Bacterial Vaginosis as a Mixed Infection
Brogden KA, Guthmiller JM, editors.
Washington (DC): ASM Press; 2002.
Bacterial Vaginosis as a Mixed Infection
Phillip E. Hay.
Department of Genitourinary Medicine, St. George's Hospital Medical School, Cranmer Terrace, London, SW17 0QT, United Kingdom.
The vagina is a unique environment for bacterial colonization. It is subject to dramatic changes over the course of a lifetime, induced by developmental and hormonal changes. At birth, it is lined by stratified squamous epithelium, which regresses as the influence of maternal estrogen wanes. In childhood, the vaginal flora contains skin commensals and bowel organisms. At menarche, the pH falls from neutral to approximately 4, and the flora becomes dominated by lactobacilli. Many other organisms may be present in lower concentrations, including anaerobic and facultative anaerobic bacteria and Candida spp. The hormonal environment alters on a monthly basis, with additional disturbances to the ecosystem produced by menstruation, washing, and hygiene. Sexual activity can introduce a number of new species and pathogens, as well as alter the pH. Pregnancy and breast-feeding produce longer fluctuations in the hormonal balance. In the climacteric, the vaginal epithelium gradually atrophies, the pH rises, and a flora more similar to that of skin may become reestablished.
Bacterial vaginosis (BV) can be thought of as a disturbance in this vaginal ecosystem in which the lactobacilli are replaced by an overgrowth of vaginal commensal organisms. It may be transient or become persistent. It is recognized as the most common cause of abnormal vaginal discharge in women of childbearing age. The symptoms of a thin, white or yellow discharge accompanied by a fishy smell are so characteristic that it is surprising that BV was not widely recognized until described as nonspecific vaginitis by Gardner and Dukes in 1955 (11). In the 1920s, Schroder in Germany described three grades of vaginal flora and changes in the bacterial composition of the vagina, which broadly correspond to our current understanding of normal, intermediate, and BV flora (36).
The reported prevalence of BV varies widely, from 5 to 51% between different populations. In the United States, Bump and Buesching reported a prevalence of approximately 13% among adolescent girls (8). We reported a similar prevalence in a gynecology clinic (16) and antenatal clinic (14) in the United Kingdom. The incidence is higher in women undergoing termination of pregnancy (28%) (5) and in a group of women having in vitro fertilization treatment (24.6%) (31). In the United States, a high incidence was reported for some populations, e.g., inner-city pregnant women (32.5%) (21). The highest incidence, however, has been reported from Rakai in rural Uganda, where 50.9% of women had BV, along with a prevalence for Trichomonas vaginalis of 23.8% (27). Eighty percent of these women were asymptomatic.
Vaginal Physiology and BV
At menarche, under the influence of estrogen, stratified squamous epithelium develops in the vagina. Lactobacilli become the dominant organism. The source of the vaginal lactobacilli in an individual woman has not been determined. Lactic acid is produced by both bacterial metabolism and that of the epithelium, and the vaginal pH falls to a level usually between 4.0 and 4.5. Physiological discharge consists of mucus, desquamated epithelial cells, and lactobacilli. The pH may rise above 4.5 at the time of menstruation, when the concentration of lactobacilli is reduced. Cervical mucus and semen have pH between 7 and 8. In mice, exogenous treatment with progesterone alters the flora to one that resembles BV (40). How much do the hormonal changes of a menstrual cycle influence the flora in women?
If BV develops, the pH rises to a level between 4.5 and 7.0. The anaerobic or facultative anaerobic organisms which are usually present in low numbers increase by between 100- and 1,000-fold, to considerably outnumber the lactobacilli, which may eventually disappear. Trimethylamine and the polyamines putrescine and cadaverine are produced by anaerobic metabolism, and they are thought to be responsible for the fishy smell. Microscopy of vaginal fluid shows multiple small bacteria and epithelial cells with large numbers of adherent bacteria. Gardner and Dukes called these clue cells, as they gave a clue to the diagnosis of nonspecific vaginitis (11).
