Definition and classification of abnormal vaginal flora · Definition and classification of abnormal vaginal flora Gilbert G.G. Donders* MD, PhD ... (‘das nativ preparat’),

Best Practice & Research Clinical Obstetrics and GynaecologyVol. 21, No. 3, pp. 355–373, 2007

doi:10.1016/j.bpobgyn.2007.01.002available online at http://www.sciencedirect.com

2

Definition and classification of abnormal

vaginal flora

Gilbert G.G. Donders* MD, PhD

Professor

Department of Obstetrics and Gynecology, Algemeen Ziekenhuis Heilig Hart, 3300 Tienen, University Hospital

Gasthuisberg, 3000 Leuven, Belgium

Department of Obstetrics and Gynecology, Citadelle Hospital, University of Liege, 4000 Liege, Belgium

Studying the vaginal microflora is not only fascinating, with many discoveries to be made, it isalso a very practical way to help women get rid of bothersome and sometimes dangerous infec-tions. Gram-stained vaginal preparations, Pap smears, specific cultures, and nucleic acid detec-tion techniques can be used to diagnose the constituents of the vaginal flora, but in trainedhands office-based microscopy of a fresh vaginal smear, preferably using a �400 magnificationphase-contrast microscope, allows almost every diagnosis and combination of diagnoses imagin-able. In this chapter I will address the pros and cons of the tools that are in use to study vaginalflora, and discuss the different types of bacterial flora and the difficulties encountered in reachingthe correct diagnosis of pathological conditions. The ‘intermediate flora’ is addressed separately,and a new entity – ‘aerobic vaginitis’ – is discussed. Future research should focus on theinteraction between infecting microorganisms and host defence mechanisms, as both togethergenerate the pathogenicity of these conditions.

Key words: abnormal vaginal flora; lactobacillary grades; intermediate flora; bacterial vaginosis;aerobic vaginitis; Trichomonas vaginal; genital infection.

Vulvovaginal infections are among the commonest reasons why women seek profes-sional help. At the same time it is the core of a multi-million dollar business ofover-the-counter medicines for self-treatment. Proper diagnosis by a trained specialistwould enable women to get timely and efficient treatment, limit the cost of diagnosticshopping, the side-effects of inadequately treated disease, and unnecessary anxiety.Although office microscopy seems the logical cornerstone to achieve such a diagnosis,its use was progressively abandoned due to reports of its limited diagnostic value and

* Tel.: þ32 16344204; Fax: þ32 16344205.

E-mail address: [email protected].

1521-6934/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved.

356 G. G. G. Donders

the upsurge of laboratory-based techniques such as the Gram stain, culture, and anti-gen or nucleic acid detection techniques.1,2 This, together with the desire to sharediagnostic responsibility, resulted in many physicians drifting towards overuse of labo-ratory services to obtain a diagnosis and even treatment advice. In this review I willdiscuss the possibilities and limitations of office microscopy in discerning differenttypes of bacterial vaginal flora.

HISTORY

The use of microscopy went hand in hand with the development of microbiology andthe awareness that parasites, bacteria and yeasts were involved in the causation ofvulvovaginal disease. Direct visualization of trichomonads and yeasts in vaginal fluidwas possible soon after the introduction of the microscope. The recognition thatthe bacterial flora may be a cause of vaginal infectious conditions started more thana century ago in 1892, when Albert Doderlein wrote his dissertation entitled ‘DasScheidensekret’ (‘vaginal discharge’ or, more precisely, ‘vaginal secretion’).3 Using sa-line without additive colouring or fixation (‘das nativ preparat’), he was able toshow lactobacilli in vaginal secretions of healthy women and a lactobacillus-deficientflora in women with postpartum endometritis. His successor, Schroder, started touse this information in a broader clinical context, and the first lactobacillary gradeswere born.4 Lactobacillary grade I, corresponding to a ‘healthy’ microflora, hadpredominant lactobacillary morphotypes of variable size. Lactobacillary grade III isa condition wherein the lactobacillary morphotypes are completely replaced by otherbacterial morphotypes. Lactobacillary grade II is an intermediate grade, with partial re-placement of the lactobacilli by other bacteria. Due to their specific link to pathology,we refined the three grades, and subdivided grade 2 (LBGII) into a less severe LBGIIaand a more severe LBGIIb variety (Figure 1).5

