Comparison of methods for diagnosing bacterial vaginosis among pregnant women

0095-1137/89/061266-06$02.00/0

Copyright C 1989, American Society for Microbiology

Comparison of Methods for Diagnosing Bacterial

Vaginosis

among

Pregnant Women

MARIJANE A. KROHN,1* SHARON L. HILLIER,2 ANDDAVID A. ESCHENBACH2

Departments of Epideiniology' and Obstetrics andGynecology,2 University of Washington, Seattle, Washington 98195

Received 21 December1988/Accepted 2 March 1989

The diagnosis of bacterial vaginosis is usually based on clinical criteria including homogeneous vaginal

discharge,anelevatedvaginalpH, thepresenceofclue cells, andanamine odor. We have evaluated the vaginal

flora and clinical signs for 593 pregnant women. Gardnerella vaginalis, Bacteroides spp., and Mycoplasma

hominiswereisolatedmorefrequentlyamong womenwith clinical signs thanamongthosewithout clinical signs

ofbacterial vaginosisinmultivariableanalyses that controlledfor other bacteria. To determine the laboratory

method that best predicted bacterialvaginosis,wecalculated thesensitivity, specificity,andpredictivevalueof

positiveandnegativetestsfor Gram-stainedvaginalsmears,gas-liquid chromatography of vaginal fluid, and

G.vaginalis cultures comparedwith clinicalsigns.G. vaginalis culturewassensitive(92%)and bothgas-liquid

chromatography (78%) and Gram-stained vaginal smears (62%) were moderately sensitive in identifying

womenwith threeof the four clinical signs of bacterial vaginosis. However, theGram-stained vaginalsmear

(95%)was morespecificthan G. vaginalis culture (69%)orgas-liquid chromatography(81%).Thepredictive

valueofa positive testwasalso higher for the Gram staining (76%)than forG. vaginalisculture(41%) or

gas-liquid chromatography (48%).

Bacterial vaginosis (nonspecific vaginitis) wasrecognized

as a vaginal syndrome over 30 years ago by Gardner and

Duke (9). They associated bacterial vaginosis with the

iso-lation of Haemophilus vaginalis, later briefly named

Corv-nebacteriumr vaginale and currently named Gardnerella

iag-inalis(10). However, the microbiology of bacterial vaginosis

is complex and involves organisms other than G. vaginalis.

G. vaginaliscanbe isolated fromthevaginas of 20to40% of

women without bacterial vaginosis (1, 4, 6), and large

quantities ofnot onlyG. vaginalis but alsoanaerobic

bacte-ria (16, 18) and Mycoplasmna hominis (13, 14) canbe

recov-eredfromwomen with bacterial vaginosis.

The diagnosis of bacterial vaginosis has usually been

based onthree or moreof thefollowing clinical signs of the

vaginal discharge: a thin homogeneous appearance, an

ele-vated pH,anamine odor after the addition of 10% KOH, and

the presence ofclue cells (vaginal epithelial cells studded

withbacteria) (1,6, 9). Four other laboratory methodshave

beenused todiagnosebacterial vaginosis: culture ofvaginal

fluid for G. vaginalis (9), gas-liquid chromatographic analy-sis ofvaginal fluidfor short-chainfattyacids believed tobe

products of anaerobic bacterial growth (16, 18),

Gram-stained vaginal smears read microscopically for bacterial

morphotypes (5, 19), and an assay for proline

aminopepti-dase(21). Each laboratory method is basedon the

assump-tion that thevaginal floraofwomenwithbacterial vaginosis

differs in some quantifiable way from the flora of normal

women. In prior reports, individual laboratory methods of

diagnosing bacterial vaginosis have been compared with

clinical signs (1, 4,6, 11, 18M 21). However, multiple labora-torymethods havenotbeencomparedwithinasinglecohort

ofwomen.

