Our understanding of the vaginal microflora has improved significantly and has allowed for the development of new approaches for the treatment and diagnosis of vaginal infections, such as bacterial vaginosis (BV), aerobic vaginitis (AV) and vulvovaginal candidiasis (VVC), that look to restore the vaginal microflora’s balance, rather than modify its components. Treatments that have shown promise in promoting healthy equilibrium in the vaginal flora, including the use of prebiotics, preparations of lactic acid, and properly selected probiotics, are of benefit in both treatment and preventative settings. Recent advances in the development of home diagnostic aids benefit both the general practitioner (GP), who may not have access to microscopy, and the patient. Alongside new diagnostics and treatment, there remains a clear need to improve awareness of the vaginal ecosystem among the general public and healthcare professionals, to promote best practice in vaginal hygiene and ultimately prevent vaginal infections.
Phillip Hay, Department of Genitourinary Medicine, St George’s Hospital, University of London, London, UK; Gilbert Donders, Department of Obstetrics and Gynaecology, Antwerp University Hospital, Antwerp, Belgium; and Femicare, Clinical Research for Women, Tienen, Belgium; Paulo Giraldo, Department of Gynaecology and Obstetrics, University of Campinas (UNICAMP), São Paulo, Brazil; Jean Pierre Lepargneur, Faculty of Medicine, Paul Sabatier University, Toulouse, France.
Healthy vaginal environment and flora – what’s new?
Healthy vaginal flora – what does it mean?
A normal healthy vaginal microflora in women of reproductive age is characterised by the presence of lactobacilli, which produce large amounts of lactic acid to give the genital tract its low pH of 3.8–4.2.1 However, this acidity, and the extent to which Lactobacillus species dominate the flora, can vary in different ethnic groups.2 Vaginal acidity is microbicidal for many pathogens and creates a niche where lactobacilli can have a competitive advantage over other taxa. Lactobacillus crispatus, L. gasseri, L. iners and L. jensenii are the predominant vaginal lactobacilli species colonising most women of reproductive age.3 Therefore, supporting the growth of endogenous lactobacilli is key in maintaining a healthy balance in the vaginal flora during the reproductive period of women. In most women, the presence of vaginal bacterial species vary very little, though slight differences can be observed during menstruation, as well as changes in relative species abundance.4 More significant changes can occur as a result of intrinsic factors (e.g. age, pregnancy) or extrinsic factors (e.g. level of sexual activity, genital hygiene), that can lead to dysbiosis and subsequently disease (Figure 1).5-13
In addition, determining which Lactobacillus species dominate an individual’s vaginal microbiome can provide an indication of the stability of the flora’s bacterial community. L. crispatus-dominant profiles are thought to be the most stable, while L. iners-dominant profiles are less stable. BV-status does not appear to be related to dominance of L. iners, as women with high levels of L. iners can be either BV-negative or positive. In contrast, women with high levels of L. crispatus are typically BV-negative.14 It has therefore been proposed that L. iners-dominant vaginal flora may confer less protection from BV than profiles dominated by L. crispatus.15 Recent studies have provided insights into the bacterial composition of the vagina in asymptomatic women. In North American women from four ethnic backgrounds, the majority of microbiomes could be placed into one of five community state types (CSTs), each differing in microbial composition and abundance.2 Four of these CSTs were dominated by a Lactobacillus species, but the final CST group lacked lactobacilli, had a highly heterogeneous flora and was most frequently associated with bacterial communities with high Nugent scores, a Gram stain scoring system used in BV diagnosis. In addition, lactobacilli were dominant in 80–90% of Asian and white women, but only 60% of Hispanic or African American women. Moreover, Hispanic and African American women were found to have higher average vaginal pH than Asian or white women, and so are more susceptible to dysbiosis and its associated complications (Figure 2). A similar analysis of Estonian women also identified one distinct cluster of microbiomes characterised by high levels of heterogeneity and no clear dominating microbial species.16 These studies recognise that increased microbial diversity and absence of lactobacilli correlate with the higher vaginal pH often associated with vaginal disease; in contrast, lactobacilli-dominated profiles are associated with a healthy vaginal environment due to lactic acid production.
What is the role of L- and D-Lactic acid in vaginal flora?