Nonspecific vaginitis was defined as a clinical entity recognizable from the symptoms of a fishy-smelling vaginal discharge confirmed by detecting thin homogenous vaginal fluid adherent to the walls of the vagina and confirmed by finding clue cells on microscopy. Initially, it was thought to be a straightforward infection by one organism, now called Gardnerella vaginalis. Subsequently, other bacteria were identified as part of the BV flora. In 1983, the term BV was coined, with the recognition that there are many bacterial spp. contributing to the condition and that inflammation is usually absent. The KOH or "whiff" test was added as a fourth criterion for the diagnosis, as shown in Table 1 (2).
Table 1. The composite (Amsel) criteria used for the diagnosis of BV in clinical practicea.
The composite (Amsel) criteria used for the diagnosis of BV in clinical practicea.
More recently, scoring systems for interpreting Gram-stained vaginal smears have been used to diagnose BV (14, 23). Self-administered vaginal swabs can be used, making noninvasive screening possible. They are smeared on a glass slide, which is air dried and subsequently read in a central laboratory. The composite criteria define a dichotomy of BV or normal flora. Scoring systems for interpreting Gram-stained smears allow a gradation to accommodate intermediate patterns.
Treatment and Complications
The reason that some women get BV frequently and others either never get BV or get it infrequently has yet to be fully defined. This will be discussed below. Treatment with antibiotics to suppress the anaerobic overgrowth is usually successful in eradicating BV but not necessarily in eliminating the underlying disturbance which allowed it to develop in the first place. In some women, BV may relapse within 2 to 3 weeks of antibiotic treatment. Standard treatments are shown in Table 2.
Table 2. Standard treatments for BV.
Standard treatments for BV.
Women with BV have an increased risk of many obstetric and gynecological complications. These include second-trimester miscarriage and preterm birth, early failure of in vitro fertilization, an increased risk of upper genital tract infection following termination of pregnancy, and an increased risk of infective complications after hysterectomy. In addition, in prospective studies, BV has emerged as a risk factor for acquisition of sexually transmitted infection, including human immunodeficiency virus (HIV) infection (18).
Natural History and Epidemiology
In many women, the vaginal ecosystem is in a state of flux, changing at different stages of the menstrual cycle. Four studies have looked at self-collected Gram-stained vaginal smears to examine the natural history of vaginal flora. Schwebke and colleagues monitored 51 women considered to be at low risk for sexually transmitted infection for up to 6 weeks (38). Only 11 of these women had normal flora throughout, with 25 having an intermediate pattern at some point and 13 developing BV. Five of the 13 reported symptoms. There were significant associations between developing BV and prior BV (44 versus 12%), mean number of lifetime partners (13.4 versus 7.15), and a higher mean number of episodes of receptive cunnilingus (3.6 versus 1.4). Of these, only cunnilingus remained significant in a multivariate analysis. Changes in flora were also associated with menses and use of vaginal medication or spermicide.
Increased abnormalities of vaginal flora occur during the first 9 days of the cycle, and studies have concluded that abnormal flora occur in most women at some time and that we might therefore need to revise our concept of what is normal. One study examined daily smears from 18 women with recurrent BV (17). Again, BV usually developed spontaneously early in the menstrual cycle and resolved spontaneously in the second half of the cycle, if it did resolve. Interestingly, in some women, the onset followed episodes of vaginal candidiasis and if anything, BV was more likely to resolve than to appear after unprotected sex with the regular partner. In some women, the onset in resolution of BV occurred within 2 or 3 days, usually after an intermediate stage. The association with resolution of candidiasis is interesting in light of an in vitro study which demonstrated inhibition of candidal growth by putrescine and cadaverine (33). These amines are produced by Gardnerella and other organisms found in BV. An earlier study also reported an association between Candida and recurrent BV (32). Changes in the vaginal flora over a period of 3 months in a woman with recurrent BV are shown in Fig. 1 (17). Another woman in the study had frequent symptomatic relapses of BV over a 10-month period (17). She received standard treatments for BV and one course of doxycycline and metronidazole for pelvic inflammatory disease during this time. She has had no relapses in the following 4 years. It is not clear what happened to prevent BV from recurring again.
Figure 1. Changes in the vaginal flora over a period of 3 months in a woman with recurrent BV (17).