In the present study, we determined the vaginal flora of

patientswithbacterial vaginosis diagnosed by clinical signs,

gas-liquid chromatography, and Gram-stained vaginal

smears to document that eachdiagnostic methodwas

asso-ciated with similarvaginal flora. Thesensitivity, specificity,

* Correspondingauthor.

and predictive value ofpositiveandnegativetestsfor three

laboratory methods (Gram-stained smear, G. vaginalis

cul-ture, and gas-liquid chromatography) were compared with clinical signs fordiagnosing bacterial vaginosis. The

impor-tanceofanaccurate, reproducible, andinexpensive

labora-tory method to diagnose bacterial vaginosis has increased

with the recent association of placental infection (12) and

prematuredelivery (13) with this vaginal syndrome.

MATERIALS ANDMETHODS

Women enrolled into the study were part ofa cohort of

pregnant women attending the University of Washington

prenatal clinics. Patients were enrolled between July 1984

and June 1986 at 23 to 26 weeks ofgestation as part ofa

National Institutes ofHealth-sponsored collaborative

multi-center study concerning vaginal infection in pregnancy. Women were excluded for the following reasons: maternal age less than 16 years; antibiotic use within the previous 2

weeks;twins;cervical cerclage; erythroblastosis;and

hyper-tension, kidney disease, heart disease, or diabetes mellitus

requiring therapy. Of716 women enrolled, Il had

unsatis-factory vaginal specimens, and 112 hadat least onemissing

result, leaving 593 women with complete data who were

used foranalysis.

At the enrollment visit, women had a vaginal speculum

inserted without lubrication. The appearance and pHofthe

vaginal discharge were determined, a vaginal smear for

subsequentGram stainingwasobtained,avaginalsmearfor

saline andpotassium hydroxidewetmountwas made,anda

vaginalwash wastakenforculture andgas-liquid

chromato-graphic analysis as previously described (13). The vaginal

vault was examined for discharge, which was described as

normal(mucoid and floccular), purulent, curdy, orthin and

homogeneous. The vaginal pH was determined by placing

the vaginal discharge on pH paper (Color pHast; MCB

Reagents, Gibbstown, N.J.), which had six comparison colors for pH 4.0 through 7.0. The saline wet mount was

examinedmicroscopicallyfor motile trichomonadsand clue

cells. The 10% potassium hydroxide wet mount was

exam-1266

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TABLE 1. Vaginal flora present in pregnant women with and without bacterial vaginosis diagnosed by three methods inaunivariateanalysis

% of womendiagnosedasfollows:

Organism Three of four clinical signs Gram stain Gas-liquidchromatography"

Vaginosis Novaginosis Vaginosis Novaginosis Vaginosis Novaginosis

(n = 122) (ti = 471) (n = 73) (n =520) (n = 117) (n =307) Facultative bacteria

G.vaginalis 83 43' 97 45"' 71 41'

Lactobacillus spp. 72 94b 59 94' 76 97'

Viridans group streptococci 38 31 51 30' 40 31

GroupB streptococci 12 15 7 16 il 16

Enterococcusspp. il 16 10 16 9 17

Diphtheroids 71 67 74 67 74 71

Coagulase-negative staphylococci 53 63 53 62 56 66

Anaerobicbacteria'

Bacteroidesspp. 62 15' 70 18' 45 14'>

Peptostreptococcus spp. 59 23b 70 25' 45 26'>

Genital mycoplasmas

U. urealyticum 94 73b 97 74"' 84 74'>

M.hominis 65 17b 74 20' 48 16'>

'A totalof169 women were notincluded because thechromatographic results were notinterpretable.

P<0.01.

'Growth inthethirdandfourthstreak zones on an agarplate.

ined for odor (15) (normal, foul, or amine) and

microscopi-cally forhyphae.