L- and D-lactic acid are the two functionally-distinct isomers of lactic acid. L-lactic acid has bactericidal effects against microorganisms associated with many vaginal infections. It is also able to stimulate the antimicrobial activity of vaginal epithelial cells, induce the release of pro- inflammatory cytokines, activate T helper 17 cells, stimulate dendritic cell maturation, and induce interferon-γ production.17 L-lactic acid has been shown to correlate with expression of extracellular metalloproteinase inducer (EMMPRIN), a major inducer of matrix metalloproteinase (MMP)-8; conversely, D-lactic acid, or specifically a low L-lactic acid/D-lactic acid ratio of approximately 0.5, appears to inhibit EMMPRIN expression.18, 19 If levels of EMMPRIN were not correctly regulated, MMPs would be overproduced, which could break down the extracellular matrix of vaginal epithelial cells and degrade the endocervical barrier. This would allow bacteria to enter the upper genital tract and increase susceptibility to infection.18 The production of lactic acid and thus the maintenance of an acidic environment creates a niche where lactobacilli can thrive and, significantly, where potentially harmful bacteria cannot. Lactobacilli acidify to a pH in the range of 3.2–4.5, which is comparable to the pH range observed in the vagina.20 In contrast, growth of three BV-associated bacteria, G. vaginalis, Prevotella bivia, and Peptostreptococcus anaerobius, leads to a more neutral pH in the range of 4.7– 5.9, which is comparable with the pH range observed in the vaginas of women with BV.20 This demonstrates the importance of lactobacilli in establishing the acidic environment associated with good vaginal health, and indeed the role of other microorganisms in the pH increase often observed in disease.
What is the role of glycogen in supporting vaginal flora?
Within the vagina, the glycogen produced by epithelial cells during anaerobic glycolysis is the primary energy source used by lactobacilli to make lactic acid. There is a correlation between levels of L. crispatus and L. jensenii and high levels of glycogen, but not L. iners,21 which may be unable to produce D-lactic acid due to a lack of D-lactate dehydrogenase.18 This limited production of lactic acid means than L. iners confers less antimicrobial protection than other Lactobacillus species. In a hormone-depleted, atrophic vagina, for example in some postmenopausal women or women with breast cancer receiving anti‑hormonal therapy, the addition of very small amounts of oestriol is required to increase the level of glycogen from desquamating vaginal epithelial cells so that sufficient nutritional glycogen is available as a substrate for probiotic lactobacilli.22, 23 Before it can be utilised by lactobacilli, glycogen first requires degradation to oligosaccharides via vaginal α-amylase. Decreased levels of glycogen breakdown products, which may occur as a result of impaired production of MMP-8 and α-amylase, is therefore likely to inhibit proliferation of lactobacilli.24 Competition for carbon sources from pathogenic bacteria may also prevent lactobacilli growth.
What happens when the vaginal flora balance is disrupted? Abnormal vaginal flora and diagnosis of vaginal infections
Lactobacillus species are found at significantly reduced concentrations or absent altogether in women with vaginal bacterial infections, which can leave other anaerobic bacteria in the flora to thrive in their place, or cause the colonisation of exogenous microorganisms. BV is a common disease characterised by a substantial reduction in vaginal lactobacilli and an increase in a wide variety of anaerobes.25 Anaerobes can increase by between 100- and 1000-fold, leaving lactobacilli absent or significantly outnumbered by the BV-associated bacteria that thrive in a less acidic environment.25 Patients present with a significantly increased vaginal pH of 4.5–7.0 and vaginal discharge with a “fishy” odour, which can distinguish BV from other vaginal infections.25 This characteristic odour is a consequence of the high trimethylamine (TMA) levels that can arise from the degradation of carnitine and choline, which is produced by many BV-associated bacteria, including Gardnerella, Mobiluncus, and Bacteroides.26-28 Often, BV can be associated with the formation of a biofilm, where G. vaginalis typically dominates.29 No inflammation, pain, dyspareunia, redness or oedema occur as a result of BV; if a BV-like flora is observed, but an inflammatory component is present, it is likely that the patient will have a mixed infection from BV/VVC or AV.30 AV is a common condition associated with a clinically significant abnormal vaginal flora.30 Both BV and AV are characterised by the absence of lactobacilli, but AV, in contrast to BV, is associated with a sparsely populated, predominantly aerobic microflora, defined by an elevated vaginal pH that is often >6 and varying degrees of vaginal atrophy, with or without inflammation.30 Some investigators refer to the most severe forms of AV as desquamative inflammatory vaginitis.31 VVC is a yeast infection that has a similar epidemiology to BV; it is associated with age of onset of sexual activity and the use of oral contraceptives.32, 33 Candida albicansis the primary species causing VVC. VVC can be divided into uncomplicated cases, which are sporadic, mild infections; or complicated cases, which may be recurrent or severe, or involve VVC during pregnancy or in an immunocompromised individual.34 Symptoms include burning, itching, and a thick, curdy vaginal discharge.34 VVC can occur in women with otherwise normal, lactobacilli-dominant flora, as well as in women with disrupted vaginal microflora.
