Aerobic vaginitis: no longer a stranger

Accepted Manuscript Aerobic vaginitis: no longer a stranger Gilbert GG. Donders, Gert Bellen, Svitrigaile Grinceviciene, Kateryna Ruban, Pedro Vieira-Baptista PII:

S0923-2508(17)30086-4

DOI:

10.1016/j.resmic.2017.04.004

Reference:

RESMIC 3592

To appear in:

Research in Microbiology

Received Date: 13 December 2016 Revised Date:

5 April 2017

Accepted Date: 5 April 2017

Please cite this article as: G.G. Donders, G. Bellen, S. Grinceviciene, K. Ruban, P. VieiraBaptista, Aerobic vaginitis: no longer a stranger, Research in Microbiologoy (2017), doi: 10.1016/ j.resmic.2017.04.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

For publication Aerobic vaginitis: no longer a stranger Gilbert GG Dondersa,b*, Gert Bellena, Svitrigaile Grincevicienea,c, Kateryna Rubana, Pedro a b c

Femicare vzw, Tienen, Belgium

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Vieira-Baptistad Department of Obstetrics & Gynaecology Antwerp University, Antwerp, Belgium

Vilnius University Institute of Biotechnology Department of Biothermodynamics and Drug Design, Vilnius, Lithuania

Department of Gynaecology and Obstetrics, Centro Hospitalar de São João, Porto, Portugal

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*Correspondence:

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Tel: +32 16 808102 Fax: +32 16 808109 [email protected]

ACCEPTED MANUSCRIPT Abstract

Aerobic vaginitis (AV) is the name given in 2002 to a vaginal infectious entity which was not recognized as such before. It is characterized by abnormal (dysbiotic) vaginal microflora containing aerobic, enteric bacteria, variable levels of vaginal inflammation and

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deficient epithelial maturation. Although AV and bacterial vaginosis (BV) share some characteristics, such as a diminished number or absence of lactobacilli, increased discharge (fishy smelling in BV, while in severe forms of AV, a foul, rather rotten smell may be present) and increased pH (often more pronounced in AV), there are also striking differences

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between the two. There is no inflammation in women with BV, whereas the vagina of women with AV often appears red and edematous, and may even display small erosions or ulcerations. The color of the discharge in BV is usually whitish or gray and of a watery

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consistency, whereas in AV it is yellow to green and rather thick and mucoid. Women with BV do not have dyspareunia, while some women with AV do. Finally, the microscopic appearance differs in various aspects, such as the presence of leucocytes and parabasal or immature epithelial cells in AV and the absence of the granular aspect of the microflora, typical of BV. Despite all these differences, the distinction between AV and BV was not

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recognized in many former studies, leading to incomplete and imprecise diagnostic workouts and erroneous management of patients in both clinical and research settings. The prevalence of AV ranges between 7 and 12%, and is therefore less prevalent than BV. Although still largely undiagnosed, many researchers and clinicians increasingly take it

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into account as a cause of symptomatic vaginitis. AV can co-occur with other entities, such as BV and candidiasis. It can be associated with dyspareunia, sexually transmitted infections (such as human papilloma virus, human immunodeficiency virus, Trichomonas vaginalis and

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Chlamydia trachomatis), chorioamnionitis, fetal infection, preterm birth and cervical dysplasia. Many other possible pathological associations are currently under investigation. The diagnosis of AV is made using wet mount microscopy, ideally using phase contrast. An AV score is calculated, according to: lactobacillary grade, presence of inflammation, proportion of toxic leucocytes, characteristics of the microflora and presence of immature epithelial cells. To circumvent the hurdle of microscopic investigation, some groups have begun to develop nucleic-acid-based and enzymatic diagnostic tests, but the detailed information obtained with phase contrast microscopy is irreplaceable. The best treatment is not yet fully determined, but it must be tailored according to the microscopic findings and the patient’s needs. There is a role for local estrogen therapy,

ACCEPTED MANUSCRIPT corticosteroids, antimicrobials and probiotics. Further research will reveal more precise data on diagnosis, pathogenesis, management and prevention.

Keywords: Aerobic vaginitis; Bacterial vaginosis; Abnormal vaginal microflora; Vaginal

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microbiome; Preterm birth; Cervix dysplasia; Desquamative inflammative vaginitis

1. Introduction

The term ‘aerobic vaginitis (AV)’ was coined for the first time in 2002 to meet the need to describe another condition of vaginal dysbiosis besides the existing entity, ‘bacterial

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vaginosis (BV)’. BV is a rather well described and over the last 100 years has become relatively well known as a condition that is very prevalent amongst women throughout the

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world. Gradually, evidence accumulated that viewed BV as a sex-associated disease, which poses a risk for acquiring sexually transmitted infections (STI) and that may have a negative influence in the outcome of pregnancy, namely, increasing the risk of preterm birth. However, analysis of these studies also revealed some shortcomings. Indeed, most of the time, clinicians and researchers would agree on the definition of full blown BV (as defined by the Nugent score of 7 or more) and of normal BV-negative microflora (Nugent score of 3 or less), but

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few would be able to tell exactly what the so called ‘intermediate microflora’ group stands for (Nugent score of 4 to 6, falling between normal and BV microflora) [1]. Moreover, it has become clear that this intermediate microflora is not a transition state from normal to BV, as is implicitly suggested by the designation. Rather, it represents a mixture of ‘partial BV’, i.e. a

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transitional state between BV and normal, with areas of BV microflora mixed with areas of normal microflora in the same slide and other vaginal microflora abnormalities [2]. Keen

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microscopists can observe more subtle differences than can be accounted for by simple overgrowth of anaerobic BV-associated bacteria alone. It was noticed that a subset of women with paucity of lactobacilli also had inflammation, different grades of unexpected atrophy and microflora that did not have the typical granular aspect of BV-associated microflora. This led to the definition of a new condition, with a particular type of microflora: AV. Based on wet mount phase contrast microscopy, a scoring system for its diagnosis was established. It encompasses the level of the presence of lactobacilli (designated ‘lactobacillary grade’, or LBG) [1], the characteristics and quantity of inflammatory cells, the microflora characteristics and the presence of immature epithelial cells originating from the lower layers of the vaginal

ACCEPTED MANUSCRIPT mucosa. This enables calculating a composite score (see below) that grades the severity of AV [3]. The most severe grade of AV encompasses desquamative inflammatory vaginitis [4, 5]. Considering the diagnosis of AV allows an explanation for several formerly unanswered questions: 1) what is “intermediate microflora”?; 2) why do women with BV according to Nugent scoring show a range of symptoms of different intensity?, 3) what is so-called

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‘inflammatory BV’?, 4) which condition can lead to DIV (severe AV)?, and most of all, 5) why did several large randomized controlled studies indicate that treatment with

metronidazole failed to prevent preterm birth in most women with BV? [6]. In summary, the presence of AV can explain why many enigmas surrounding abnormal vaginal microflora and

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BV have remained unresolved for so long. As a consequence, as we will discuss below, AV seems to be a major player in several associations formerly thought to be exclusively linked to BV: infection-related preterm birth, HPV infection and cervical cancer, and risk of acquiring

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STIs. Importantly, as the pathogenesis and diagnostic modalities of both infections are so diverse, treatment is likewise different. Hence the differential diagnosis between AV and BV is crucial, and most likely this need will become more and more important as more detailed

2. Etiology of AV

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information about epidemiology, pathogenicity and treatment gathers.