Avaginalsmear wasobtained byrolling a swab across the vaginal wall and then onto a glass slide. The Gram stain was counterstained with safranin and evaluated by the method of

Spiegeletal. (19).Briefly,ifLactobacillus spp. morphotypes

werefewer than five peroilimmersion field and if there were

five or more G. vaginalismorphotypes together with five or

more other morphotypes (gram-positive cocci, small

gram-negative rods, curved gram-variable rods, or fusiforms) per oil immersion field, the Gram stain was interpreted as

indicatingbacterial vaginosis. If five or more Lactobacillus

spp.andfewer than five othermorphotypeswerepresent per

ou

immersionfield, the Gram stain was considered normal.

The smears were interpreted by microbiologists without

knowledge of the clinical examination or of the bacterial

isolation.

After the cervix was wiped free of vaginal discharge,

specimens for Chlamydia trachomatis were taken with a

Dacron swabon aplasticshaft andtransportedinchlamydia

medium until it was inoculated onto cycloheximide-treated

McCoy cells (20). Vaginal specimensweretaken with sterile cottonswabs andinoculated into thefollowingmedia: mod-ified Diamond medium for the isolation of Trichomonas

vaginalis (8), Sabouraud agarfor the identification of

Can-didaalbicans, T (2) and M broth and A7B (17) agar for the

isolation of Ureaplasma urealyticum and M. hominis.

Spec-imens forfacultative and anaerobic bacteria were obtained

by instillinga prereduced sterile balanced salt solution (13)

into the vagina; after the vaginal fluid mixed with the

solution, itwas removed witha sterile syringe. The vaginal

fluidwasinjectedintoasterilestoppered Hungate tube filled

with 85% N2, 10% H2, and 5% C02 for transport to the

microbiology laboratory and was inoculated onto

prere-duced medium inananaerobicglove box.Asampleof100,ul

ofvaginal washwas inoculatedonto each agarplate,which

was then streaked for isolation into four zones with

decon-tamination ofthe loop after inoculating each streak zone.

Anaerobic rods and cocci were identified on the basis of

Gram stain, production of volatile and nonvolatile fatty

acids, the API Anident and 20A systems (Analytab

Prod-ucts, Plainview, N.Y.), andbiochemical tests. G. vaginalis

and Lactobacillus spp. were identified by Gramstain,

cata-lase reaction,beta-hemolysis onhumanbilayerTween agar

(22), and typical Gram stain morphology. Beta-hemolytic

streptococci were identified by colony morphology on 5%

sheepblood agar andwereplaced intoLancefield groupsby

the StrepTex (Welicome Diagnostics, Research Triangle

Park, N.C.) typingsystem. Enterococciand viridans group

streptococci were differentiated by growth in a broth

con-taining 6.5% salt and by bile-esculin hydrolysis.

Gas-liquid chromatographic analysis of vaginal fluid for the diagnosis of bacterial vaginosis was performed by the

method of Spiegel et al. (18) with a Varian 3700 gas

chro-matograph fitted with achromasorb column(Supelco, lnc.,

Bellefonte, Pa.). Agaschromatographicpatternwas

consid-eredabnormal and consistent with bacterial vaginosis if the

peak ratio of succinate to lactate was -0.4, if the acetate

peak was -3 mm, or if the proprionate, isobutyrate, or

isovalerate peak was -1 mm inheight.

Univariate relationships were tested for statistical

signifi-cance by chi-square or Fisher exact tests. The odds

ratio,

alsocalled across-product ratio, was used as a measureof

association for cross-classified categorical data (7).

Multi-variable logistic regression analyses were performed to

estimate adjusted relationships (3). Confidence intervals of

95% were reported for the odds ratios from multivariable

analysesand verifiedbylikelihoodratiotestsforsignificance

(3).