What are the consequences of abnormal vaginal flora?
The underlying dysbiosis seen in conditions like BV and AV can leave patients at risk of a number of diseases and complications, particularly relating to pregnancy. Both BV and AV have been associated with abnormal pregnancy outcomes, including chorioamnionitis, pre- term birth and miscarriage.35-37 Prior to pregnancy, vaginal infections may also affect clinical pregnancy rates in women undergoing in vitro fertilisation treatment.38 In addition, BV has been associated with risk of acquiring STIs39-42 and is also thought to be correlated with onset of pelvic inflammatory disease.43 While still under investigation, AV has also demonstrated an association with cervical dysplasia in women with human papillomavirus,42 and may be linked to other STIs,41 sexual dysfunction and vulvodynia [data on file].44
The introduction of patient self-tests in the diagnosis of vaginal infections
Traditional diagnostics for vaginal infections are dominated by microscopy assessments, but the required equipment may not be routinely available to GPs and gynaecologists, so other diagnostic methods including patient self-tests may be useful. Traditional diagnostics usually consist of a wet mount or Gram stain for BV, wet mount or culture on Sabouraud media for VVC, and a wet mount or polymerase chain reaction (PCR) for trichomoniasis. For gonorrhoea and Chlamydia, the typical causes of cervicitis, a nucleic acid amplification test (NAAT) of the cervix, vagina or urine is used45. AV is diagnosed using a scoring system in wet mount microscopy that evaluates inflammatory and host defence markers, together with microflora patterns and cytology.30 If microscopy is available, diagnosis of BV is commonly based on the Nugent score or the Amsel criteria, a clinical and microscopic examination that comprises an evaluation of discharge, a potassium hydroxide test, a pH test (>4.5), and the presence of “clue” cells on microscopy.46 The Amsel criteria has a sensitivity of 99% and a specificity of 89%, whereas the Nugent score had a sensitivity of 96% and a specificity of 94%.47 Both wet mount and Nugent score perform well in the diagnosis of BV and AV and are comparable to NAATs.48,49 However, lack of a microscope, laboratory support and reimbursement mean that GPs are not always able to perform microscopy examination. In its absence, diagnosis of common vaginal infections focuses on symptoms and medical history, gynaecological examination, vaginal pH and vaginal odour. In addition, point-of-care diagnostic tools are also available to clinicians to obtain a diagnosis. Patient self-tests have been developed to serve as quick, easy‑to‑perform diagnostic assessments that can be used at home. These typically work by assessing vaginal pH; a negative result (normal acidic pH of 3.8–4.2), indicates that BV is unlikely to be present, and that a vaginal Candida infection is more likely, while a positive result (pH > 4.5) indicates BV or Trichomoniasis. These tests help women to diagnose their vaginal condition through a combination of a pH test swab and their symptoms, which can guide them when seeking advice from a medical practitioner. A recent study found that a self-test swab could diagnose vaginal infections with 92.9% specificity and 91.8% sensitivity, and was able to detect all severe cases of BV and AV tested,50 but was unable to diagnose or exclude VVC. Patient self-tests also address some limitations associated with more traditional diagnostic methods. For example, an important barrier to consider in the diagnosis is patient reporting. Many women with vaginal symptoms often do not present to a healthcare professional,51 due to embarrassment, cultural factors or differences in the perceived severity of symptoms. A self-test would provide women with an opportunity to diagnose themselves at home and could also reassure women if the test result is negative. The use of such diagnostic tests may be less appropriate in instances where symptoms of vaginal infections are more complex or inconclusive. For example, microscopy testing may be required for an accurate diagnosis in cases of partial BV or mixed infections, which can affect approximately 30% of patients.52 The advent of high-throughput, multiplex PCR platforms may also have applications in the diagnosis of vaginal infections. Studies have demonstrated the accuracy of PCR-based DNA amplification to effectively diagnose both BV and VVC.53, 54 While promising, it is important to note that such approaches need to be developed further to be suitable for use in the clinic. Additionally, the cost associated with PCR platforms remains its primary and arguably most significant limitation, particularly when compared with more cost-effective options such as microscopy and diagnostic aids.