In a healthy vaginal microenvironment, the balance and interaction between microbes are critical. This balance can transform into a disturbed state named ‘abnormal vaginal

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microflora’ (AVF) or ‘dysbiosis’, resulting in conditions such as bacterial vaginosis, aerobic vaginitis or vaginal candidiasis [6, 7]. The microflora in AV is composed of commensal aerobic microorganisms of intestinal

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origin [3, 7, 8]. The most frequently encountered bacteria are Escherichia coli, Staphylococcus aureus, coagulase-negative staphylococci such as S. epidermidis, group B Streptococcus (Streptococcus agalactiae) and Enterococcus faecalis [3][8-11]. According to some authors, viridans streptococci are also frequently recovered [10,11], but this finding is not generally accepted. It is as yet unclear whether the presence of aerobic microflora is of a causative rather than of an associative nature, as some think of AV as an immunological disorder with an influence on the vaginal microflora rather than as a strict bacterial infection [12]. Indeed, within AV, there exist variable degrees of inflammation (not only measured by the number of leucocytes, but also by the proportions of leucocytes with toxic granulation -

ACCEPTED MANUSCRIPT the so-called ‘toxic leucocytes’) and a variable presence of parabasal or immature epithelial cells (Table 1) [3, 7, 13].

3. Clinical presentation

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AV is a form of vaginal dysbiosis [14] presenting with a purulent discharge, significant inflammation and epithelial disruption [15]. In AV, the transition of the healthy lactobacillus-dominated microflora to vaginal dysbiosis is accompanied by symptoms of inflammation, introital and vaginal redness, stinging and burning sensations, the presence of

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sticky, yellow vaginal discharge and dyspareunia [2, 3]. The typical vaginal discharge is described as homogeneous and purulent, yellowish or yellow–green in color, with a foulsmelling rotten odor, but, unlike in the case of BV, without a fishy smell. Symptoms can be

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present for a long period of time and fluctuate in terms of severity.

Upon speculum examination, the vaginal mucosa and the vestibule is variably red and inflamed and, in case of severe AV, ecchymotic bleeding points and erosions can be encountered.

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Severe AV is indistinguishable from desquamative inflammatory vaginitis [4, 5], and both are usually chronic conditions, characterized by severe vaginal enantema and purulent discharge. Many patients have long-standing symptoms of profuse discharge, vestibulevaginal irritation, dyspareunia and vaginal inflammation or erythema for 12 months or more.

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4. Diagnosis

Fresh wet mount microscopy is the preferred diagnostic method [3]. Upon microscopic evaluation, using phase contrast at 400x magnification, LBG (I, IIa, IIb or III) [16] can be

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identified (Fig. 1). In brief, LBG I corresponds to normal microflora, i.e. predominantly lactobacillary cell types, with absent or very few coccoid bacteria present. However, care has to be taken not to misidentify the cellular cytoplasmatic debris from lysed epithelial cells [epitheliolysis] as coccoid bacteria and/or the bare nuclei from lysed epithelial cells as leucocytes. LBG II corresponds to diminished numbers of lactobacilli, mixed with other bacteria. It is subdivided into slightly disturbed, fairly normal (LBG IIa) and moderately disturbed, rather abnormal (LBG IIb) microflora, according to the relative dominance of lactobacilli or other bacteria, respectively. Finally, the grossly abnormal LBG III microflora consists of numerous other bacteria, with no lactobacilli present. By having different investigators in different countries reading a similar set of slides, it was elegantly

ACCEPTED MANUSCRIPT demonstrated that the use of phase contrast instead of the traditional bright-field microscopy provides

increased accuracy (from a kappa index of 0.45 to 0.93) for

determination of

LBGs [3]. Also, the number of leucocytes compared to epithelial cells should be scored, as well as the proportion of leucocytes with toxic appearance. Toxic leucocytes are swollen and contain granules showing lysosomal activity (Fig. 2, panel C) [17]. The microflora is

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evaluated for the presence of cocci and/or chains of cocci, or small coliform bacilli (Fig. 2, panel A and C). Finally, the proportion of parabasal epithelial cells (small round epithelial cells with a high nuclear to cytoplasma ratio), must also be taken into account (Fig. 3, panel A). A score ranging from 0 to 2 is assigned to each of the five above-mentioned

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characteristics, added together to a composite score. AV is then diagnosed according to the composite score as follows: a score of 0-2 means no AV; a score between 3 and 4 indicates light AV, between 5 and 6 represents moderate AV and a score from 7 to 10 represents severe

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AV. In some studies, moderate/severe AV (msAV, AV score 5-10) is considered as pathological (Table 2) [18]. In severe cases, other culture or PCR tests can be used to detect Streptococcus pyogenes (group A streptococci) and/or Trichomonas vaginalis and, more rarely, measurement of the estrogen content in blood and histology has been used [19].

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According to Macklaim et al., microscopic evaluation of vaginal fluid for the diagnosis of AV is rarely performed in routine examination, even in symptomatic women [7]. Moreover, in the US and Canada, the diagnosis of AV is rarely performed outside of specialized centers [7]. In Europe, on the other hand, a new wave of microscopy training

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courses and centers seems to be gradually improving the level of diagnostic skills amongst physicians and research centers, In Asia, AV is investigated and picked up at routine visits at an increasing rate [20, 21].

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Tempera et al. found that patients with AV have depressed levels of hydrogen-

peroxide-producing lactobacilli, but increased levels of aerobic bacteria (group B streptococci, S. aureus, E. coli and enterococci) [22]. Compared with the normal vaginal microflora, these aerobes increased by three- to fivefold and were associated with inflamed vaginal mucosa [4, 17] and with evidence of local cytokine-mediated inflammation [3]. As decreased H2O2 would not be specific to AV, because it is also diminished in BV, an isolated decrease in H2O2 production has never been introduced as a diagnostic feature of AV. Wang et al. developed a bedside test for AV diagnosis. AV was diagnosed based on five enzymatic indicators: (1) hydrogen peroxidase activity, which reflects the growth status of hydrogen peroxide-producing lactobacilli; (2) leucocyte esterase (LE) activity, which

ACCEPTED MANUSCRIPT indicates the presence of inflammation [23]; (3) sialidase activity, which is produced in high quantities by BV and AV associated bacteria, such as Atopobium vaginae, Gardnerella vaginalis, Prevotella bivia and S. agalactiae [24-26]; (4) beta-glucuronidase activity, specific for E. coli infection; and (5) coagulase activity, which indicates the presence of S. aureus and E. faecalis [6]. The authors also found increased presence of E coli, but the methodology used

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for detection was not clear. The reported sensitivity of their test was 90 %, but the study lacked analysis of specificity [6].