Up to the 1950s, symptomatic women with lactobacillary grade III were diagnosedwith ‘non-specific vaginitis’, as the microbial aetiology of lactobacillary deficiency wasstill uncertain at that time. This was resolved by Gardner and Dukes, a gynaecologistand a microbiologist, who together discovered a new genus of bacteria held respon-sible for the condition we now know as bacterial vaginosis (BV).6 At first, Gardnerand Dukes thought their newly discovered bacterium belonged to the Haemophilusgroup (‘Haemophilus vaginalis’), but soon afterwards the unique properties of thebacteria isolated necessitated the creation of a new genus: Gardnerella. So it had tobe proved that G. vaginalis was the cause of the foul-smelling watery vaginal dischargein symptomatic women suffering from BV. Although inoculation of young healthywomen with vaginal fluid from women with BV caused symptoms typical of BV in13 of 15 volunteers, inoculation with purified G. vaginalis resulted in only a singlecase of BV., It was therefore recognized that although G. vaginalis was present in largequantities in almost all women with BV, it is not the cause of its symptoms. Progres-sively, other organisms were discovered in the vaginal fluid to explain the complaintsof the foul-smelling discharge, such as the anaerobic Bacteroides sp, peptostreptococciand others. Subsequently, mycoplasmas, especially Mycoplasma hominis, and alsoMobiluncus species were encountered more frequently in BV flora.7 Partly due tothe fact that most of these bacteria cannot be visualized on wet-mount microscopy,many attempts were made to cast the diagnosis in microbiological terms; quantitativebacteriology was used to try and explain symptoms in terms of numbers of differentbacteria.8 Furthermore, bacterial products – volatile short carbon chains such as

Abnormal vaginal flora 357

lactate, succinate and triethylamine – could be detected by the use of gas–liquid chro-matography. Bacterial vaginosis could now be diagnosed by the chemical properties ofthe anaerobic bacteria involved: a succinate/lactate ratio of �4 was found indicativefor BV.9

The diagnosis of bacterial vaginosis based on Gram-stained specimens was firstdone by Carol Spiegel et al10 and later refined and quantified by Nugent et al11,thereby progressively moving the diagnosis of a common clinical condition into thelaboratory (Table 1). Older studies show the superiority of Gram-stained specimensover clinical diagnosis and fresh wet-mount microscopy in routine settings12, butmore recent studies have challenge this (Platz-Christensen et al, submitted for

Figure 1. Lactobacillary grades (LBGs). (a) LBGI without cyolysis of epithelial cells. (b) LBGI with cytolysis of

epithelial cells, with numerous bare epithelial nuclei and cytolyic debris clearly visible (cytolytic vaginosis).

(c) LBGIIa: lactobacilli prominent, but mixed with some other bacteria. (d) LBGIIb: lactobacilli still present,

but more bacteria of other types present. (e) LBGIII: coccoid aerobic vaginosis (AV) flora. (f) LBGIII: bacterial

vaginosis (BV) flora.


publication). For Trichomonas, polymerase chain reaction (PCR) is marching in, pro-gressively replacing microscopy as a preferred diagnostic tool13, but at the sametime failing to offer the patients an immediate diagnosis and treatment.

DIAGNOSTIC TOOLS FOR ABNORMAL VAGINAL FLORA

Clinical criteria

Bacterial vaginosis may be diagnosed clinically by the presence of three of four ofAmsel’s criteria, namely: (1) homogenous watery discharge; (2) pH> 4.5; (3) clue cellspresent on fresh wet mount; and (4) fishy odour after addition of 10% KOH in water.9

The great benefit of the clinical diagnosis of Amsel et al is that it successfully convertedthe former exclusion diagnosis of ‘non-specific vaginitis’ into a positive, recognizableentity, nowadays known as ‘bacterial vaginosis’.14 A positive whiff test has a specificityof 87% with sensitivity of 34%.15 In other words, fishy odour is not always present inbacterial vaginosis, even after the application of KOH. As may be expected, the pres-ence of a thin, homogenous discharge clinging to the vaginal epithelium has the lowestsensitivity (56%) and specificity (49%).

Fresh wet-mount microscopy

When compared with the diagnosis of BVaccording to the Nugent score on Gram stain-ing, the presence of clue cells on wet mounts is both highly sensitive (77%) and specific(92%).15 When experienced microscopists also take the typical granular flora into ac-count (Figures 2b and 3d), the diagnosis is even more accurate and more rapid thanwith the Gram stain. Bacterial vaginosis and abnormal lactobacillary grades (LBGs)were diagnosed reliably and with great concordance by six international expertswho were blinded for each other’s data, especially when phase-contrast microscopeswere used (Platz-Christensen et al, submitted for publication). There is evidence thatthe Gram-stain procedure harms part of the lactobacillary flora and favours the non-lac-tobacillary flora.16,17 This leads to a false overemphasis of abnormal flora in Gram stainswhen compared with wet mounts, wherein normal flora is better visualized.

Vaginal pH

The normal pH in the vagina of a woman of reproductive age is about 4 (range3.8–4.4). Extreme acid pH makes the epithelial cells vulnerable to cytolysis, a condition

Table 1. Nugent criteria for the diagnosis of bacterial vaginosis (score� 7), normal flora (score� 3), or

‘intermediate flora’ (score 4–6). Other floral types, such as that of aerobic vaginitis, cannot be diagnosed

in this system. Intermediate flora is not equal to partial bacterial vaginosis (see text).