Changes in the vaginal flora over a period of 3 months in a woman with recurrent BV (17). The day of the menstrual cycle, treatment with metronidazole (Met) or clotrimazole pessaries (Canesten), menstruation (Period), and unprotected sexual intercourse (more...)
For the results shown in Fig. 1, self-collected vaginal smears were prepared by the subject daily. They were subsequently Gram stained. The Nugent score is used, in which 0 to 3 is considered normal flora, 4 to 6 is intermediate, and 7 to 10 is considered BV. An arbitrary scoring system has been used for candidiasis, with 0 being no Candida, 2 being presence of spores, and 4 being presence of hyphae. Overall, the subject presented with BV, which resolved with metronidazole treatment. During her next period, candidiasis developed. This resolved with treatment, only to be followed by BV. The BV spontaneously resolved in midcycle, but candidiasis and BV recurred shortly afterwards, and BV recurred again in the final month.
During pregnancy, the vagina is not subjected to the frequent changes in hormone levels associated with menstrual cycles. Few observational studies have been performed during pregnancy, but it appears that BV resolves spontaneously in approximately 50% of women in whom it is present at around 16 weeks gestation and may develop in 2 to 3% of those who did not have it at 16 weeks gestation (15).
Most studies have found an increased prevalence of BV in women of black race compared to those of white race and in those who report cunnilingus, smoking, and use of an intrauterine contraceptive device. In community-based studies, BV is more common in women with chlamydial infection and also those undergoing termination of pregnancy. It has also been associated with changing sex partners and high-risk lifestyle (22). These associations suggest that it behaves as a sexually transmitted disease (STD), but the early study by Bump and Buesching found no difference in the prevalence between virgin and nonvirgin adolescent women, with clue cells detected in 9% of both groups (8). Thus, BV appears to be associated with sexual intercourse and risk of STDs but appears to not be an STD itself. Moreover, BV seems to be common in lesbians, a group at low risk for most STDs. In one study, there was a trend for both members of couples to have either normal flora or BV, suggesting transmission of an etiological agent (4).
A large prospective study of 1,248 women who did not have BV at baseline has recently been reported (M. A. Krohn et al., International Society for Sexually Transmitted Disease Research Meeting, presentation, June 2001). Development of BV was associated with smoking, douching, and no contraceptive use. In the multivariate analysis, independent risk factors were nonwhite race, intermediate flora on Gram stain, a lack of H2O2-producing lactobacilli, two or more sex partners in the previous 4 months, and intercourse more than 3 times/week.
Factors That Reduce Lactobacilli
Vaginal douching or other washing practices are frequently cited as a cause of disturbance of the vaginal flora leading to the onset of BV. In a prospective study, douching was associated with loss of protective H2O2-producing lactobacilli and acquisition of BV (13). A case-control study of 200 women attending a genitourinary medicine clinic in London, United Kingdom, investigated associations between vulval washing, vaginal washing, and douching and BV (30). BV was more common in black Caribbean women than in white women (odds ratio, 2.1; 95% confidence interval, 1.1 to 4.1). Use of bubble bath, antiseptic solution, and douching was more common in women with BV. Prior history of BV was the strongest predictor for current BV (odds ratio, 13.4; 95% confidence interval, 5.5 to 32.6). In the multivariate analysis, after controlling for washing practices, there was no ethnic difference in the incidence of BV. In contrast, in a study of 842 women early in the third trimester of pregnancy in North Carolina, it was reported that race remained associated with BV after controlling for many possible confounding variables, including douching (35). BV was found in 22.3% of black women compared to 8.5% of white women. Black women were also more likely to have the highest Nugent scores of 9 or 10, weighted by the presence of high counts of Mobiluncus morphotypes.
An elegant new hypothesis to explain the occurrence of BV is that lactobacilli are killed by a phage infection (6). Lactobacillus phages are known to affect yogurt cultures in the food industry. They can remain in a temperate (inactive) state or become lytic, when up to 99% of the lactobacillus population may be killed. These phages have now been isolated from human lactobacilli from the vagina (25) and gut and from lactobacilli in yogurt. The last were shown to inhibit vaginal lactobacilli (39). Blackwell hypothesizes that phages might be transmitted by sexual intercourse, dairy products, or feco-oral spread (6). Moreover, carcinogens such as benzo[a]pyrene diol epoxide (26), which is present in cigarette smoke, can induce lysogeny in cultures. If a male partner has triggered BV by transmitting a lactobacillus phage, it is not surprising that treatment with antibiotics makes no difference to the subsequent relapse rate for his partner. Clearly, further prospective studies are warranted.