Rumyantseva et al. evaluated the use of quantitative PCR (qPCR) for the diagnosis of AV. Using mathematical formulas, including the concentration of lactobacilli, aerobic and

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anaerobic microorganisms as variables, they were able to accurately detect the cases of AV [10]. However, the authors recognized the limitations of qPCR for diagnosis AV, because inflammation and immaturity of the epithelial cells were not accounted for. A shortcoming of

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the study was its lack of sensitivity and specificity analysis.

Fettweis et al. recommended next-generation sequencing for vaginal microbiome analysis [14]. This technique allows use of mid-vaginal samples for microbiome analysis targeting the V1–V3 hypervariable region of the 16S rRNA gene employing a deepsequencing approach (i.e. ~30,000 reads per sample) that permits detection and quantification

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of less abundant microbes, which are often important constituents of the vaginal microbiome [14]. However, although this method, amongst others, enables investigating the composition of the vaginal microbiome, it is not developed as a diagnostic tool. Despite the evidence of low vaginal estrogen (suggested by the presence of parabasal

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or immature epithelial cells), determination of its serum levels is not clinically relevant [19]. Finally, for screening purposes, pH (self) testing could be proposed as a proxy test for AV due to its high sensitivity of 90% [27]. However, this technique lacks specificity, since

BV,

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Trichomonas infection, inflammatory Candida infection, menstrual blood and sperm are also usually associated with a higher pH. pH testing can only be used as a surrogate screening tool for abnormal vaginal microflora, in general, in developing countries with no other diagnostic means [28]. Similarly, the microscopic feature of coccoid microflora can be used as a proxy of AV [29], which is a rapid screening technique. However, this feature only singles out a specific subgroup of AV, and does not include information on the inflammatory and atrophy components.

5. Prevalence and epidemiology

ACCEPTED MANUSCRIPT Reliable data on the prevalence of AV in the general population are not very abundant. A 2013 meta-analysis showed an AV prevalence of 5 to 10% in non-pregnant women, but this was based on only two papers, and the grade of AV was not mentioned [9]. Based on microscopy reading, Donders et al. found moderate or severe (ms) AV in 11% of women presenting for a routine gynecological examination in Kampala, Uganda [27]. In a

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retrospective study in Bulgaria, the overall prevalence rate of msAV in the study population was 11.8% [8]. In Moscow, Russia, Rumyantseva et al. described the presence of light, moderate and severe AV in 13.4%, 7.2% and 3.1% of asymptomatic women, respectively, accounting for a mean general prevalence of AV of 13.1% in non-pregnant women in Russia

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[10]. Finally, in a population-based study using wet mount, Vieira-Baptista et al. found 7.4 % msAV in Portuguese women presenting for routine Pap smears [18]. In China, Fan et al. found AV in 23.7% of symptomatic women, while mixed infections with bacterial vaginosis,

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candidiasis or trichomoniasis were also frequently encountered [11]. Hence, in most studies across Europe, Asia and Africa, the prevalence of msAV can be estimated at around 7 to 13% of non-pregnant women, similar to the prevalence of BV (at least in Europe) (Table 2). In a Belgian study on 759 low-risk pregnant women, an overall prevalence of 8.3% of coccoid microflora was found [29], while in pregnant women in Russia, only 4.3% was found [8].

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Hence, despite scarce data in the literature thus far, the prevalence of AV during pregnancy seems to be lower, in the range of 4 to 8%.

Only very few studies have been carried out thus far to evaluate risk factors for the development of AV. In China, being unmarried (odds ratio (OR): 2.6, 95% confidence

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interval (CI): 1.3–5.4), use of an intra-uterine device (OR: 5.0, 95% CI: 1.9–12.9), long-term use of antibiotics (OR: 11.2, 95% CI: 1.4–91.7) and frequent vaginal douching (OR: 4.7, 95% CI: 1.4–16.1) were found to be independent risk factors [13] and are, in fact, comparable to

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risk factors for BV. Consistent condom use and higher education were protective factors in that same study [13], but not in another [18]. Consistent condom use as a protective tool could be interpreted as a sign that AV may be due to a sexually transmitted agent, or its use could be linked to the absence of sperm in the vagina, influencing the vaginal pH, but both hypotheses are highly speculative. In their study on pessary users, Collins et al. did not use the term “aerobic vaginitis”, but they described more Nugent-positive BV (p=0.03), more leucocytes (> 10 per high power field (hpf), p < 0.001) and increased numbers of parabasal cells (p < 0.01) in new and longterm pessary users. Pessary users with complaints of vaginitis were found to be carriers of enterococci, viridans streptococci and GBS more often, while new users more often had E.

ACCEPTED MANUSCRIPT coli, viridans streptococci and enterococci. Diagnosis of AV using microscopy instead of cultures could help pessary users to better support the devices and better manage

the

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vaginal discharge they sometimes have [30].

6. Pathogenesis

As is the case for BV, the pathogenesis of AV is not yet unravelled. However, it seems

6.1.Decrease in lactobacillus-dominated microflora

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coincide with different factors, which will be discussed separately.

The healthy microbiome of the vagina predominantly consists of lactobacilli.

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Lactobacilli can inhibit adherence of various urogenital pathogens, including group B Streptococcus and S. aureus [31]. Furthermore, L. crispatus colonization [32] and the surface proteins of L. crispatus and L. jensenii are associated with strong inhibition of the growth of E. coli [33]. Direct physical contact is important in the reduction by lactobacilli of toxic shock syndrome toxin-1 (TSST-1) secretion by S. aureus [34, 35] and of the staphylococcus-induced

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secretion of inflammatory cytokines by epithelial cells [34]. Lactobacillus species are thought to compete for binding to receptors on host epithelial cells, and Spurbeck et al. (2008) have demonstrated that different species and strains vary in their ability to adhere to host cells [36]. The competition theory is further supported by the finding that women only benefitted from

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using probiotic lactobacilli to prevent cystitis when they achieved high-quantity vaginal colonization (> 106 16S rRNA gene copies/swab) [37]. However,

other mechanisms may

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also be involved, as outlined below.