Lactobacilli Gardnerella Mobiluncus

0 4þ 0 0

1 3þ 1þ 1e2þ2 2þ 2þ 3e4þ3 1þ 3þ4 0 4þ


called cytolytic vaginosis, and it may also produce symptoms such as burning and in-creased discharge (see below). More commonly, pH is increased above 4.5, promptingfurther evaluation. Even in the absence of symptoms, routine pH testing increases thedetection of trichomoniasis and bacterial vaginosis in a primary-care setting byprompting microscopy in a significant proportion of asymptomatic cases.18 The sensi-tivity of vaginal pH> 4.5 for the diagnosis of bacterial vaginosis is 88.3%, specificity ismuch less: 58.6%.15 In cases of Trichomonas vaginalis infection or severe aerobic vagini-tis, the pH may be vastly increased to 6.5 or more.19

Theoretically, many non-infectious conditions may alter the normal vaginal pH:menstruation, recent unprotected sexual intercourse with deposition of semen, useof local antifungal agents or antibiotics. Therefore, the finding of an increased pHshould be followed by microscopy and/or cultures to confirm a presumptive diagnosis,in order not to erroneously treat a common non-infectious condition with antimicro-bial agents. The pH of the vagina should be measured directly in the vagina, on thespeculum, on the swab or on the glass slide prepared for microscopy, but additionof saline for fresh microscopy causes the pH to rise and should be discouraged(Donders et al, submitted for publication). Office dipstick tests can be used andshow a good correlation of increased pH with lactobacillary grades20, cervicitis,Trichomonas infection, bacterial vaginosis and aerobic vaginitis.19 Merck’s as well asMachery Nagel’s dipstick can be used efficiently in this pH range, but in difficult casesthe latter are more user-friendly and less time-consuming.21

Gram stain

Most studies comparing wet mount with Gram stain favour the latter because of itshigher sensitivity in diagnosing BV. In the most commonly used scoring system, a scoreof 1–4 of lactobacillary morphotypes, a score of 1–4 of Gardnerella morphotypes, anda score of 1 or 2 for Mobiluncus morphotypes has to be added to obtain a global Nugentscore.11 Nugent score is well suited to diagnosing BV (score of �7) and normal flora(score of �3), but the interpretation of the so-called ‘intermediate flora’ (score 4–6)remains controversial. In many studies the intermediate flora was associated withundefined microbial correlate22 and a different set of complications during pregnancy23,and classic therapy for BV (metronidazole) did not cure most cases with this type offlora.

Figure 2. Images of phase-contrast microscopy (�400) of vaginal fluid from patients with bacterial vaginosis.

(a) Lactobacillary grade IIb (LBGIIb) with partial bacterial vaginosis (BV). (b) LBGIII with full-blown BV.


Pap smear

Pap smears can be used for the detection of clue cells and bacterial vaginosis flora.24 ThePap smear was 78% sensitive and 87% specific in detection of BV in one study15 and 89%and 90% in another.25 The problem is that Pap smears are used for the purpose ofscreening for cervical dysplasia, and are not designed for detecting BV or other genitalinfections. As a result, pathologists focus mainly on the issue of cervical epithelial dis-ease, thus reducing the sensitivity of the cervical smear for detecting BV. Attending phy-sicians will have difficulty in tracing and persuading women to get treatment for a benign,asymptomatic disease discovered incidentally. Furthermore, it is questionable whethertreatment is required for an asymptomatic disease which is harmless in most women.

Culture and PCR

Cultures of Gardnerella vaginalis are not useful for BV diagnosis, as up to 50% of healthywomen have positive cultures due to low numbers of G. vaginalis in the vagina withoutany sign of BV. However, when no wet mounts or Gram stains are available and a clin-ical diagnosis is doubtful, massive growth of BV-associated bacteria or of Escherichiacoli, group B streptococci or Staphylococcus aureus can help in distinguishing AV from

Figure 3. Images of phase-contrast microscopy (�400) of vaginal fluid from patients with aerobic vaginitis

(AV). (a) A microflora devoid of lactobacillary morphotypes (lactobacillary grade III) and coccoid bacteria.

(b) Apparently rod-like organisms, but on closer inspection they appear to be chains of cocci, a typical fea-

ture of AV caused by group B streptococci. (c) The ‘toxic’ leukocytes, full of lysozymic granules. (d) The

typical AV flora, also illustrating the parabasal cells.


BV. Also, cultures for T. vaginalis and Candida may be extremely helpful in doubtfulcases and in cases with mixed infections.

Mycoplasma cultures may help to delineate the pathogenicity of certain types ofabnormal vaginal flora, especially in pregnancy, where there is evidence that the con-comitant infection of M. hominis and U. urealyticum with BV may cause a more severeset of complications such as miscarriage or preterm birth.26–28

Enzymology and immunology

The products of anaerobic infection responsible for the fishy smell – putrescine, cadav-erine, diethylamine and succinate – are increased in the vaginal washings of womenwith bacterial vaginosis, and the lactate/succinate ratio has been used as a biochemicalmarker for bacterial vaginosis.9 Detection of bacterial enzymes such as mucinases,proteinases, G. vaginalis haemolysins and sialidases are correlated with bacterial vagi-nosis but not with candidiasis.28 Approximately 50% of women produce IgA immuno-globulins against G. vaginalis, but some women are infected with G. vaginalis strains thatproduce anti-IgA activity by cleaving the immunoglobulins. The presence of enzymessuch as sialidase or mucinase may change the pathogenicity of the abnormal vaginalflora, while microscopy can by no means detect the difference. Sialidase-positive preg-nant women with BV have a higher likelihood of preterm delivery.29 Interleukins can bemeasured to assess the host response to vaginal intruders and normal constituents ofthe vaginal flora.30 Interleukin 1 is increased in bacterial vaginosis, but even more so inaerobic vaginitis.19 IL8, a pro-inflammatory cytokine responsible for the attraction ofleukocytes, is dramatically increased in aerobic vaginitis, but not in bacterial vagino-sis.19,31 All of these tests can be helpful in studying the pathogenicity of abnormalvaginal flora, but are not suitable for diagnosing the different floral types, eitherbecause they are too laborious to perform or because they are non-specific.