RESULTS

The frequency of recovery of microorganisms from

womenwith bacterialvaginosis diagnosed byclinicalcriteria

wascompared withthatofwomenwithout bacterial

vagino-sis by univariate analysis (Table 1). The vaginal floras of

womenwithand without bacterialvaginosiswere compared

by two other methods of diagnosing bacterial

vaginosis:

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TABLE 2. Microorganisms associated with bacterial vaginosis diagnosed by three methodsin alogistic regression analysis' Oddsratio(95% confidence interval) forwomendiagnosedasfollows:

Organism Threeof four Gas-liquid

clinicalsigns

Gram

stain chromatography

G. vaginalis"' 2.5(1.4-4.6) 13.3(3.0-58.9) 2.2(1.3-3.9)

Facultative Lactobacillus spp. 0.4(0.2-0.9) 0.4(0.2-0.7) 0.2 (0.1-0.5)

Viridans group streptococci 0.5(0.3-0.8) 0.9 (0.5-1.7) 0.7 (0.4-1.3)

Bacteriodes spp.b 4.1(2.2-7.6) 4.4(1.7-10.8) 2.6(1.3-5.2)

Peptostreptococcus spp.b 1.8(1.0-3.0) 2.4(1.2-4.7) 0.9(0.5-1.7)

U. urealyticurm 2.5(1.0-6.0) 2.4(0.5-11.6) 1.0(0.5-1.9)

M.hominis 3.7(2.3-6.2) 3.7(1.9-7.2) 2.8(1.6-4.9)

aAdjustedfor C. trachomnatis,T. vaginalis,C.albicans,and eachoftheother tabulated bacterialisolates.

bGrowthinthe third and fourth streak zones on an agarplate.

Gram-stained vaginal smears and gas-liquid

chromatogra-phy. Bacterialisolates that occurred in fewer than 10% of the

women in the cohort (Escherichia coli, Micrococcus spp.,

Moraxella spp.,Actinomyces spp., Eubacteriwn spp.,

Pro-pionibacterium spp., and Mobilhncus spp.) were not

tabu-lated because they occurred at such low frequencies.

Women with C. trachomatis, T. vaginalis, and C. albicans

were not excluded from the univariate analyses. Women

with bacterial vaginosis diagnosed by any of the methods

hadincreased frequencies ofG. vaginalis, Bacteroides spp.,

Peptostreptococcus

spp., U. urealyticurn, and M. hominis

and decreased frequencies of facultative Lactobacillius spp.

compared with women without bacterial vaginosis (for all

comparisons, P < 0.01). Group B streptococci,

Enterococ-cus spp., diphtheroids, and coagulase-negative

staphylo-cocci were not associated with bacterial vaginosis as

diag-nosedbyanyofthe methods. Bacterial morphotypessuchas

G. vaginalisandLactobacillusspp. wereidentifiedaspartof

theGram stain interpretation, so it would be expected that

these species would be associated with the diagnosis of

bacterial vaginosis made by Gram stain. TherecoveryofG.

vaginalis washigher and therecovery ofLactobacillus spp.

wasloweramongwomenwho hadthe diagnosis of bacterial

vaginosis made by Gram stain than by other methods.

However, the presence of G. i'aginalis and Lactobacillus

spp. was similar among women without bacterial vaginosis

identified by all three methods. Although the frequencies of theother microorganisms varied slightly for the three

diag-nostic methods, a similarflora wasstatistically significantly

associated with each of the threediagnostic methods.

Logistic régression analysis was used to determine

whether bacterial isolatesassociated with bacterialvaginosis

by univariate analyses would still be associated after

adjust-mentfor(i) coinfections suchasC. trachomatis, T.

vagina-lis, or C. albicans or (ii) the other bacterial isolates also

associated with bacterial vaginosis. G. vaginalis,

Bacteroi-des spp., and M. honinis remained positively associated

with bacterial vaginosis, and Lactobacillus spp. remained

negatively associated with bacterial vaginosis after

adjust-ments in the multivariable analysis (Table 2). These four

bacterial isolates were interpreted to be independently

re-latedtobacterial vaginosis.Peptostreptococcusspp. and U.

urealyticumwere nolonger statistically significantly related

tobacterial vaginosis aftertheseadjustments. ExceptforG.

v'aginalis,thesemicroorganisms hadsimilar relative risksfor

bacterial vaginosis identified either bythree of fourclinical

signs or Gram-stained vaginal smears. G. vaginalis was

strongly independently related to bacterial vaginosis

diag-nosed byGram-stained smears because itspresence is part

of the Gram staininterpretation.Ingeneral,the relativerisks

of the microorganisms werelowerwhen bacterial vaginosis

was diagnosed by gas-liquid chromatographic criteria than

whenitwas diagnosed bythe othertwomethods.