Indeed, besides

competitive exclusion through adherence to vaginal epithelial cells,

two other general strategies have been proposed to explain how lactobacilli inhibit growth of other microorganisms in the vaginal niche in premenopausal women: 1) competition for nutrients; and 2) production of antimicrobials, such as bacteriocins, hydrogen peroxide (H2O2) and lactic acid [38]. Bacteriocin production by some isolates of lactobacilli inhibited the growth of E. coli, while its production in other isolates prevented growth of S. aureus [39]. In in vitro studies, L. Crispatus- and/or L. jensenii-derived proteins inhibited the growth of E. coli in cervicovaginal fluid [33]. O’Hanlon and colleagues demonstrated that in vitro H2O2 antimicrobial activities can be blocked by both semen and cervicovaginal fluid, which would

ACCEPTED MANUSCRIPT indicate that the mechanism may be ineffective in vivo [40]. Another reason why H2O2 bactericidal activity could be questioned is the low concentration of the substance under in vivo anaerobic conditions, while high concentrations of H2O2 would be fatal for both pathogenic bacteria and lactobacilli. Hence the rationale for why H2O2-producing lactobacilli seem to be associated with vaginal health has yet to be conclusively determined. A recent,

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interesting hypothesis suggests an important role of H2O2 as a mediator of the SOS response (global response to DNA damage) and prophage induction [41]. As different Lactobacillus species are commonly lysogenized by intermittent presence of bacteriophages, this may trigger a transition to a less defensive microbiome profile [42]. Production of H2O2, on the

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other hand, may select for lactobacilli strains harboring defective prophages [41], rendering these strains more resistant to cell lysis and becoming more stable, thereby reducing the likelihood of transition to the abnormal vaginal microbiome state.

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The concentration of vaginal interleukin (IL)-1β is related to the lactobacillus grade [43]. Presence of H2O2-producing lactobacilli reduces the levels of IL-1β, even in cases with abnormal microflora and BV [44], leading to the conclusion that H2O2-producing lactobacilli function as immune modulators despite the presence of numerous other bacteria in the vagina. L. crispatus and L. Jensenii, in particular, were shown to express this effect on IL-1β

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depression, while L. iners failed to do so [44]. Other authors found lactobacilli to also decrease IL-6 [45] [46], but Mitchell et al. could not confirm this finding [44]. A probiotic strain L. reuteri RC-14 alone and in combination with L. rhamnosus GR-1 upregulated IL-8 and interferon-γ-inducible protein-10 (IP-10) secretion by VK2 epithelial cells in vitro [47].

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Therefore, although several immunoregulatory mechanisms still remain to be elucidated, an H2O2-regulated cytokine pathway seems to be in place. Loss of these protective lactobacilli

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can help to lead to the hyperinflammatory state found in AV.

6.2.Imbalance in local immune modulation An imbalance in local immune modulation in women with AV has been described [17,

22, 48]. In AV, there is a rise in IL1β which is much higher (178.8 pg/mL) than in BV (71.2 pg/mL) and significantly higher than in normal controls (5.0 pg/mL, p < 0.001) [25]. There is also a dramatic increase in IL-6 and IL-8, which is not seen in BV according to most [25, 48, 49], but not all authors [50]. This increase in pro-inflammatory cytokines is directly related to the decrease in the number of lactobacilli in the vagina [43], [48], although it should be noted that a comparable decrease in lactobacilli is not pro-inflammatory in case of BV. It is not clear

ACCEPTED MANUSCRIPT from these papers whether endocrine influences were taken into account, as the menstrual cycle, contraceptive use and pregnancy also influence cytokine responses.

High IL-8 levels and sialidase activity present in AV oppose the idea that microbial sialidases may interfere with the cytokine cascade leading to diminished IL-8 levels, as was

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suggested as a mechanism to explain its depression in BV [25]. While IL-6 is an essential trigger of the inflammation cascade [51], IL-8 is a chemoattractant cytokine that attracts and activates neutrophils in infected regions [25]. Besides the migration of cells, the response of neutrophils to IL-8 is also characterized by the release of granule enzymes, the induction of

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phagocytosis and several intra- and extracellular changes. Some overlap of the symptoms of severe AV, i.e., DIV, with erosive lichen planus and the comparable response of these conditions to corticosteroids, makes it appealing to consider immune alteration as a possible

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cause of AV or DIV, but this remains to be further confirmed [19]. On the other hand, most authors think that the present aerobic bacteria are opportunistic and do not trigger the disease [19]. Other authors disagree, suggesting that S. aureus producing TSST-1 or similar organisms could induce a ‘vaginal toxic syndrome’ resulting in AV and DIV [52]. However, compared with other infections, such as BV, Candida vaginitis and urinary tract infection,

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spontaneous resolution of AV has not been properly investigated [7]. Its chronic persistence strongly suggests that host immunity is an important factor in the course of AV.

6.3.Aerobic bacteria

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The third important finding in AV is significant colonization with aerobic bacteria [3, 4, 7, 9, 17, 39, 53-55, 56-59]. Streptococcus spp., S. aureus and coagulase-negative staphylococci were the most commonly identified Gram-positive pathogens among

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symptomatic women with presumptive diagnosis of AV, with prevalence rates of 58.7%, 41.7%and 37.4%, respectively, while E. coli was the most common Gram-negative pathogen identified, with a prevalence of 23% among symptomatic women [9]. E. faecalis is also frequently encountered in studies [9]. Not only the species composition, but also the concentration of the bacterial species may be of importance. In one study, the sum of the concentrations of aerobes was tenfold higher in AV-positive than in AV-negative cases [log7.30 vs. log6.06 (p = 0.02)] [10]. Concentrations of aerobes in severe, moderate and light AV cases did not vary significantly (p = 0.14), but the concentration of lactobacilli was 1,000-fold lower in AV-positive cases compared to women with normal vaginal microflora (log5.3 vs log8.3, p < 0.0001) and

ACCEPTED MANUSCRIPT Streptococcus spp. dominated in the majority of AV-positive cases [19/22 (86.4 %) of samples].

6.4.Production of sialidases Some bacterial species or some strains within a species produce sialidases, which

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degrade host defense molecules such as IgA and which can remove sialic acid from mucosal epithelial cells and mucins. Removal of sialic acid from secretory IgA is the mechanism by which sialidase leads to IgA proteolysis, and consequently, a lowered local immune response [58]. High sialidase activity is considered a risk factor for preterm birth [49]. Although a marker for BV, it is also produced by at least some AV-

6.5.The role of local estrogen levels

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associated species, such as S. agalactiae.

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sialidase activity is considered

A vaginal smear of normal vaginal fluid contains only mature epithelial cells. A lack of estrogen leads to a shift of exfoliating epithelial cells towards intermediate, or in worse cases, parabasal cells (Fig. 3) [4, 59], as seen in AV cases. Estrogen plays an important role in AV treatment, in conjunction with other components supporting the role of locally lowered

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estrogen levels in AV pathogenesis [60]. Because Reichman et al. could not demonstrate low serum estrogen levels in DIV cases, and therefore proposed that, rather, the immune/autoimmune-induced inflammation, and not estrogen deficiency, was primarily involved in

causation of the disease [19]. However, wet mount microscopy of smears of

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women with DIV also show increased numbers of parabasal cells, as is the case in AV, indicative of insufficient estrogen impregnation of the vaginal mucosa, even in the presence of high estrogen serum levels.