DIFFERENT TYPES OF ABNORMAL VAGINAL FLORA

Lactobacilli

Lactobacilli are the most well-known markers of normal vaginal flora. Their ability toproduce an acid pH in the vagina (mainly due to the acidification enzyme hydrogenperoxidase) and bacteriocins that kill off other bacteria makes them prime candidatesfor the surveillance of vaginal health. There are many different strains of lactobacillipresent in the vagina, the most frequent being L jensenii, L gasseri, L iners and L crispatus,and there is a wide variation in species and relative numbers of species according tothe population studied.32,33 In general, where lactobacilli predominate, other bacteriaand parasites such as Trichomonas are not abundant. On the other hand, lactobacillus-deficient conditions are associated with the development of numerous infectiousconditions such as bacterial vaginosis and aerobic vaginitis, and promote the transmis-sion of sexually transmitted diseases such as gonorrhoea, Chlamydia, syphilis, tricho-moniasis, HIV, and HPV which may lead to cervical cancer.

Normal and abnormal lactobacillary flora are divided into three or four floral types,also depicted as lactobacillary grades (see above). Lactobacillary grade 3 (LBGIII), andto a lesser extent lactobacillary grade IIb (LBGIIb), are more likely to be linked withpathological conditions, and are said to be ‘abnormal vaginal flora’. This condition isa screening tool that should not be confused with bacterial vaginosis. Bacterial


vaginosis is a condition associated with abnormal vaginal flora, but abnormal vaginalflora is not always bacterial vaginosis. Some studies demonstrate that the absenceof lactobacilli is a more powerful predictor of preterm birth than the presence of bac-terial vaginosis.23,34 In order to diagnose such abnormal lactobacillary grades, the useof the wet mount is preferred to the Gram stain due to its superior accuracy16 andbetter correlation with vaginal lactate35, accepted by most as the best functionaltest for lactobacillary defence function.36

Bacterial vaginosis

Ecological disorder

Bacterial vaginosis is an ecological disorder of the vaginal flora in which the normallactobacillus-dominant flora is replaced by a 100–1000-fold increase in the numbersof anaerobic bacteria.37 Symptoms are few, and most women do not realize theyhave the condition. If symptomatic, a fishy smell and watery vaginal discharge arethe most common symptoms. These together with a pH > 4.5 and typical clue cellson microscopy suggest the clinical diagnosis according to Amsel et al.14 An experi-enced microscopist, however, will not only look for clue cells, but will be moreconvinced of the diagnosis if the typical granular vaginal microflora with uncountablecocci are present and so numerous that they cannot be seen as separate bacteria.This allows the recognition of bacterial vaginosis flora in a slide with an otherwise pre-dominant flora. This type of flora is called partial bacterial vaginosis, i.e., a mixture ofnormal flora with zones of typical BV flora, as opposed to ‘intermediate flora’ (seelater), which is seen in Gram-stained specimens scored according to Nugent (Table 1).Intermediate flora is a misnomer and should be replaced by ‘undetermined flora’, as itcorresponds to a floral type which is neither normal nor BV, and usually not ‘partialBV’ either. Hence ‘intermediate flora’ is not an entity but includes ‘partial BV’ aswell as other types of abnormal flora such as aerobic vaginitis (see below).

Absence of inflammation

A typical feature of bacterial vaginosis is the absence of inflammation. In BV there isonly a slight increase in interleukin 1 and an unexpectedly low production of interleu-kin 8, preventing the attraction of inflammatory cells such as macrophages and neutro-phils.15,31 Hence, if severe inflammation is present – e.g. when more than 10leukocytes are present per epithelial cell – one must be suspicious, and another diag-nosis has to be considered. Indeed, concomitant cervicitis, trichomoniasis, candidiasisand/or aerobic vaginitis are all known to present with an increased immune responsewith increased numbers of monocytes and leukocytes in the vast majority of cases.Therefore, the finding of increased leukocytosis in a vaginal smear with bacterialvaginosis must prompt a more intensive search for another diagnosis.

