduction in the recurrence rate of BV. No other regimens, including clindamycin-based or oral metronidazole, have been studied for suppression of recurrent BV. Another randomized trial of STI pre-exposure prophylaxis evaluated other vaginal infections. It assessed the effect of directly observed oral treatment with 2 g of metronidazole plus 150 mg of fluconazole compared with metronidazole placebo plus fluconazole placebo administered monthly in reducing vaginal infections among Kenyan women at risk for HIV-1 acquisition. Of 310 HIV1-seronegative female sex workers enrolled (155 per arm), 303 were included in the primary endpoints analysis. Compared with control subjects, women receiving the intervention had fewer episodes of BV (hazard ratio (HR) 0.55, 95% CI 0.49–0.63) and more frequent vaginal colonization with any Lactobacillus species (HR 1.47, 95% CI 1.19–1.80) and hydrogen peroxide-producing Lactobacillus species (HR 1.63, 95% CI 1.16–2.27). The incidences of vaginal candidiasis (HR 0.84, 95% CI 0.67–1.04) and trichomoniasis (HR 0.55, 95% CI 0.27–1.12) among treated women were less than those among control subjects, but the differences were not statistically significant. The authors concluded that periodic presumptive treatment reduced the incidence of BV and promoted colonization with normal vaginal flora (McClelland et al., 2008). Another trial randomized women with asymptomatic BV to observation or treatment and prophylaxis with twice-weekly intravaginal metronidazole gel. Women in the metronidazole gel arm had fewer chlamydial infections over the subsequent 6 months (Schwebke & Desmond, 2007). Because BV recurrence is so common even with excellent anti-anaerobic antibiotic therapy, patients and investigators have attempted a wide range of other treatments. As noted above, commercially available Lactobacillus preparations do not contain lactobacilli that adhere to human vaginal epithelial cells, nor do they usually produce hydrogen peroxide, a probable key component for the protective benefit conferred by human vaginal lactobacilli. They are also frequently contaminated with other enteric organisms, presumably of bovine origin. For these reasons, neither oral nor intravaginal therapy with over-thecounter Lactobacillus preparations is recommended. Yogurt may contain several species of lactobacilli, but none adhere to vaginal epithelial cells or reliably produce hydrogen peroxide; thus, it has no role in BV treatment. Most recently, advances in the field of probiotics have infused new energy into therapeutic

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studies for BV. Probiotics are defined as ‘a product containing viable, defined microorganisms in sufficient numbers, which alter the microflora (by implantation or colonization) in a compartment of the host and by that exert beneficial health effects in this host.’ BV is a logical target for treatment with probiotics aimed at establishing sustained vaginal recolonization with appropriate Lactobacillus species. At least one human-derived strain of Lactobacillus crispatus is currently under study as treatment for BV, but is not commercially available at this time. Substances that act as vaginal acidifiers have been evaluated in small studies. BufferGel, a spermicidal microbicide that acidifies semen and vaginal fluid, showed a modestly beneficial effect, and is undergoing further study. However, another acidifier, Aci-Jel, failed to improve rates of BV cure in one study, so the effects of these products may be unpredictable. Further, none offer a method to sustain low vaginal pH over time. However, an absorptive tampon that also releases lactic and citric acids has been approved by the FDA. Because this product was not categorized as a new drug, evidence to support any efficacy in preventing or treating BV was not included in the approval process. Finally, some investigators have recommended vaginal instillation of H2O2, either as a douche or in a saturated tampon. Concerns with this approach include that it involves douching (a procedure most experts feel should be generally discouraged), that simple application of H2O2 may produce short-term disinfection but will not sustain resolution of abnormal flora over time, and that H2O2-producing lactobacilli may be killed by high concentrations of H2O2. Incorporation of our knowledge about key risk factors into client-centered counseling may positively impact women’s ability to prevent future episodes of BV. First, douching is a major risk factor for BV and for the loss of the vaginal lactobacilli that are associated with the clinical development of symptomatic BV. For this reason, because douching has also been epidemiologically linked to pelvic inflammatory disease (PID), ectopic pregnancy, and chlamydial cervical infection, and because douching provides no known prophylactic benefit against genital tract infection, women should be advised not to douche. The observation that BV is associated with acquisition of a new sex partner, multiple sex partners, and sex with female partners implies that use of barrier methods (condoms) may protect against its acquisition and against its persistence or recurrence if they are used in the first month after treatment for BV. Since concurrent treatment of male partners has not yet been shown to prevent recurrent BV, treatment of male sex partners cannot be recommended. Recolonization of the vagina with H2O2 lactobacilli may protect against infection by BV-associated organisms, but this has not yet been adequately evaluated. The role of IUD usage as a risk factor for BV requires further study to confirm or refute the association and to assess the importance of IUD type to any relationships found. Some data suggest a role for receptive anal and oral sexual behaviors; limiting these behaviors around the time of BV treatment might offer some protection, though data in this area are lacking.