Bacterial vaginosis wasdiagnosed by findingthree offour clinical criteria in 122 (21%) of 593 pregnant women. In

comparison, bacterial vaginosis was diagnosed by

Gram-stained smears in 12%, by gas-liquid chromatography in

28%, and by G. vaginalis culture (third and fourth streak

zones on anagarplate)in41% of thewomen.Thefrequency of each individual clinical sign was compared for the three

TABLE 3. Frequency of clinical signs of bacterial vaginosisamong pregnant womenidentified with bacterial vaginosisby Gram-stained vaginalsmear. gas-liquidchromatography,and G. i'aginalisculture"

1%rof womendiagnosedasfollows:

Clinicalsign

Grarm-stained

smear Gas-liquidchromatography G.î'aginalis

culture"'

Vaginosis Novaginosis Vaginosis Novaginosis Vaginosis Novaginosis

(n =73) (n = 520) (n = 117) (n=307) (n= 243) (n =350)

Homogeneous discharge 71 24 51 21 44 20

pH - 4.7 84 21"' 55 15' 50 14"

Amine odor after KOH 74 22' 50 22 47 16'

Clue cellson wetmount 80 24"' 54 22' 48 18"

Three of theabove four clinical signs 77 13 47 9 40 7

`Statisticalsignificanceindicates that theindividualclinicalsignwasindependentlyrelated to bacterialvaginosis by logisticregressionanalysisafteradjusting

forthe othersigns.

"Growthin the third and fourth streak zones only.

A totalof 169womenwere not includedbecausethechronatographyresultswereuninterpretable. <'P<0.001.

P< 0.01. JP<0.05.

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TABLE 4. Correlation of thediagnosisofbacterial vaginosis madeby laboratorytests with clinical criteria for diagnosisofbacterialvaginosis"

Clinical criteria for diagnosis of bacterialvaginosis, no./total(%) Method

Sensitivity Specificity _predictivePositive Negative value predictivevalue

Gram-stained smear 38/61 (62) 249/261 (95) 38/50 (76) 249/272 (92)

Gas-liquidchromatography 31/40 (78) 149/183 (81) 31/65 (48) 149/158(94)

Single microorganismS identifiedby culture

G.vaginalisb 56/61 (92) 179/261 (69) 56/138 (41) 179/184(97)

M.hominis 42/61(69) 222/261(85) 42/81(52) 222/241 (92)

Bacteroidesb 40/61(66) 227/261(87) 40/74(54) 227/248(92)

Peptostreptococcusb 39/61(64) 199/261 (76) 39/101 (39) 199/221(90)

aWomen with T.vaginalisor C. albicans by culture, Gram stain, or wet mountwere excluded. For gas-liquidchromatography, n = 201; for all other variables,

n =271.

bGrowth inthethird and fourth streak zones on an agar plate.

laboratory methods of identifying bacterial vaginosis (Table 3). Each of the four clinical signs was present among 71 to

84% ofpatients with bacterial vaginosis and 21 to 24% of those without bacterial vaginosis diagnosed by Gram stain (P <

10-'

for each determination). Individual clinical signs

wereless frequent (44 to 55%) when bacterial vaginosis was

diagnosed by gas-liquid chromatography or G. vaginalis culturethan whendiagnosis was made by Gram stain. Based

uponthepresenceofthreeof fourclinical criteria, bacterial

vaginosis was present among 77% of women diagnosed by

Gram-stained smear compared with only 47% of women

diagnosedby gas-liquid chromatography and40%of women

diagnosed by G. vaginalis culture.