Gardnerella vaginalis

With newer techniques for the isolation of G. vaginalis, mycoplasmas and anaerobicbacteria, it was felt in the 1980s that the future of the diagnostic work-up would liein the qualitative and quantitative description of the microbial content of the vagina.Numerous attempts to quantify bacteria in vaginal lavage led to complicated theories,none of which related to or evolved into useful diagnostic clinical tools. The sensitivityof G. vaginalis cultures, for instance, is so good today, and the organism may be


detected in 70–80% of women, half of whom have no signs or symptoms of bacterialvaginosis.1 On the other hand, it was recognized that BV samples contained 100–1000times more bacteria than in normal controls, and that this overgrowth is a typical/char-acteristic feature of anaerobic BV.36 Recently developed PCR techniques can detectgenomic DNA or RNA coding for structural proteins, allowing detection of extremelysmall numbers of microorganisms, and this led to the discovery of some new specieslinked to the condition.38

Mobiluncus

BV as diagnosed nowadays remains a very confusing and heterogeneous condition. Forinstance, women with BV may have a completely different risk profile during pregnancy,depending on co-infection with M. hominis, Bacteroides sp, or both26,39, and according toother studies U. ureaplasma is a necessary cofactor to induce preterm birth.27 Asdiscussed above, some women have immune defence against G. vaginalis by means ofproducing G. vaginalis-specific IgA, while in others vaginal bacteria may produce sialidaseand cleavage enzymes that attenuate this protective action.28 While all these differencescannot be diagnosed by microscopic appearance alone, other obvious markers – such asthe presence of Mobiluncus – do not seem to have any pathogenic meaning. Mobiluncusare small, vibrating, comma-shaped bacteria that are present in about 15% of BV cases.Although the presence of Mobiluncus establishes an important component of the Nugentscore, it has never been related to any sort of pathology and does not cause any symp-toms that are different from women without this organism.

Atopobium vaginalis and other new discoveries

As most women harbour low numbers of potentially pathogenic bacteria and yeastswithout symptoms, one may question the value of highly sophisticated methods fordetecting low numbers of such microorganisms. Even more strikingly, of some micro-organism often recovered in vaginal fluid, such as the recently discovered Atopobiumvaginalis, it is not clear whether they constitute a pathogenic risk40, or are rathermarkers of abnormal or even normal vaginal flora.36 Also, the presence of lactobacillimay not always have the same beneficial influence on vaginal health. Some lactobacillido not produce the hydrogen peroxide or bacteriocins that contribute to vaginaldefence against overgrowth of pathogens, and can cause rather than prevent disease.Such lactobacilli often have slender morphotypes and are probably of anaerobic origin(vaginal lactobacillosis).41

The ‘intermediate flora’

Intermediate flora on Gram stain

As is generally acknowledged, Nugent score >7 on Gram-stained specimens corre-sponds well with bacterial vaginosis, and is nowadays accepted as the gold standardfor the diagnosis of BV in most clinical trials. Compared to this method, wet mountis said to be less sensitive. However, some constraints have to be taken into consid-eration. First of all, on a continuous scale of 1–10, there is no consensus on whatthe intermediate group with a score of 4–6 stands for. If Nugent were an ideal scoringsystem for bacterial vaginosis, with score 1–3 being normal and �7 being full-blownBV, score 4–6 should be transitional, partial or intermediate BV, but in reality it isnot. Ideally this ‘intermediate flora’ state represents a turning point from a normal


state into BV, or from BV to normal. In reality, however, most of the women with so-called intermediate BV according to Nugent will have neither BV nor a normal flora. Infact, this category represents a ‘garbage can’, even though it may well include impor-tant pathology. In fact, in almost all studies addressing the importance of BV and theintermediate group as a separate category, it was clear that the intermediate groupwas linked to a different and usually more serious range of complications, includingmid-trimester pregnancy loss, than the ‘classic’ full-blown BV.23,34,42

Concordance in difficult slides

In a large international project, many researchers in the field of vaginal infections madethe effort to read BV slides, normal slides and those so-called ‘difficult slides’ in orderto measure the concordance among researchers and to see whether the diagnosis ofBV by use of phase-contrast wet-mount microscopy correlated well with Nugent’sdiagnosis on Gram stain. It was confirmed that wet mount, even after later rehydrationof air-dried samples, was as accurate in the diagnosis of BV as the Gram stain.43 How-ever, at the same time it was clear that most diverse opinions prevailed when the ‘dif-ficult slides’ were studied: some did not read them and discarded the difficult slides asunreadable or non-classifiable, others classified them as partial BV, and others pro-posed classifying some of them in a completely different category: aerobic vaginitis(see below). In another study, the inter-observer concordance of wet-mount readingwas tested by six independent vaginal disease specialists in Europe (Platz-Christensenet al, submitted for publication). An excellent k-index was obtained in the diagnosis ofbacterial vaginosis and lactobacillary grades, reflecting the good inter-observer agree-ment, at least when phase-contrast microscopy was used.

Partial BV

Finally, an intermediate abnormal flora does not respond to treatment as one wouldexpect if it were partial BV. Even full-blown BV (Nugent >7) does not always respondto repetitive courses of metronidazole, leaving some 15% of cases unchanged, suggest-ing that a condition other than BV may be involved in such cases.22 Therefore, in theintermediate group, partial BV as well as other abnormal conditions may be present.‘Partial BV’ is by our definition a transient state between normal flora and full-blownBV, as is illustrated in Figure 2. It is obvious that there is a mixed flora, but the abnor-mal flora are of the anaerobic Gardnerella-morphotype-like microflora. In full-blownBV, granular flora is omnipresent and covers the epithelial cells, which are called‘clue cells (Figure 2b). This condition, which we call ‘partial BV’, should not beconfused with other states of intermediate or abnormal flora such as AV, which isdiscussed below.