Misdiagnosis of vaginal infections is common
Although treatment for vaginal infections is typically straightforward with high levels of success, misdiagnosis is common55 and can lead to unnecessary costs, persistent symptoms, complications, incorrect treatment and the over-use of antimicrobials. AV is not always considered or recognised in research or professional settings, leading to frequent misdiagnoses and consequently inadequate conclusions about pathogenicity and poor treatment success.30 Moreover, the epidemiology of BV and VVC is similar, yet BV is not as well-known or accepted as VVC in the general public, to the extent that patients may perceive BV as more embarrassing than VVC. This, coupled with women who either are hesitant to report symptoms of AV or BV to their healthcare professional or have a limited understanding of the condition, means that BV and AV are often misdiagnosed as VVC,51 resulting in unnecessary use of antimycotic drugs and persistent symptoms. Consequently, it is important that awareness of these conditions is increased among both healthcare professionals and the general public.
Best practices in managing vaginal infections
Best practices in managing conditions associated with an abnormal vaginal flora are centred on adopting a combined physiological approach; this would promote good intimate hygiene and interventions to support a healthy vaginal flora, rather than those that actively modify the microbiome, such as repeated use of antibiotics.
Asymptomatic vaginal infections – to treat or not to treat?
A key and somewhat controversial factor in the management of vaginal infections is the treatment of asymptomatic cases. Approximately 50% of women with BV are asymptomatic, and it is estimated that Candida species can be isolated in the vaginal tracts of 20–30% of healthy asymptomatic, non-pregnant women.34, 56 While asymptomatic VVC does not require treatment, opinion on whether or not asymptomatic BV should be treated is subject to debate. Guidelines state that women with asymptomatic BV only require selective treatment in some high-risk pregnancies,57 where it is essential to prevent complications. However, some experts consider that, due to the recurrent nature of BV, if a woman has no symptoms and does not require preventative measures, treatment would have no long-term benefit to her vaginal health, and is therefore unnecessary. On the contrary, others argue that BV does not have to be symptomatic to be considered a disease; pathogenic factors associated with BV, such as sialidase, can impair mucosal immune defences regardless of whether or not symptoms manifest, which can favour the development of other infections.58 Further investigations are required to confirm the value of interventions in asymptomatic cases of BV.
Treating BV, AV and VVC
Clindamycin and metronidazole are first-line antibiotics prescribed to treat BV, which can effectively resolve the condition in the majority of cases, but their broad-spectrum activity can reduce taxa diversity.59 In cases where BV involves the presence of a biofilm that antibiotics are unable to penetrate, these standard drugs have limited efficacy and their use can lead to high recurrence rates of BV.60 Other antibiotic and acidifying treatments are being studied, including tinidazole, rifaximin, nitrofurans, dequalinium chloride and ascorbic acid.60 Efforts have been made to develop antimicrobial peptides and quorum sensing inhibitors, which, while yet to be tested in humans, have shown promise in other bacterial infections characterised by biofilm formation.61 For AV, treatment should be tailored to address a patient’s specific clinical needs. Treatment strategies are informed by microscopy analysis and, depending on the individual, may involve one or a combination of local antibiotics, such as clindamycin, co-amoxiclav or moxifloxacin, to target infection; steroids to target inflammation; and oestrogen to target atrophy.30 The use of pre- and pro-biotics for the treatment and prevention of AV is also under investigation. Intravaginal treatments, such as over the counter clotrimazole, and oral preparations, such as fluconazole, can effectively resolve VVC infection. Treatment with azoles provide relief from symptoms and negative cultures in 80–90% of patients, whether administered orally or topically.62 Biofilm formation has also been reported in some cases of VVC, which may be of significance when considering patients with recurrent infections.63
How physiological treatment approaches to vaginal infections can help
Lactic acid can provide quick resolution of BV-associated symptoms, particularly “fishy odour”. While beneficial in the treatment of short-term, sporadic BV, lactic acid treatment does not appear to result in changes to the bacterial composition of the vaginal flora, and symptoms can recur. Therefore, lactic acid could be best placed in a preventative setting, to maintain a normal vaginal flora when risk of BV is increased, or as an adjunct therapy to either clindamycin or metronidazole to support the growth of lactobacilli. Clinical studies are underway to investigate the benefit of using such preparations to resolve vaginal infections and restore eubiosis.64 To maximise the efficacy of lactic acid as an adjuvant intervention, it is necessary to identify environmental conditions that best support growth of lactobacilli. For example, it is important that possible preparations include oligosaccharides to serve as a suitable energy substrate for lactobacilli. The use of a synbiotic vaginal insert containing the probiotic strain L. crispatus BC5, the prebiotic substrate fructo-oligosaccharide, and the antioxidant agent ascorbic acid to target genitourinary infections was able to support survival of L. crispatus and control its release, as well as exhibit antimicrobial activity against a number of pathogens, including C. albicans.65