Logisticregression analysis was performed to determine

which of the individual clinical signs was independently

related to bacterialvaginosis afteradjustment forthe other

clinical signs. ApH of-4.7 and clue cells present on wet

mount were independently related to bacterial vaginosis

identified by any ofthe three methods in the multivariable analysis (Table 3). Homogeneous discharge was not inde-pendently related to bacterial vaginosis as determined by

any ofthe three methods after adjustment for pH, amine

color,and cluecells.Amineodorafteraddition ofpotassium hydroxide was independentlyrelated to bacterial vaginosis diagnosed by Gram-stainedsmear or G. vaginalisculture. A

pH of

.4.7,

amine odor, and clue cells provided unique information that independently contributed to the clinical

diagnosis ofbacterial vaginosis.

Becausecoinfectionwith T.vaginalisor C. albicans could

influence the clinical signs of bacterial vaginosis, only

women negative forthesemicroorganisms by culture, Gram

stain,or wet mount wereusedto evaluate thesensitivity and

specificityof Gramstain, gas-liquid chromatography,and G.

vaginalis

culture in comparison with clinical criteria for

diagnosing bacterial vaginosis (Table 4). Women with C.

trachomatisor Neisseriagonorrhoeaeoccurredinfrequently (3 and 0.5%, respectively) and occurred rarely without T.

vaginalis

or C. albicans. G.

vaginalis

culture was more

sensitive(92%)thaneithergas-liquid chromatography(78%)

or Gram stain (62%) in predicting the clinical diagnosis of

bacterial vaginosis. However, the Gram-stained vaginal

smear wasmore specific for diagnosis (95%) thangas-liquid

chromatography (81%) or G.

vaginalis

culture (69%). The

Gram-stained vaginal smearhad ahigher

predictive

valueof

a positive test(76%) than gas-liquid chromatography (48%)

or G.

vaginalis

culture(41%). All threediagnostic methods

hadhigh

predictive

values ofa negative test. In summary,

for predicting the clinical criteria of bacterial vaginosis, the

Gram-stained vaginal smear had a higher specificity and

predictive value ofapositive testcompared withgas-liquid chromatography and G. vaginalis culture. The Gram stain was only moderately sensitive in diagnosing women with the

clinical criteria for bacterialvaginosis.

To determine whether vaginal bacteria other than G.

vaginalishave greaterspecificity for the diagnosis of

bacte-rial vaginosis, further analyses were performed for single

microorganisms associated with bacterial vaginosis by univariate analysis in this report (M. hominis, Bacteroides

spp., and Peptostreptococcus spp.). The sensitivity ofM.

hominis (69%), Bacteroides spp. (66%), and

Peptostrep-tococcus spp. (64%)inpredictingwomen with threeoffour

clinical signswas lower than the

sensitivity

of G. vaginalis (92%), whereas the specificity ofdiagnosing bacterial vagi-nosis was higher for the isolation of any of these three

microorganisms (76% to 87%) than for G. vaginalis (69%) (Table 4). Thepredictivevalueofapositiveornegativetest

based on the isolation of these single microorganisms was

similar to that with G. vaginalis isolation (Table4).

DISCUSSION

The first goal of this report was to describe the vaginal flora associated with bacterial vaginosis both by clinical

signs and laboratory methods. Three microorganisms

con-sistently occurredmorefrequentlyamong women with

bac-terialvaginosisinmultivariableanalyses: G. vaginalis,

Bac-teroidesspp., and M. hominis. Facultative lactobacilli were

isolated consistently less often from women with bacterial

vaginosis diagnosed by any method.Peptostreptococciwere

isolated morefrequentlyfrom women with bacterial

vagino'

sis than from women without bacterial vaginosis identified

by clinical signs or Gram-stained smears but not

by

gas-liquid chromatography. The consistency of vaginal flora in

bacterial vaginosis identified by differentmethods supports

theconclusionthatthedifferentdiagnostic methodsidentify

a similar microbiologic condition.