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For women who have sex with women, those who share vaginal sex toys should either use condoms on these toys between shared use or wash them thoroughly. Presumptive treatment of female sex partners has not been evaluated, and cannot be recommended but, given the high rate of concordance for BV within sexual partnerships, evaluation of female partners and treatment of BV if present might be prudent, particularly for women with persistent or recurrent BV. Limiting exchange of vaginal fluid from one woman with BV to another would seem to be a logical recommendation to address the possibility of this behavior in facilitating BV. However, a study of a behavioral intervention that led to lower rates of unprotected digital–vaginal contact and of shared sex toy use did not observe a reduction in the rates of persistent BV at 1 month following the intervention (primary study outcome) or in recurrent/ persistent BV over the course of longer term follow-up (secondary outcome) (Seck et al., 2001). Of note, the study did not address a potentially causative role for oral sex in promoting BV, and participants engaged in this behavior with equal, and not uncommon, frequency in both study arms. Receptive oral sex has been associated with a trend towards abnormal vaginal microbiota or frank BV, and several of the bacteria associated with BV have been identified in the oral cavity (Kumar et al., 2005). Although the intervention did result in behavior change during the time between BV treatment and assessment of cure at 1 month, a longer duration of behavior change may be required to substantially impact the likelihood that sexual practices affect the vaginal microenvironment.

COMPLICATIONS OF BV BV is associated with serious sequelae related to the upper genital tract. The exact means by which BV promotes adverse reproductive tract sequelae is not clear. Possible explanations include loss of antimicrobial compounds produced by lactobacilli (lactic acid, H2O2); destruction of mucin gel coating the vaginal/ cervical epithelium via inhibition of glycosidase-producing anaerobes; degradation of secretory leukocyte protease inhibitor (SLPI); induction of a cervical proinflammatory environment; and alteration in the immune cell environment of the cervix. In non-pregnant women (Sweet, 2000), BV increases risk of post-hysterectomy infections (Larsson et al., 1991; Soper et al., 1990) and PID (Hillier et al., 1996; Wiesenfeld et  al., 2002), and risk of acquiring Neisseria gonorrhoeae (Hawes et al., 1996; Martin et al., 1999; Wiesenfeld et al., 2003) and C. trachomatis (Brotman et al., 2010). BV itself may cause endocervical inflammation that manifests as mucopurulent cervicitis (Marrazzo et  al., 2006; Seck et  al., 2001). Finally, BV enhances women’s likelihood of sexual acquisition of HIV (Martin et al., 1999; Schmid et al., 2000; Taha et al., 1998; Wawer et al., 1999), possibly through inducing reversible changes in the cervical or other mucosal immune environment (Rebbapragada et al., 2008).