So it appears that the most obvious reason for discordance in most studies may notbe the lack of diagnostic power of the microscope but rather the use of different def-initions of what is being studied. If a patient has no lactobacilli but other flora mimick-ing clue cells and an increased vaginal pH, it does not necessarily mean she is sufferingfrom BV. It may also mean that we have been overlooking another condition that hassome similarities with BV but is not at all the same condition. The distinction betweenall these forms of abnormal flora is a major challenge for new treatment studies.


Aerobic vaginitis

Diagnosis of AV is based solely on microscopy, and in that respect it is comparable tothe Nugent’s method on Gram stains to diagnose BV (Table 2). Lactobacillary grades(LBGs, see above) are the basis for a composite score to which the following four vari-ables have been added19: (1) proportional numbers of leukocytes; (2) the presence oftoxic leukocytes; (3) the presence of parabasal epithelial cells; and (4) the type of back-ground flora. Therefore in this classification the immune reaction of the host is alsotaken into account for the diagnosis (Figure 3). Parabasal cells are considered a signof severe epithelial inflammation not usually seen in uncomplicated BV. They are en-countered only in moderate or severe forms of aerobic vaginitis, such as in desquama-tive inflammatory vaginitis.44,45 Background flora was allocated a score of 0 if it wasunremarkable or showed debris and bare nuclei from lysed epithelial cells (cytolysis),a score of 1 if the lactobacillary morphotypes were very coarse or resembled smallbacilli (rather than lactobacilli), and 2 if prominent cocci or chained cocci were visible.Leukocytes were scored according to their proportional number when compared withepitheliocytes; more than ten per epithelial cell is assigned 2 points, while less than tenper epithelial cell but more than ten per high-power field corresponds to 1 point. Add-ing these points together comprises a composite score, the ‘AV’ score. A compositescore of 1–4 represents normal flora, a score of 5–6 moderate AV, and a score above6 (to a maximum of 10) to severe AV. In practice, a score of 8–10 matches the defi-nition of ‘desquamative inflammatory vaginitis’.

The use of this AV criterion enables us to divide the flora in a more detailed and com-prehensive way, avoiding undefined and unclear categories. Bacterial flora is predominantlylactobacillary type (normal) or it is abnormal. If abnormal, the flora can be disturbed by an-aerobic overgrowth (bacterial vaginosis) or by aerobic microorganisms such as E. coli, groupB streptococci, enterococci etc (aerobic vaginitis), or can be a mixture of both (mixed ab-normal flora). Therefore one has to be constantly aware that concomitant infectious con-ditions such as candidiasis, trichomoniasis, bacterial vaginosis or cervicitis may occur.46

Table 2. Criteria for the microscopic diagnosis of aerobic vaginitis (AV) (400x magnification, phase-

contrast microscope).19

AV

score

Lactobacillary

grades (LBG)

Number of

leukocytes

Proportion of

toxic leukocytes

Background

flora

Proportion of

parabasal

epitheliocytes

(PBCs)

0 I and IIa �10/hpf None or

sporadic

Unremarkable

or cytolysis

None or

<1%

1 IIb >10/hpf and

�10/epithelial cell

�50% of

leukocytes

Small coliform

bacilli

�10%

2 III >10/epithelial cell >50% of

leukocytes

Cocci or chains >10%

LBGI, numerous pleiomorphic lactobacilli, no other bacteria; LBGIIa, mixed flora, but predominantly

lactobacilli; LBGIIb, mixed flora, but proportion of lactobacilli severely decreased due to increased num-

ber of other bacteria, LBGIII, lactobacilli severely depressed or absent because of overgrowth of other

bacteria; hpf, high-power field (400 times magnification). A composite AV score of <3 corresponds to

‘no signs of aerobic vaginitis (AV)’, 3–4 to ‘light AV’, 5–6 to moderate AV, and >6 to ‘severe AV’. The

latter group corresponds well to the entity ‘desquamative inflammatory vaginitis’.44,45


Trichomonial vaginitis (TV)

Trichomonas vaginalis is one of the most frequent sexually transmitted pathogensworldwide.47 Cultures in specific medium and PCR have the highest sensitivity forthe diagnosis of Trichomonas (80–85%), but direct microscopy is a very powerfultool for office diagnosis in many cases. An experienced microscopist can easily detectTV with a sensitivity of at least 70% and a specificity of 100%. It is crucial not to delaythe examination as the sensitivity drops by 20% if delayed for only 10 minutes.48,49 Thespecimen should be diluted with physiological solution, and the warming effect of themicroscopic lamp can be beneficial in seeing the jerky motile flagellated parasiteswhich are usually the size of leukocytes. Typically many leukocytes are present,parabasal epithelial cells and cocci may be seen (aerobic vaginitis flora), but the typicalgranular flora of bacterial vaginosis is also frequently present. Invariably the lactobacilliare depressed, and in most cases TV is found in an LBGIII flora. Gram stains and Papsmears have been tested for use in screening, but the latter are only 57% sensitive.50

Also the more modern liquid-based Papanicolaou medium does not offer bettersensitivity (61%).49 During menopause the diagnosis of TV must always be questionedas the rate of false-positive diagnoses is extremely high.