In other reports that identified bacterial vaginosis by

gas-liquid chromatography (18) or by clinical signs (16), it

was concluded that G. vaginalis, Bacteroides spp., and

Peptostreptococcus spp. were morefrequently isolated and

Lactobacillusspp. werelessfrequentlyisolated fromwomen

with bacterialvaginosis comparedwith womenwithout it. G.

vaginalis,

black-pigmented Bacteroides spp., and M.

homi-niswerealsopositively associated and facultative

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cillus was negatively associated with bacterial vaginosis among pregnant women as determined by multivariable

analyses(13).The presentreport confirmedtherelationship of Bacteroides spp., G. vaginalis, and M. hoministo bacte-rial vaginosis after adjustment for coinfections and

con-firmed the decreased frequency of Lactobacillus spp.,

among women with bacterial vaginosis. Another report studying nonpregnant women from a sexually transmitted

diseases (STD) clinic related Mobiluncus spp. to bacterial

vaginosis(11).Thepresentreportfailedtoverifythis finding

because the culture methods were not adequate for the optimal detection of Mobiluncus spp., and only 3% of the

specimens yielded this microorganisminthethirdandfourth streak zones. However, whenmore sensitive methodswere

performed for 120 of the women included in this study,

Mobiluncusspp.wererecoveredfrom 10of23(43%)women

with bacterial vaginosis compared to 4 of 97 (4%) women

withoutbacterialvaginosisidentified byGram stain(datanot shown). In addition, curved rods resembling Mobiluncus

spp. weredetected by Gram stain in 34of122(28%)women

withbacterial vaginosisidentified byclinical signsand in 14 of 471 (3%) women without clinical signs of bacterial

vagi-nosis. Thus the lower frequency of Mobiluncus spp.

de-tected by culture reflects the difficulty in recovering these

fastidious microorganisms from routine genital cultures.

The second goal of this report was to examinethe sensi-tivity, specificity, and predictive value of a positive or

negative laboratorytestincomparisonwithclinicalsignsfor the diagnosis of bacterial vaginosis. We first examined the

relationship of individual clinical signstothediagnosismade byGram-stained vaginal smear,gas-liquid chromatography,

and G. vaginalis culture. Individual clinical signs and the

presenceof three of the fourclinicalsignswere moreclosely

relatedtothe diagnosis ofbacterial vaginosis byGram stain than by either gas-liquid chromatography or G. vaginalis culture. The close association between clinical signs and

Gram stain diagnosis ofbacterial vaginosis has been noted

previously among women attending an STD clinic (6).

Pa-tients with bacterial vaginosis diagnosed in the STD clinic

population andthepregnantwomeninthisreporthad similar

frequencies of individual clinical signs, although among

those without bacterial vaginosis the STD clinic population had lower frequencies of homogeneous discharge, amine

odor, and clue cells andhigher frequencies of pH .4.7 than

did the pregnant women. These differences may be due to

pregnancy ortheinclusion ofwomenwith T. vaginalis in the

analysis done in Table 3 of this report. Amongnonpregnant

women, Bump et al. (4) found frequencies of each clinical sign among women with or without bacterial vaginosis that

werelower than the frequencies found in this report.

The isolation ofG. vaginalis wasexaminedas apredictor

ofbacterial vaginosis defined by clinical signs; it had

excel-lent sensitivity but low specificity and a poor positive

predictive value. These results are not surprising in view of

the high frequency of recovery of G. vaginalis among

women without clinical signs of bacterial vaginosis in this

and other reports (1, 6, 16, 22). None of the other single microorganisms associated with bacterial vaginosis (Bac-teroidesspp.,Peptostreptococcus spp.,and M. hoininis)had agood positive predictive value foridentifying patients with

clinical signs of bacterial vaginosis. The isolation of onlyone

microorganism does not reliably predict women with

bacte-rial vaginosis. Thus, the value of vaginal culturesforany of

these microorganisms is doubtful for the identification of

bacterial vaginosis in women.