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While most studies of vaginal acidification have focused on the role of lactobacilli,

host epithelial cells may significantly contribute to vaginal acidification as well [38, 59]. Two related host roles have been suggested: 1) upregulation of proton secretion at the surface of the cervicovaginal epithelium in response to estrogen [59]; and 2) host production of lactic acid and fatty acids from cellular glycogen [38]. Thus, it is likely that host epithelial cells, in concert with lactic-acid-producing bacteria, contribute to vaginal acidification and create an environment that is more selective for lactic acid bacteria (lactobacilli, streptococci) and yeasts. Another important issue involved in the vaginal defense system is the kallikrein (KLK)-related peptidase. The KLK family consists of 15 genes located on chromosome

ACCEPTED MANUSCRIPT 19q13.4. These genes encode for the secretion of serine peptidases, which are important for skin desquamation [61]. These peptidase levels are elevated in desquamative diseases such as psoriasis, atopic dermatitis and peeling skin syndrome [61]. KLK expression is also regulated by vaginal epithelial cells, especially as a response to estrogens and glucocorticoids [61]. However, to what extent this increased expression is related to desquamation of vaginal

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epithelial cells is still unknown. Shaw et al. found that KLK levels in DIV are elevated, which could contribute to over-desquamation, but similar studies have not yet been performed in msAV cases [61]

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6.6.Vitamin D deficit

In patients with impaired absorption due to Crohn’s disease, vitamin D deficiency was seen as a possible reason for DIV, the most severe form of AV. Several cases presented with

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slightly decreased 25-hydroxyvitamin D (25-HD) [62]. However, in another study, there was no improvement in DIV after treatment with vitamin D and adequate normalization of its level [19].

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7. Clinical pathogenicity

Aerobic vaginitis is characterized by inflammation of the vaginal epithelium. The intensity of the clinical symptoms may vary and erosions may occur. Therefore, the risk of

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acquiring HIV or other STIs can increase as a result of AV.

7.1. Sexually transmitted infections and HPV-induced cervix lesions in relation to AV Jahic et al. found low-grade squamous intracellular lesions (LSILs) of the cervix to be

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associated with AV [63]. Findi et al. published similar findings, extending this association to all dysplastic lesions: out of 56 women with dysplastic lesions, 32 had aerobic infection, and 27 of these 32 had a cervical lesion [64]. Examining changes in vaginal microflora in cervical dysplastic lesions, Bernad et al. reported that microscopic and culture findings of vaginal infections, such as the presence of Candida, G. vaginalis or Trichomonas in the absence of lactobacilli, and heavy growth of aerobic vaginitis-associated bacteria such as E. coli, Klebsiella, S. aureus and enterococci, were present in vaginal secretions in 14 of 22 (64%) of patients with high grade SIL (HSIL) versus 29/74 (39%) of patients with LSIL and 23% with atypical squamous cells of undetermined significance (ASCUS)and assumed a causative role for bacterial vaginal infection in the development of HSIL [65]. In other studies, the presence

ACCEPTED MANUSCRIPT of msAV was found to be associated with increased risk of major cytological abnormalities of the cervix in high-risk HPV-infected women [21, 66]. The probability of having a major Pap smear abnormality was higher if the patient had msAV (OR: 1.87 (CI: 95 % 1.02-3.43, p = 0.045) and msAV or BV (OR: 1.84, CI: 1.21-2.78; p = 0.005). In this series, AV was not found to be associated with HPV infection itself [18]. BV was not associated with cervical

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abnormalities, and it was suggested that the formerly described association between cervical dysplasia and BV could at least in part be explained by the association of cervical dysplasia with AV that was not checked for in those older studies [67]. Thus, possibly not BV, but rather increased pH and lack of lactobacilli are key promotors of HPV propagation in the

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human cervix [18]. Likewise, in another study, Chlamydia was found to be associated with AV but not with BV [25]. Trichomonas infection, which is often found in association with

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BV, may also be present in AV microflora and can even mimic it [3].

7.2. Pregnancy and AV

Although it is usually only BV that is mentioned as a major infectious cause for adverse pregnancy outcome, AV can also be linked to the increased risk of preterm pre-labor rupture of membranes, chorioamnionitis and preterm delivery [17], probably linked to the

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increased concentrations of IL-1β, IL-6 and IL-8 found in AV, which are, amongst other, known risk factors for adverse pregnancy outcome. Also, most culture-based studies, demonstrating the presence of E. coli and Klebsiella, confirm the association of AV and preterm birth [17, 68-71], although Krauss-Silva et al. failed to find such an association [28].

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Early in pregnancy, not only BV, but also AV, seems to be associated with spontaneous miscarriage [29, 64].

Rezeberga et al. showed an increased risk of chorioamnionitis and fetal funisitis at birth

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when AV was detected before 12 weeks of gestation [72]. Isolation of S. aureus (a common agent associated with AV) in the vagina and a diagnosis of AV could be linked to fetal growth restriction [72, 73]. Donders et al. have emphasized that AV, with or without the presence of concomitant BV, may be a better candidate than BV for causing pregnancy complications, such as preterm delivery [17]. Using wet mount microscopy, those authors, as well as Nenadic et al. [74],

hypothesized that some patients with a common mild to moderate form of AV

were often misdiagnosed as having BV or intermediate microflora. On the other hand, in one recent study, only 5.2% of cases with intermediate Nugent scores were re-classified as AV, and 3.7% of the BV-positive cases were classified as moderate AV [28]. It is, however, difficult to compare these data, because the authors relied

ACCEPTED MANUSCRIPT on Nugent scoring of Gram stains.

8. Treatment of AV Treatment of AV may include antibacterial (antiseptic and antibiotic), hormonal, nonsteroidal anti-inflammatory and/or probiotic therapy and can be prescribed as local or

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systemic treatment.

8.1.Antiseptic therapy

Two studies [75, 76] showed that vaginal treatment with dequalinium chloride (DQC)

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resulted in clear reduction of the clinical symptoms of AV, reduction in the presence of relevant facultative pathogenic microorganisms in vaginal cultures, and was well tolerated. Della Casa and colleagues demonstrated the in vitro antimicrobial activity of DQC against

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different pathogens that are relevant to vaginal infections, including aerobic bacteria such as staphylococci, streptococci and E. coli [76]. Petersen and colleagues [77] included in their clinical study 73 women diagnosed with ‘fluor vaginalis’, whereby signs and symptoms of vaginitis and a disturbed vaginal microflora were present and BV, vulvovaginal candidiasis and T. vaginalis were excluded. The total symptom score of these women with AV-like

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vaginitis decreased under therapy with DQC from 5.0 ± 1.9 at entry to 1.9 ± 1.5 and 1.3 ± 1.3 at subsequent follow-up visits, respectively. The percentage of women with more than 10 leucocytes per microscopic hpf decreased in the DQC group, from 42.2 % at entry to 9.3 % and 8.6 % at follow-up visits 2 and 4 weeks later, respectively. Furthermore, DQC therapy

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resulted in a decrease in the positive cultures for streptococci, enterococci and E. coli by 36 %, 49 %and 73 %, respectively. Also, other clinical studies reported good clinical efficacy of DQC in treatment of aerobic bacterial vaginal infections [20, 77, 78].