Cytolytic vaginosis

Cytolytic vaginosis is a non-inflammatory condition in which hydrogen peroxidase-producing lactobacilli cause an extreme vaginal acidity (below pH 4), leading to epithe-liolysis.51 Usually the abundant presence of coarse, equal-sized lactobacilli is evident(LBGI), together with bare nuclei, patches of cytoplasmic debris of lysed epithelialcells, paucity of leukocytes and absence of bacterial vaginosis, aerobic vaginitis,Trichomonas and Candida morphotypes (Figure 1b). The condition can typically causea burning sensation and increased vaginal discharge, and is often confused with Candidavaginitis.52 In one study, in 7% of cases with signs and symptoms suggestive of candi-diasis, no Candida was found but cytolytic vaginosis was diagnosed.53

THE FULL PICTURE: HOW TO SCREEN THE VAGINAL FLORA

Appropriate diagnosis and distinction between these infectious conditions is crucial astheir treatments are different; for example, AV does not respond well to metronida-zole, the treatment of choice for Trichomonas vaginitis and BV. In order to get to sucha diagnosis, the use of lactobacillary grades is essential. Lactobacillary grades wererecently refined into 4 grades5 (Figure 1) that corresponded well with many bacterialinfections of the vagina and host response, such as vaginal leukocytosis and expressionof cytokines30, and are hence the cornerstone in the decision of whether or not a bac-terial flora is normal. In the case of an abnormal flora, extra criteria are added todistinguish between BV and AV, and a further scrutiny is required to find other path-ogens such as Trichomonas or Candida.

There is evidence that the determination of LBG may be more reliable on wetmounts than on gram stains.16 In a study using smears of 183 pregnant women, itwas shown that fewer lactobacilli were found more often in Gram-stained specimensthan in fresh wet-mount specimens (RR 2.6; 95%CI 1.7–4.1), and six times more of-ten when the Gram stain was performed in a delayed examination after transportfor 6–12 hours in Amies modified Stuart medium.17 Similarly, in a later study on


non-pregnant women attending a vulvovaginitis clinic, higher lactobacillary grades(more disrupted flora) were diagnosed in Gram-stained specimens: 2.9 timesmore often than in wet mounts (95%CI 2–4, P< 0.0001), a difference even morepronounced after transportation in Stuart medium (RR 4.2, 95%CI 3.3–5.2,P< 0.0001). Even when patients with BV were excluded, Gram-stained specimensperformed less well in the detection of lactobacilli compared to wet mounts, asthe relative risk of having a higher LBG was 3.6 (95%CI 2.5–5.2) in this group, sug-gesting that the presence of clue cells and BV microflora was not responsible for thelack of visualization of lactobacillary morphotypes on Gram staining.16 Furthermore,a better correlation exists between the lactobacillary grades on wet mounts and theconcentration of lactate in vaginal lavage than is the case with Gram-stained find-ings.54 It appears that the technique of Gram staining tends to overemphasize theloss of lactobacilli, leading to the over-diagnosis of the most severe abnormalities

I IIa IIb III

Normal LB

Short/long LB

Leptotrix

AV

AV/BV

BV

Figure 4. General overview of types of flora.

Infectious agents in vaginal flora

G. Donders, 19999

CocciSmall rods(Light/Mod AV)

Partial BVflora

Full AV

Full BV

GBS

Enteric Gr-rods

Mycoplasma

Anaerobics

Mobiluncus

I IIa IIIIIb

Cervicitis

Trichomonas

Candida

Aerobic

Candida

Candida

Candida

AnaerobicAnaerobic

Figure 5. Vaginal infectious disease in different flora types.


of the lactobacillary flora, as compared to wet mounts. As the gradual loss of lacto-bacilli is a common characteristic of both BV and AV, it is logical to use this criterionas a first screening tool in order to enable further diagnostic workout as depicted inFigure 1. Some overlap will exist, as well as mixed infection (Figure 2). If neither Tri-chomonas nor cervicitis is diagnosed, and it is unclear whether the diagnosis of BV orAV should be withheld, vaginal cultures or PCR for aerobic flora (E. coli, enterococci,group B streptococci, Candida), G. vaginalis, T. vaginalis and mycoplasmas should bedone. As abnormal lactobacillary grades are also associated with Chlamydia trachoma-tis, gonorrhoea and syphilis, a screen for sexually transmitted pathogens should alsobe considered.46

If the progressive loss of lactobacilli is seen as one dimension (x axis) and the grad-ual redox potential (aerobic–anaerobic) as the other (y axis), it is readily possible tofit all the different pathogenic conditions of vaginal flora disturbance in Figure 4. Onthe anaerobic side, normal flora will gradually become partial BV, which containssome anaerobic flora but not enough to cause the appearance of clue cells, and fur-ther down the line full-blown BV. On the aerobic side normal flora may be progres-sively altered into moderate AV and further deteriorates to severe AV ordesquamative inflammatory vaginitis (DIV). Trichomonas vaginalis will usually be foundin cases with abnormal lactobacillary flora, but can also be accompanied by anaerobic-dominant or with aerobic-dominant flora, or both (Figure 5). Candida will be morefrequently found in the aerobic rather than the normal lactobacillary flora (lower

Figure 6. Images of phase-contrast microscopy (�400) of vaginal fluid from patients with Candida vaginitis.