In thisreport, gas-liquid chromatography was 78%

sensi-tive and 81% specific for diagnosing women with clinical

signs of bacterial vaginosis. Others have reported higher

specificities (90 to98%)(1, 4,16, 18, 21)andbothhigher (1,

18) and lower (4, 21) sensitivities. These differences may

result from differences ingas-liquid chromatographic

tech-niques,differences in the clinicalsignsusedtodefine

bacte-rialvaginosis, and differences in thepopulationorthestudy

designs. Reports with cohort designs (4, 18, 21), including

the present report, have, in general, obtained lower

sensi-tivities than reports with a case-control design (1). Women

identified as having bacterial vaginosis among a cohort of

women may have a wider spectrum of disease that is less

closely associatedwithgas-liquid

chromatographic changes

thanamong womenidentified in acase-control study.

The Gram-stained smear had moderate sensitivity and

predictive value ofa positivetest with excellent specificity

andpredictivevalueofanegativetest. Thepositive

predic-tive valueof the Gram stain method was

76%,

representing

the highest predictive value of a

positive

test among the

methods evaluated. In a small case-control

series,

Gram-stainedvaginalsmears were more

closely

relatedtobacterial

vaginosisasidentifiedby clinicalsignsthanwasfoundin this

report (19). The cohort study design used in this report

involvedexaminations performedby severalclinicians, and interobserver differences may have led to less controlled

results than exist in a case-control study performed

by

a

single clinician.

Inthis report, pregnant women were evaluated to

deter-mine thevaginal flora associated with bacterial

vaginosis

and

the laboratory test that best identified women with the

clinicalsignsof bacterial

vaginosis.

Manycomponents of the

findingsinthis report have been studied among nonpregnant

women in other reports with similar results. Otherreports

based on theevaluation of nonpregnant women have

asso-ciated bacterial

vaginosis

with an increased

frequency

of

isolating G. vaginalis, Bacteroides spp., and

Peptostrep-tococcusspp. andadecreased

frequency

of

isolating

Lacto-bacillus spp. (16, 18). The close association of

identifying

bacterial vaginosis by Gram stain smear with the clinical

signs ofbacterial

vaginosis

hasbeen noted among

nonpreg-nant women (16). The high frequency of

isolating

G.

vagi-nalis from nonpregnant women without bacterial

vaginosis

has beenreported(1,6, 16).Thussubstantialevidence exists

in this and other studies that the

vaginal

Gram stain is

superior to vaginal cultures for the

diagnosis

of bacterial

vaginosisin both pregnant and nonpregnant women.

Inaddition tothe scientificconsiderations in

choosing

an

accurate laboratory method of

diagnosing

bacterial

vagino-sis, there are considerations of

complexity,

cost, and the

frequency of

uninterpretable specimens.

Vaginal

cultures andgas-liquidchromatographymethodsto

identify

bacterial vaginosis require skilled personnel and costly

equipment.

Up to30% of

specimens analyzed by

gas-liquid

chromatog-raphy may yield results that are not

interpretable.

Further,

thesemethods hadapoorpredictivevalueofa

positive

test.

Thesefindingssupport the conclusion that theGram-stained

vaginal smear identifies women with the clinical

signs

of

bacterial vaginosisfroma cohortofwomen betterthan other

laboratorytests. Combined with its high

frequency

of

inter-pretable results, low cost, ease of transport, and ease of

storage, the Gram-stained smear provides a

good

screening

test for bacterial vaginosis. In our

experience,

both

clini-cians and clinical

microbiologists

are able to

accurately

interpret the Gram smears after a short

period

of

training.

These findings also support the conclusion that the

interpre-tation of the Gram-stained smear can

identify

women with

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the same group ofvaginal microorganisms as identified by clinical criteria.

ACKNOWLEDGMENTS

This work was supported by Public Health Service contract HD3-2832 and grantAI12192from the National Institutes of Health.

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