The organisms most commonly associated with BV are G. vaginalis, Bacteroides (Prevotella) spp., Mobiluncus spp., and Mycoplasma hominis. High concentrations of Gardnerella, >100-fold greater than normal, are found in up to 95% of women with BV, but Gardnerella was also found in more than 50% of women without BV, so culture has a poor specificity. Quantitative culture showing high concentrations correlates better with BV in research studies, but culture should not be used for routine diagnosis. The reported prevalence of other organisms often reflects the sensitivity of the culture method for the specific organism. For instance, Fusobacter spp., peptostreptococci, and non-viridans group streptococci have also been associated with BV.
One study looked at the changes in bacterial flora that occur as it passes from normal to intermediate and BV (34). At the intermediate stage, Gardnerella and Bacteroides were present in moderate concentrations, but high concentrations of those organisms and of M. hominis were not seen until full BV had developed. The presence of high concentrations of Mobiluncus, visible on a Gram stain, have been used to define the most abnormal flora, with a Nugent score of 9 or 10. Recently, by use of PCR, Mobiluncus spp. were detected in 84.5% of women with BV and 38% of those without BV (37).
After isolating Haemophilus vaginalis (now called G. vaginalis), Gardner and Dukes investigated its role in inoculation experiments (11). Thirteen volunteer subjects were inoculated with a pure culture of H. vaginalis. Ten of them failed to develop clinical evidence of the disease or positive cultures subsequently. Cultures were positive for two subjects for 2 or 3 months, but neither developed the disease. One patient developed the clinical manifestations and had the organism recovered in pure culture. A further 15 women, of whom 4 were pregnant, were inoculated directly with material obtained from infected patients. Eleven (73%) of these subjects developed clinical signs of "nonspecific vaginitis," and the organism was recovered from all of them. In eight of these women, the syndrome developed within 1 week of inoculation. This suggests that inoculation with the full cocktail of bacteria found in BV is more likely to induce the condition than is inoculation with a single organism. This is supported by in vitro studies.
Anaerobic metabolism produces succinic rather than lactic acid. One study looked at the symbiotic relationship between Gardnerella, a facultative aerobe, and Prevotella bivia (29). The former produces amino acids that are utilized by the latter. In turn, Prevotella produces ammonia, which is utilized by Gardnerella in a symbiotic fashion. The same group also demonstrated another symbiotic relationship in vitro, with P. bivia making amino acids available for Peptostreptococcus anaerobius (28).
A host of enzymes which break down mucus are produced by the different bacteria involved in BV. Thus, mucinases, sialidases, and neuraminidases are present. These break down cervical and vaginal mucus. This is probably the explanation for the thin homogenous discharge, which lacks the cohesion normally induced by mucus. Additional virulence factors cleave immunoglobulin A (IgA) and IgM, reducing the ability of the host to prevent infections (9). Other protective mediators, such as secretory leukocyte protease inhibitor (SLPI), are also reduced (10).
Lactobacilli produce a variety of substances, such as bacteriocins and lactocins, which are toxic to other bacterial species and lactobacilli, respectively. Acidification of the vagina is an important defense mechanism. H2O2 is also an important inhibitor of anaerobic growth, and it is thought that Lactobacillus spp. producing high levels of H2O2 provide protection against BV and acquisition of sexually transmitted infections. Thus, in vitro at a pH of <4.5, H2O2-producing lactobacilli inhibit the growth of BV-associated organisms effectively, but at higher pHs the effect wanes (20). The inhibition is also reduced by addition of myeloperoxidase, which breaks down H2O2.