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Pelvic Inflammatory Disease Anaerobes have long been linked with salpingitis (Sweet, 2000). Many of the bacteria recovered from the endometrium and Fallopian tubes of patients with PID are those present in the vagina in high numbers among women with BV (Soper et al., 1994). However, likely interactions between Chlamydia trachomatis, N. gonorrhoeae, and facultative bacteria in producing PID have not been well defined, and the relationship between BV or intermediate flora and acquisition of C. trachomatis and N. gonorrhoeae is probably complex and may depend in part on the microbiologic composition of BV itself. In one study that prospectively followed more than 1000 women for 3 years, BV at baseline was associated with concurrent chlamydial or gonococcal infection (adjusted OR 2.8, 95% CI 1.81–4.42) but not significantly with subsequent detection of either of these pathogens at follow-up visits (RR 1.52, 95% CI 0.74–3.13). However, among women whose BV at baseline was characterized by dense growth of pigmented, anaerobic Gram-negative rods, risk of subsequent detection of chlamydia or gonorrhea was increased (RR 1.93, 95% CI 0.97–3.83) (Ness et al., 2005). In a study of symptomatic women presenting for care, endometrial biopsies were obtained on 178 consecutive women with suspected PID (Hillier et  al., 1996). Endometrial specimens and cervical swabs were tested for N. gonorrhoeae and C. trachomatis. Eighty-five of the patients also underwent laparoscopy to confirm a clinical diagnosis of salpingitis. Among women with endometritis, 27% had N.gonorrhoeae present in the endometrium, while 13% had endometrial C. trachomatis. Approximately 50% of the patients with endometritis had anaerobic Gram-negative rods in their endometrial samples. Endometrial N. gonorrhoeae was independently associated with a fivefold increase in endometritis, which was similar for endometrial C. trachomatis (OR 4.8) and anaerobic Gram-negative rods (OR 2.6), respectively. BV was not an independent risk factor for endometritis in the absence of endometrial anaerobes. More recently, Wiesenfeld performed endometrial biopsies on women with BV (and other lower genital tract infections, including chlamydia and gonorrhea) but without symptoms or signs of acute PID (Wiesenfeld et al., 2002). Among 377 women with BV, 58 (15%) had evidence of subclinical PID (defined as the presence of 5 neutrophils per 400× field and 1 plasma cell per 120× field of endometrial tissue), while plasma cell endometritis (defined as the presence of at least 1 plasma cell per 120 of endometrial tissue) was present in 90 (24%). Subclinical PID, but not plasma cell endometritis, was more common in women with BV than in women without BV. Women with intermediate vaginal flora on Gram stain were at intermediate risk for subclinical PID (P < 0.05, test for trend), but not plasma cell endometritis. In a multivariate model that included proliferative phase of the menstrual cycle, previous pregnancy, black race, and current infection with N. gonorrhoeae, C. trachomatis, BV, or T. vaginalis, subclinical PID remained significantly associated with BV (adjusted OR 2.7, 95% CI 1.02–7.2).