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In Ugandan women, similar satisfactory improvement

in the vaginal microflora,

notably AV, was maintained for up to 1 month after a 6-day treatment course with vaginal rifaximin (a non-absorbable broad spectrum antibiotic) and DQC [27]. A randomized, open-label cohort study of treatment with nifuratel 500 mg

intravaginally for 10 days showed good clinical efficacy for AV, particularly in cases of mixed infections (AV + BV, 43 %; AV + Candida, 31%; AV + T. vaginalis, 26%). In the former case, 500 mg proved to be superior to the 250 mg dosage [20]. No other studies have been performed thus far, to our knowledge. The use of povidone iodine rinsing of the vagina may have some logic, as the product inhibits growth of most major groups of Gram-positive and Gram-negative enteric bacteria,

ACCEPTED MANUSCRIPT involved in AV. This technique has merit as a disinfectant to prevent post-procedure infectious morbidity in women undergoing in vitro fertilization, abortion or cesarean section [20, 78, 79]. However, as the product rapidly disappears from the vagina after its application, it is uncertain as to whether any prolonged effect can be expected in women with an already established vaginal aerobic infection, and whether any short-term improvement is not merely

properties of the product itself. Although

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a consequence of the mechanical cleaning effect of the rinsing rather that the antimicrobial regrowth of lactobacilli is rather swift in women

with BV treated with povidone iodine [80], we found no validated, controlled studies

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documenting the usefulness of povidone iodine in women with AV.

8.2.Antibiotic therapy

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Mulu et al. investigated antibiotic susceptibility of aerobic bacterial isolates from the vagina in women of reproductive age in a hospital-based cross sectional study [81]. Group B Streptococcus had a 20 % resistance rate against ciprofloxacin, norfloxacin, erythromycin and clindamycin. Gram-negative rods (E. coli, Pseudomonas spp. and Enterobacter spp.) showed high percentages of resistance to cotrimoxazole (57.1–80 %), tetracycline (57.1–73.3 %) and

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amoxicillin (80–85.7 %). Similarly, S. aureus showed resistance rates from 67 to 83 % to tetracycline, amoxicillin and cotrimoxazole. These data indicate that great care must be taken whenever antibiotics are used for vaginal infections, and that inadequate use of any antibiotic should be discouraged in order not to even further increase resistance rates [81].

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Per definition, the use of metronidazole is unlikely to improve the AV condition, as the bacteria associated with it are not anaerobic species, as seen in BV, but rather a limited number of aerobic (enteric) bacterial microorganisms. Thus, treatment failure of

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metronidazole in women with symptomatic vulvovaginitis is a fair reason to suspect AV as an underlying diagnosis. Clindamycin, on the other hand, with its broader spectrum, also active against several aerobic Gram-positive cocci [82], and its inherent anti-inflammatory effect, seems to have a much more profound effect on women with severe forms of AV [4]. However, clindamycin as first choice may have its shortcomings: infection control may be short-lived and may not cover all species involved in AV and, most disturbingly, therapy with clindamycin is prone to resistance development in patients treated repeatedly for recurrent disease, especially with methicillin-resistant S. aureus (MRSA) and group B streptococci [8284]. Focusing on the Gram-negative bacilli, rather than the Gram-positive cocci, Tempera et

ACCEPTED MANUSCRIPT al. studied the use of local 35 mg meclocycline or 100 mg kanamycin to treat AV, as these drugs are not absorbed and, like quinolones, spare vaginal lactobacilli [56]. After 6 days of treatment, 80 % of the meclocycline and 100 % of the kanamycin-treated patients were cured when examined on day 7, whereas, after 13-16 days, only the kanamycin-treated women remained in remission with normal vaginal pH and LBG I [22]. In a larger study, 81 patients

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with AV were randomized to intravaginal 6-day treatment with either 100 mg kanamycin ovules or 35 mg meclocycline. One to 2 days following completion of treatment, the authors found a stronger reduction in vaginal leucocytes, stronger reduction in isolation of Enterobacteriaceae (97 vs. 76 %) and less vaginal mucosa burning and itching in the group compared to the meclocycline-treated group [85]. At the second

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treated with kanamycin

follow-up, vaginal homeostasis (normalization of pH and presence of lactobacilli) was more evident in the kanamycin-treated group.

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Wang et al. investigated the therapeutic efficacy of oral moxifloxacin for AV [53]. A total of 102 women were treated with oral moxifloxacin 400 mg once daily for 6 days (one course). Women who improved microscopically, but not clinically, received a second course of therapy. After one course of therapy, 65.7 % of women were cured, 29.4 % improved and 4.9 % failed to respond to therapy. After two courses of therapy, 85.3 % (87/102) of women

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were cured, 9.8 % improved and 4.9 % failed to respond. Most women with AV were cured with one course of moxifloxacin, but the authors suggest that two courses of therapy should be considered for those with a vaginal pH ≥ 5.0 before treatment [6]. In the study of Mumtaz et al., a significant growth in aerobic pathogens was seen in 76.6

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% of 731 patients with AV. S. aureus was the most prevalent vaginal pathogen in 31–40-yearold patients, followed by enteric Gram-negative bacilli and other Gram-positive cocci [86]. There were very few antibiotics among the conventionally available aminoglycosides, third-

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generation cephalosporins, penicillins, quinolones, sulfonamides and tetracyclines that possessed sufficiently potent activity (more than 80 % of strains killed) against these common aerobic vaginal pathogens. The most effective chemotherapeutic agents belong to the groups of carbapenems and the clavulanic-beta-lactam combinations (amoxyclav) [86]. In a study of Fan et al. whereby 39 patients with AV received one course of 400 mg of moxifloxacin for 6 days and 21 received two courses of 400 mg for 12 days, the cure rate one month after treatment was 90% in the 6-day group and 75% in the 12-day group [11]. The decrease in efficacy with longer treatment and/or a higher dose of an antibiotic may have been due to loss of protective viable lactobacilli due to intensive treatment, as was also shown when treating BV patients with different doses of rifaximin [87, 88].

ACCEPTED MANUSCRIPT 8.3.Non-antibiotic therapy and combination therapy However, it is very unlikely that oral administration of any of the above antibiotics will have a long-term positive effect on the vaginal milieu. Hence, it should only be considered for initial use, with short courses, to control acute symptoms in complicated and severe cases

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such as staphylococcal or macular streptococcal vaginitis, which are rare and specific subcategories of severe AV [89-92]. Sobel first used clindamycin to treat DIV [4], which showed efficacy in two-thirds of women after 1 or 2 courses of treatment. In the remaining one-third of women, further addition of topical estrogen led to improvement. In a later study,

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the same author studied the outcome of women with DIV treated with topical application of either 2% clindamycin or 10% hydrocortisone [91]. However, as this was an open-label, noncontrolled observation study, not much can be concluded as to whether one therapy is better

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than the other. The only outcome that can be retained from this study is a high early failure rate (37%) and a high recurrence rate of 32, 43and 74% after 6 weeks, 6 months and 1 year, respectively [91], although continued therapy often resulted in control, but not cure.