The use of prebiotics and probiotics in vaginal infections
Using either probiotic lactobacilli or a prebiotic to stimulate growth of lactobacilli has been the subject of significant research interest. For example, women with a history of recurrent BV received either the vaginal probiotic capsule (containing L. rhamnosus, L. acidophilus and Streptococcus thermophiles) or placebo every day for 7 days, followed by no treatment for 7 days, continually for 11 months. Probiotic prophylaxis was well-tolerated and associated with significantly lower BV recurrence rates and G. vaginalis incidence, compared with placebo.66 Supplementation of antibiotics with a probiotic has also proven effective in AV, where an oral probiotic preparation could delay clinical relapse by up to 31 days (76%).67 However, use of probiotic lactobacilli has a number of limitations. The most widely applied criterion in selecting probiotic strains of lactobacilli is their ability to produce hydrogen peroxide (H2O2), which acts cooperatively with lactic acid in vitro to kill vaginosis-associated and uropathogenic bacteria.68 However, this is not the case in vivo, where H2O2’s microbicidal activity appears to be inhibited, and high concentrations of H2O2 are more toxic to the vaginal lactobacilli than the BV‑associated bacteria.69, 70 Selection of lactobacilli based on their ability to produce lactic acid may be a more desirable criterion, but effective producers of lactic acid, such as L. crispatus, can be difficult to grow sufficient quantities in vitro and to stabilise in a preparation. The use of prebiotics to treat and prevent vaginal infections is a less-explored area than the use of probiotics, but the available literature seems to support its therapeutic potential. Further randomised controlled trials will help to draw definitive conclusions on the efficacy of prebiotics in the treatment and prevention of vaginal infections.
How can women eliminate risk factors for vaginal infections?
As well as supporting a patient’s endogenous flora, it is important to consider extrinsic risk factors that may leave patients vulnerable to recurrent vaginal infections. Providing information to patients and the general public regarding factors influencing the vaginal ecosystem and immune response is essential, so that extrinsic factors can be monitored or controlled as appropriate. Guidance on what constitutes good vaginal hygiene is particularly important in this context. For example, women should be encouraged to avoid vaginal douching or use of heavily-perfumed products in the vaginal area. Cleaning the internal vaginal compartment with a lactic acid solution, particularly in patients with recurrent BV, may also be a suitable option. However, a proper diagnosis is imperative in this context, as such interventions can worsen conditions such as cytolytic vaginosis or VVC. Management through both lifestyle and treatment interventions will allow for a holistic approach to treat and prevent vaginal infections most effectively.
Expert panel consensus recommendations
Our understanding of the vaginal microflora and its complex dynamics has informed contemporary approaches to the management of vaginal infections. Rather than attempting to modify the vaginal microbiome, new approaches to treatment recommend a holistic physiological approach that supports the existing ecological balance of the microflora and promotes the endogenous growth of lactobacilli. In addition to promoting good intimate hygiene, such an approach could incorporate probiotics or application of a lactic acid preparation, to restore microbial balance in the vaginal flora. Provided that these preparations are safe and efficacious in restoring the balance of the vaginal flora, it could present a viable option for the treatment of vaginal infections, as well as their prevention during preconception or the early stages of pregnancy. Patient self-testing diagnostic aids have provided women with useful tools to inform a diagnosis of a possible vaginal infection. While microscopy remains the standard for obtaining a full diagnosis of BV, AV and other vaginal infections, patient selftests can aid a diagnosis where microscopic examination is not available and, with proper guidance and support material, help women to manage a positive or negative test result effectively. For optimal management of these infections, it is important to increase awareness of the vaginal ecosystem, good vaginal hygiene and vaginal infections among both the general public and healthcare professionals. In doing so, clinicians and patients will have a better understanding of the value of physiological interventions in preventing and resolving vaginal infections, as well as the treatments and diagnostic tools available to them to ensure that they can be managed optimally.
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