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BV in Pregnancy Pregnant women with BV have an increased risk of preterm delivery (Hillier et al., 1996; Hillier et al., 1995; Meis et al., 1995; Goldenberg et al., 1996b), first trimester miscarriage in women undergoing in vitro fertilization (Ralph et al., 1999), amniotic fluid infections (Silver et al., 1989), chorioamnionitis (Hillier et al., 1988), postpartum and postabortal endometritis (Watts et al., 1990), and postabortal PID (Larsson et al., 1992). Observational studies that have included women from different ethnic and socioeconomic groups have consistently reported an increased risk for preterm delivery and/or low birthweight among women colonized by BV-associated pathogens (anaerobic Gram-negative rods and M. hominis), and among those with clinical or Gram stain evidence of BV, and some have noted a decrease in prematurity among those vaginally colonized by Lactobacillus spp. Several studies link BV with infection of the fetal placental membranes (chorioamnion) and amniotic fluid, suggesting that ascension of vaginal microorganisms into the decidua, chorioamnion, or amniotic fluid, resulting in infection and inflammation at these sites, represents a probable mechanism by which BV can initiate labor and result in preterm delivery. Amniotic fluid infection results most commonly from invasion of lower genital tract bacteria through the placental membranes. The isolates most frequently recovered from the amniotic fluid of women with intact membranes are the microorganisms associated with BV, and women with BV are twice as likely to have invasion of the amniotic fluid as women with Lactobacillus-predominant vaginal flora. Watts reported a relationship of BV diagnosed at the time of Cesarean section to clinically diagnosed amniotic fluid infection, which occurred among 22% of women with a Gram stain diagnosis of BV compared to 4% of those with a Lactobacillus-predominant vaginal smear (Watts et al., 1990). Hitti evaluated 197 afebrile women in preterm labor with intact fetal membranes and found that women having a Gram stain consistent with BV were more likely to have amniotic fluid infection. High concentration of IL-8 in the vagina and anaerobic flora were both associated with amniotic fluid infection (Hitti et al., 2001). Multiple studies have evaluated whether treatment of BV in pregnancy prevents preterm birth (McDonald et al., 2011). Numerous agencies have evaluated the available data on treatment of BV in pregnancy and concluded that routine screening, and consequent treatment, of asymptomatic women with BV cannot be justified (Nygren et  al., 2008). A Cochrane review published in 2011 concluded that: 1) there was good evidence that antibiotic therapy was effective at eradicating BV; 2) treatment did not reduce the risk of preterm birth before 37 weeks or the risk of preterm premature rupture of membranes; 3) treatment before 20 weeks’ gestation may have reduced the risk of preterm birth at less than 37 weeks; and 4) treatment of women with a previous preterm birth did not affect the risk of subsequent preterm birth. Although some authors have asserted that BV may be more of a stimulant for preterm delivery at earlier gestational ages, an analysis of 12 937 deliveries failed to demonstrate that the

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risk of preterm birth was increased when BV was identified at earlier gestational ages (Klebanoff et al., 2005). In summary, BV during pregnancy has consistently been related to chorioamnion infection, histological chorioamnionitis, and amniotic fluid infection. These three entities are strongly interrelated and are associated with preterm delivery. The mechanisms by which BV causes prematurity and low birthweight are not completely understood, and some experts argue that BV may be a biomarker for other factors that lead to preterm birth. As discussed above, BVassociated microorganisms ascend to cause infections of the decidua, placenta, and/or amniotic fluid. Although some experts have postulated that BV could lead to initiation of preterm birth through local production of proinflammatory cytokines such as IL-8, treatment of BV has been shown to normalize levels of proinflammatory cytokines and chemokines in the cervical fluid of women with BV. The association of infection with preterm birth is complex, and likely attributable to numerous interrelated factors. A greater understanding of these complex interrelations between infection, the immune response in pregnancy, and genetic differences in response to infection will be needed to develop the insights required for more effective approaches to prevention of preterm birth. Determination of causality will be dependent not only on more precise categorization of the vaginal microbiota, but also on variations in the host environment that may be associated with changes in bacterial communities over time. Continued development of the transcriptomic and metabolomic technology will be of critical importance to the success of these endeavors.

BV and HIV A growing body of evidence suggests that the presence of BV, or absence of vaginal lactobacilli, may increase a woman’s risk of acquiring HIV via heterosexual intercourse. A direct association between HIV infection and BV has been seen in numerous cross-sectional studies, and supported by prospective studies (Atashili et  al., 2008). Taha et  al. followed 1196 pregnant women in Malawi for a median of 3.4 months in the antenatal period and 2.5 years in the postnatal period, with monthly assessment of vaginal flora and HIV serostatus (Taha et al., 1998). They observed that the risk of HIV acquisition as measured by seroconversion increased proportionally to increasingly abnormal vaginal flora as measured by clinical criteria (P = 0.04), and that, overall, BV conferred more than a twofold increase in risk of HIV acquisition (P = 0.04). Among Kenyan sex workers seen monthly, absence of any vaginal Lactobacillus and absence of H2O2 + Lactobacillus and abnormal vaginal flora were independently associated with increased risk of HIV acquisition (Martin et al., 1999). Absence of H2O2-producing Lactobacillus strains conferred the highest risk of HIV acquisition (adjusted HR 2.8, 95% CI 1.1–4.7). In a large, prospective, nested case-control study of women participating in a cervical cancer screening trial in Cape Town, South Africa, HIV acquisition was significantly associated