In clinical practice, therapy is decided based upon

microscopic findings:

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hydrocortisone if predominance of inflammation, estrogens in case of low maturation indexes and antimicrobials for the more infectious types (as evidenced by large numbers of cocci). Hence, comparing therapies in such heterogeneous groups is unlikely to produce any conclusion about which therapy is superior.

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In most women with a prominent atrophy component, as diagnosed by an increased number of parabasal cells (more than 10% of epithelial cells) by microscopy of the vaginal fluid, local intravaginal application of estrogens (e.g., 0.1 % estradiol valerate) is very helpful

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in most cases. This is typically effective in young women in their postpartum period, or when on low-dose combined contraceptive pills, or progestin-only regimens, or in elderly women in their peri-menopausal or untreated menopausal period. However, in women with contraindications for use of steroid hormones, such as breast cancer patients or patients with previous thromboembolism, this approach is not possible, and other options have to be explored. Recently, excellent treatment success was achieved with the use of a vaginal product combining a probiotic (107 viable cells of Lactobacillus acidophilus) with an ultralow dose of 30 µg estriol (E3) for

treatment of severe symptomatic vaginal atrophy in

menopausal women [93], and in women with hormone-receptor-positive breast cancer taking antihormonal aromatase inhibitors [94, 95]. In a multicenter, randomized, double-blind,

ACCEPTED MANUSCRIPT placebo-controlled, parallel-group study, Herczko et al. demonstrated a prolonged time to relapse of BV and/or AV clinical symptoms with vaginal probiotics compared to placebo. In addition, the effect of this adjuvant probiotic therapy was superior after targeted antibiotic therapy compared to the per-protocol population [96]. In summary, local estradiol or estriol administration, with or without probiotic

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lactobacilli, can be considered in cases where atrophy, i.e., increased numbers of parabasal cells, predominates [97, 98]. Local application of a corticosteroid can be used when inflammation, i.e., > 20 leucocytes per epithelial cell, predominates, whereas primary antimicrobial treatment is indicated in cases with clear infectious abnormalities, i.e.,

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lactobacillary grade IIb or III, with cocci or cocci chains on wet mount microscopy and/or heavy growth of E. coli, group A streptococci, group B streptococci or staphylococci on

8.4.Treatment in pregnancy

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culture.

AV is a notorious cause of complications in pregnancy. The broad-spectrum effect of clindamycin versus the narrow anaerobic spectrum of metronidazole can explain why clindamycin, but not metronidazole, is capable of stemming the complications of preterm

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delivery and premature preterm rupture of the fetal membranes (PPROM) in women with an abnormal vaginal microflora [17, 99]. Accordingly, preterm delivery can be caused by abnormal vaginal microflora, both BV and AV, or a combination thereof, but metronidazole will only treat for BV, leaving the dangerous inflammatory cytokine-producing condition AV

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untreated, while clindamycin is active against both. To date, four randomized controlled studies have shown a substantial reduction in the occurrence of preterm birth in women with AV treated with clindamycin [100-103]. However, two other randomized studies could not

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find an improved outcome after clindamycin treatment, and concluded that some cases of AV are refractory to treatment or cause frequent recurrences [104, 105], leading some authors to advise rescreening and retreatment of cases in which no cure has been achieved with initial antimicrobial treatment [105, 106]. Of course, potential causes other than AV or BV, like M. genitalium, may have remained untreated after clindamycin treatment. In 50 patients with proven intra-amniotic infection (IAI) due to ascending AV microorganisms, Curzik et al. were able to improve the pregnancy outcome by treating with erythromycin, cefuroxime and local tetracycline [55]. The cervical swab became negative in 30 of the patients with IAI and, in these women, perinatal loss was only 6.7 %, while in cases with persistent infection, loss was 55.0%.

ACCEPTED MANUSCRIPT A randomized, controlled study was performed to compare the clinical and microbiological results of patients with infectious vaginitis receiving vaginal irrigation with saline, or no irrigation before standard antibiotic therapy [107]. The findings of the study showed that vaginal irrigation with saline significantly reduced the patients’ self-reported symptoms in the short term. These findings can be explained by the rapid decrease in the

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amount of vaginal discharge and bacterial loads in vaginal secretions after irrigation. However, as expected, the reduction in the number of bacteria is not sufficient for long-lasting improvement [107]. We do not recommend such a treatment. A good review of topical

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treatment possibilities for women with vaginitis was provided by Frey [108].

9. Conclusions

There currently exist several shortcomings in the diagnosis and treatment of bacterial

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vaginitis. Mainly, the categorization of the vaginal microflora of women into ‘normal’, ‘intermediate’ or ‘bacterial vaginosis’ causes a failure to discover women with significant infectious conditions and, as a consequence, leads to improper and incomplete treatment. Especially in pregnancy, the lack of an appropriate diagnostic workout seems to have led to numerous misconceptions and treatment failures in studies aiming to decrease infection-

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related pregnancy complications such as preterm delivery, chorioamnionitis and PPROM. By adding an extra diagnostic scoring system to the current diagnosis of BV, many of these discrepancies can be explained and addressed in a more comprehensive way. The diagnosis of AV is based on wet mount microscopy by the presence of variable grades of: 1) disturbed

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lactobacillus microflora, 2) the presence of coccoid or bacillary aerobic bacteria, 3) leucocytes, 4) active, toxic leucocytes, and 5) parabasal epithelial cells. The composite score of these 5 findings differentiates in light, moderate or severe AV.

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While, formerly, only the relation of different pathologies to BV was reported, it is

gradually becoming more and more clear that moderate/severe (ms)AV is involved in a wide number of diseases as well. The presence of msAV is linked to STIs and to an increased risk of having a major Pap smear abnormality in HR-HPV-infected patients. During pregnancy, women with msAV early in pregnancy have an increased risk of having a shorter cervix from 20 weeks onward and more preterm births, and are at greater risk of chorio-amnionitis and funisitis. Several mechanisms can explain the pathogenesis and pathology of AV, including decreased lactobacillary protection, increased inflammatory cytokine reaction, the presence of opportunistic or pathogenic bacteria and the production of sialidases and estrogen deficiency.

ACCEPTED MANUSCRIPT Acknowledgements. The authors are very thankful for the extensive and precise review of

Prof. M

Vaneeckhoutte and Prof. T Crucitti, whose great efforts and valued comments significantly

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improved the quality of the manuscript.

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Fig. 1. Lactobacillary grades. A) LBG I; B) LBG IIa; C) LBG IIb; D): LBG III

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Fig. 2. Phase contrast microscopy images; 400-fold enlargement of vaginal fluid from patients with aerobic vaginitis. A) Microflora devoid of lactobacillary morphotypes (lactobacillary grade III)and coccoid bacteria. B) Chains of cocci, a typical feature of AV caused by streptococci. C) 'Toxic' leukocytes, full of lysosomic granules.