(a) Candida in a normal bacterial microflora (Lactobacillary grade I). (b) Candida together with bacterial

vaginosis. (c) Candida in a microflora devoid of lactobacillary morphotypes (lactobacillary grade III) and

coccoid bacteria. This type of AV flora often disappears spontaneously after treatment with antimycotics.


left in the diagram), but it cannot be overemphasized that Candida can also be encoun-tered in all other compartments of the diagram (Figures 5 and 6). On the aerobic side,Candida may often grow together with group B streptococci, a finding in which the path-ogenesis is not entirely clear (unpublished data) but has also been reported by others.55

In such cases the abnormal AV-type flora spontaneously normalizes as a result of erad-ication of Candida by antifungal agents. Hence concomitant administration of antibioticsis not advised and probably even contraindicated. On the other hand, it is as yet unclearwhether the co-colonization by GBS increases the risk of recurrent Candida disease.Hopefully this relationship will be unravelled by ongoing research.

CONCLUSIONS

In conclusion, microscopy of fresh or rehydrated vaginal fluid has lower sensitivity thanthe Gram stain in detecting bacterial vaginosis (according to the definition used) andcandidiasis. Whether this means that the use of office microscopy is less powerful asa diagnostic tool in the diagnostic work-up of vaginal infectious disease, however, isa matter of debate. First of all, the purpose is not to detect asymptomatic carriers(with the possible exception of Trichomonas, because of its transmissibility), but tooffer adequate management to symptomatic women before their disease becomessevere or erroneous self-treatment is started. Treatment of asymptomatic bacterialvaginosis during pregnancy with metronidazole is inefficient56, but the use ofbroader-spectrum antibiotics – such as the combination of erythromycin with metro-nidazole57, or the use of vaginal or oral clinamycin early in pregnancy58–60 – have beenshown to be very effective in the prevention of pregnancy complications such aspreterm delivery and preterm rupture of the membranes. The influence of aerobicvaginitis and intermediate flora in pregnancy has not yet been fully elucidated, butsome studies suggest that it may cause as much harm as full-blown anaerobic vagino-sis.61 Recently, in a magnificent overview, Roberto Romero et al summarized thecombined effects of vaginal microorganisms, host-cell immune responses and geneticpolymorphisms, and concluded that none of them individually, but rather the co-occurrence of all them, was responsible for adverse pregnancy outcome.62 Outsidepregnancy, bacterial vaginosis needs treatment only if symptomatic, or during intra-uterine instrumentation. In these circumstances, bacterial vaginosis will most readilyand efficiently be diagnosed by wet-mount microscopy. When phase-contrast micros-copy is used, inter-observer variability is low, and compared to the Gram stain suc-cessfully diagnoses full-blown BV provided that it is performed by experiencedtherapists. Furthermore, wet-mount microscopy allows the diagnosis of conditionswith abnormal vaginal flora other than BV. AV is one of these conditions and takesinto account the inflammatory reaction of the host, a sign that is typically missing inBV. AV may also be a missing link that explains why the so-called intermediate florain the Nugent score may in some women lead to disastrous complications.

FUTURE RESEARCH

The new insights in the differential diagnosis of all types of abnormal vaginal flora havecreated renewed interest in screening and treating pregnant women in order to pre-vent pregnancy complications such as preterm labour, neonatal infection and intrace-rebral haemorrhage causing cerebral palsy, and chorioamnionitis. As genetic variationsin the expression of cytokines and cytokine action that result from these infections


become clearer (genetic polymorphisms), further research is necessary on whichwomen and what types of vaginal flora compose the highest risk profiles for such com-plications. It will be clear that vulnerable (genetically predisposed) women should bemonitored much more closely and may need repeated or prophylactic treatmentrather than the current unifocal screening and treatment. Also, in pregnancy it shouldbe better established whether treatment enables the flora to normalize, and the indi-cations for repeat treatment.

For many subtypes of abnormal vaginal flora, such as aerobic vaginitis, no evidence-based management has been proposed so far, but this needs to be addressed urgentlyin future studies. Also the epidemiology needs to be elucidated further in order tobetter understand the condition. What is the influence of long-term contraceptivepill use? Is the condition contagious for the partner? Is viral infection of the lactobacilli(bacteriophages) involved? Or should it rather be seen as an autoimmune disorder?These are all questions that so far remained unanswered.

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Practice points

� during pregnancy, an abnormal vaginal flora is an important risk factor for pre-term delivery and neonatal complications, and should be screened for andtreated early in pregnancy� an abnormal vaginal flora is easy to diagnose by detecting a lack of lactobacilli in

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Documents

Definition and classification of abnormal vaginal flora · Definition and classification of abnormal vaginal flora Gilbert G.G. Donders* MD, PhD ... (‘das nativ preparat’),