Is a change in pH such as occurs at the time of menstruation and following unprotected intercourse sufficient to trigger BV? Alternatively, if sufficient numbers of bacteria are inoculated into the vagina from a partner, will BV develop? Another possible trigger is anything that reduces the number or quality of lactobacilli. Interestingly, broad-spectrum antibiotics that inhibit lactobacilli do not seem to trigger BV (1), possibly because they also inhibit some of the BV organisms.
An absence of healthy H2O2-producing lactobacilli might contribute to frequent recurrences of BV in some women. One approach under study is to recolonize the vagina with healthy lactobacilli. A recent study of 215 sexually active women used whole-chromosome DNA probes to determine the most common strains of vaginal lactobacilli. The most prevalent species was Lactobacillus crispatus (32%), followed by L. jensenii (23%) and a previously undescribed sp. designated Lactobacillus sp. strain 1086V (15%) (3). An in vitro study investigated the rate of acid production by Lactobacillus spp. (7). The growth medium was acidified to an asymptotic pH of 3.2 to 4.8. In contrast, BV-associated organisms such as G. vaginalis, P. bivia, and Peptostreptococcus anaerobius reached an asymptotic level at a pH of 4.7 to 6.0, consistent with the pH levels found in BV. The authors calculate that 3 ml of semen would be acidified at a rate of 0.56 to 0.75 pH units/h. Unfortunately, they do not speculate on whether a transient pH change induced by a single episode of unprotected intercourse is likely to favor the growth of BV organisms sufficiently to produce a change in the bacterial flora. However, repeated episodes of intercourse within 24 h might produce a longer period of favorable growth conditions.
In a small study from Belgium, 32 women with BV or intermediate flora were treated with vaginal tablets containing 50 mg of a lyophilisate of viable, H2O2-producing L. acidophilus and 0.03 mg of estriol for 6 days (24). There was a significant benefit, with a cure rate after 6 weeks of 88% in the treatment group compared to 22% in the placebo group.
A full discussion of the complications of BV is outside the scope of this chapter. The pregnancy complications of late miscarriage and preterm birth are mediated through the development of chorioamnionitis (12). Once again, this is a mixed infection with the various organisms associated with BV producing a mixture of enzymes which break down cervical mucus, invade the membranes, and produce enzymes which can weaken the membranes, increasing the risk of premature rupture. In vitro studies have assessed the levels of such virulence factors caused by different bacteria. Although in many studies only one organism may be isolated from the membranes, it is likely that with improved means of detection a multiplicity of organisms would be found.
BV has also been associated with acquisition of HIV. H2O2 reduces the ability of HIV to infect cells in vitro (19), and its absence in BV is one putative mechanism. Cleavage of IgA, reduction in SLPI, and inhibition of leukocyte chemotaxis are other possible factors (18).
Our understanding of BV is improving through observational studies and study of the interactions between bacteria in the laboratory. The vaginal ecosystem is subjected to a variety of hormonal changes that affect the balance between lactobacilli and anaerobes. It is likely that the normal lactobacillus flora may be overwhelmed by factors such as a prolonged alteration in the pH of the vagina following vaginal douching or frequent sexual intercourse. Instillation of large numbers of organisms from a male or female sex partner might trigger BV. Alternative hypotheses include the introduction of a lytic bacteriophage infection, reducing the lactobacillus population. Once the BV organisms are allowed to flourish, they utilize each other's metabolites in a symbiotic manner and continue to maintain a pH of >4.5.
Possibly, the lack of H2O2-producing lactobacilli in the vagina makes it easier for sexually transmitted pathogens, including HIV, to gain a foothold, although it may be that the same risk factors for acquiring STDs are in fact precipitating BV. Failure to reestablish a H2O2- producing lactobacillus flora after antibiotics have suppressed anaerobic growth might account for frequent relapses in some women. There are still many women who are troubled by frequent symptomatic relapses of BV, and we need improved understanding of the triggers for it so that we can help them. There is an urgent need to determine how to effectively prevent the adverse outcomes of pregnancy associated with BV. If we could control BV, we might additionally be able to reduce the risk of HIV infection, for which BV is an important risk factor. A promising approach is to recolonize the vagina with lactobacilli that are high-level H2O2 producers and may be better able to inhibit the growth of anaerobes than the native lactobacillus flora.
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