ACCEPTED MANUSCRIPT Fig. 3. Types of epithelial cells

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A: Superficial cell, B: intermediate cells, C: Parabasal cells

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Fig. 4. Treatment of AV adapted to the presence of three different components: infection, inflammation or atrophy. In severe cases, all components can be present.

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DIV: desquamative inflammatory vaginitis. Adapted from [75].

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Table 1. Aerobic vaginitis (AV) score calculation

Proportion of toxic No. of leucocytes

leucocytes

I and 0

≤10/hpf

None or sporadic

IIa >10/hpf and 1

IIb

III

>10/epithelial cell

of PBC

Unremarkable or

None or

cytolysis

<1%

Small coliform ≤50% of leucocytes

≤10/epithelial cell 2

Proportion Background flora

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LBG

≤10%

bacilli >50% of leucocytes

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Score

Cocci or chains

>10%

AV-score = sum of all sub-scores

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LBG = Lactobacillary grade ; hpf = high-power field; PBC = parabasal cells

A composite AV-score of <3 corresponds to ‘no signs of AV’, 3-4 to ‘light AV’, 5-6 to ‘moderate AV’

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and any score >6 to ‘severe AV’.

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Table 2. Prevalence of aerobic vaginitis in different pregnant and non-pregnant populations.

Population age

Pregnant / Nonpregnant

Time frame

N° of women

Country

Tibaldi, C. 2016

Childbearing age

Pregnant

11.219

Italy

Grinceviciene, S. 2016

21-60 years

Nonpregnant

2000 2010 2015

101

Donders, G. 2009

Childbearing age

Pregnant

759

VieiraBaptista, P. 2016 Rumyantseva, T. 2016

21-75 years

Nonpregnant

622

Portugal

8.5

Microscopy

More than 18 years

Pregnant and nonpregnant Pregnant and nonpregnant Pregnant

2000 2001 2014 2015 2011

São Tomé and Príncipe Belgium

100

Russia

10.3

Microscopy

2013

101

Chile

2.0

Microscopy

14-44 years

Zodzika, J. 2011

≥18 years

Tomusiak, A. 2013

20-40 years

Donders, G. 2011 Donders, G. 2016

≤25 years

Wang, Z.L. 2016

17-71 years

Pregnant

Nonpregnant

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Nonpregnant Pregnant and nonpregnant Nonpregnant

Diagnostic technique

25.8

Microscopy

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Gondo, F. 2011

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15-54

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Villaseca, R. 2015

% of women with msAV 7.4

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Publications

Microscopy

Microscopy

2006 2007 2009 2010 20112012 (?) 2008

245

Brazil

2.9

Microscopy

139

Latvia

10.1

Microscopy

161

Poland

12.0

876

Finland

9.0

Microscopy and microbiology Microscopy

2009

338

Uganda

11.0

Microscopy

2011

1948

China

15.4

Enzymatic tests

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[33] Kalyoussef S, Nieves E, Dinerman E, Carpenter C, Shankar V, Oh J, et al. Lactobacillus proteins are associated with the bactericidal activity against E. coli of female genital tract secretions. PLoS One

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2012;7:e49506.

[34] Younes JA, Reid G, van der Mei HC, Busscher HJ. Lactobacilli require physical contact to reduce staphylococcal TSST-1 secretion and vaginal epithelial inflammatory response. Pathog Dis 2016;74:19.

[35] MacPhee RA, Miller WL, Gloor GB, McCormick JK, Hammond JA, Burton JP, et al. Influence of the vaginal microbiota on toxic shock syndrome toxin 1 production by Staphylococcus aureus. Appl

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Figure 4. Treatment of aerobic vaginitis (AV) adopted to the presence of three different components: infe DIV: desquamative inflammatory vaginitis. Adapted from [75].

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Table 1. Aerobic vaginitis (AV) score calculation

LBG

No. of leucocytes

Proportion

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Proportion of toxic Score

Background flora

of PBC

I and 0

≤10/hpf

None or sporadic

IIa >10/hpf and 1

None or

cytolysis

<1%

≤50% of leucocytes

>10/epithelial cell

≤10%

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III

Unremarkable or

Small coliform

IIb ≤10/epithelial cell

2

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leucocytes

>50% of leucocytes

Cocci or chains

>10%

AV-score = sum of all sub-scores

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LBG = Lactobacillary grade ; hpf = high-power field; PBC = parabasal cells

and any score >6 to ‘severe AV’.

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A composite AV-score of <3 corresponds to ‘no signs of AV’, 3-4 to ‘light AV’, 5-6 to ‘moderate AV’

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2. Prevalence of aerobic vaginitis in different pregnant and non-pregnant populations.

Tibaldi, C. 2016

Childbearing age

Pregnant

11.219

Italy

Grinceviciene, S. 2016

21-60 years

Nonpregnant

2000 2010 2015

101

Donders, G. 2009

Childbearing age

Pregnant

759

VieiraBaptista, P. 2016 Rumyantseva, T. 2016

21-75 years

Nonpregnant

622

More than 18 years

Villaseca, R. 2015

15-54

Gondo, F. 2011

14-44 years

Pregnant and nonpregnant Pregnant and nonpregnant Pregnant

2000 2001 2014 2015 2011

São Tomé and Príncipe Belgium

Zodzika, J. 2011

≥18 years

Pregnant

Tomusiak, A. 2013

20-40 years

Nonpregnant

Donders, G. 2011 Donders, G. 2016

≤25 years

Nonpregnant Pregnant and nonpregnant

Wang, Z.L. 2016

17-71 years

18 - 44 years

Nonpregnant

% of women with msAV 7.4

Diagnostic technique

Microscopy

25.8

Microscopy

8.3

Microscopy

Portugal

8.5

Microscopy

100

Russia

10.3

Microscopy

2013

101

Chile

2.0

Microscopy

2006 2007 2009 2010 20112012 (?) 2008

245

Brazil

2.9

139

Latvia

10.1

161

Poland

12.0

876

Finland

9.0

Microscopy and microbiology Microscopy

2009

338

Uganda

11.0

Microscopy

2011

1948

China

15.4

Enzymatic tests

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N° of women

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Time frame

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Pregnant / Nonpregnant

Microscopy

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Population age

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Publications

Microscopy

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B

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A

Figure 1 Lactobacillary grades. A: LBG I, B: LBG IIa, C: LBG IIb, D: LBG III

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Figure 2 . Images of phase contrast microscopy at 400 times enlargement of vaginal fluid from patients with aero A. microflora devoid of lactobacillary morphotypes (lactobacillary grade III), and coccoid bacteria. B. chains of cocci, a typical feature of AV caused by streptococci. C. 'toxic' leukocytes, full of lysosomic granules. B C

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A

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Original foto from Fig 2 A

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Original foto from Fig 2 C

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B

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Figure 3. Types of epithelial cells A: Superficial cell, B: Intremidiate cells, C: Parabasal cells C