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with interim BV (adjusted OR 2.01, 95% CI 1.12–3.62) after adjustment for sexual behavior and acquisition of other STDs (Myer et al., 2005). The relationship between BV and HIV acquisition may be most synergistic – and most alarming – in areas of the world where both are highly prevalent. The prevalence of BV among many women in sub-Saharan Africa has been 50% or greater in many studies; with HIV seroprevalence up to 38% in some of these countries, even if BV confers only a small increase in the relative risk for HIV acquisition, the population-attributable risk could be considerable (Atashili et al., 2008). Critically, new data show that BV in women infected with HIV confers an increased risk of HIV transmission to male partners. BV may elevate these risks in several ways, including upregulation of relevant T cell populations by the high burden of associated vaginal anaerobes; reduced levels of protective factors (defensins, SLPI) and increased levels of inflammatory factors (certain cytokines); and loss of H2O2, which is virucidal. A small study demonstrated reversible alterations in cervical immune cell populations (reduced total number of CD4+ T cells, including those expressing the HIV coreceptor CCR5 and the activation marker CD69) among HIV-infected women with BV; these changes could conceivably enhance local HIV replication (Rebbapragada et al., 2008; Cohen et al., 2011).

CONCLUSIONS AND FUTURE DIRECTIONS Despite considerable research effort and recent advances, BV remains an enigmatic condition. Efforts to link BV to a single cultivated bacterial pathogen have been unconvincing. Molecular tools have revealed the complex microbiology of BV, but again have not found a credible single causal pathogen. BV is most likely a heterogeneous syndrome caused by different communities of vaginal bacteria, as has been described for the syndrome of periodontitis linked to changes in oral microbial communities (Socransky et al., 1998). If this view is correct, BV can be considered a dysbiotic condition caused not by a single pathogen but by a change in microbial composition and community structure. A woman’s individual risk for acquiring a particular etiologic vaginal bacterial community might depend on specific practices, such as unprotected sex or douching. Although the Nugent and Amsel criteria have been useful research tools, research focused on the vaginal microbiome may provide more precise classification of vaginal microbiota into distinct categories. Future studies of BV and its associated adverse outcomes should determine if specific combinations of organisms are more pathogenic than others, and causally associated with different adverse events. A recent workshop sponsored by the US National Institutes of Health on the state of BV-related research concluded that the data collected from well designed longitudinal studies will be critical for answering some of the unresolved questions stated above (Marrazzo et al., 2010a). Ideally, studies will collect vaginal samples, such as swabs, on a daily basis from hundreds of women to determine how the vaginal microbiota

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changes over short timescales in response to hormonal and behavioral factors. Women with diverse ages, races, sexual orientations, sexual practices, geographic representation, and underlying health status should be enrolled. For instance, there is a paucity of data on preadolescent women. Twin studies would help illuminate the role of genetics in vaginal bacterial colonization. Comprehensive information in several domains – now often referred to as ‘metadata’ – should be collected along with the vaginal swabs, including data on sexual practices, vaginal pH, antibiotic use, and periodic Gram stains of vaginal fluid. These studies will shed light on the changes in vaginal microbiota that correlate with BV and the dynamic microbial ecology of the human vagina. Are there keystone species in BV that form the initial wave of bacteria that facilitate colonization with other species, or does the entire community of BVAB become established simultaneously? What changes in the vaginal environment precede the development of BV? Finally, determination of causality will depend not only on more precise categorization of the vaginal microbiota, but also on variations in the host environment that may be associated with changes in bacterial communities over time.

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