JOURNAL OF CLINICAL MICROBIOLOGY, June 1989, p. 1266-12710095-1137/89/061266-06$02.00/0Copyright C 1989, American Society for Microbiology

Comparison of Methods for Diagnosing BacterialVaginosis among Pregnant Women

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

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

Received 21 December 1988/Accepted 2 March 1989

The diagnosis of bacterial vaginosis is usually based on clinical criteria including homogeneous vaginaldischarge, an elevated vaginal pH, the presence of clue cells, and an amine odor. We have evaluated the vaginalflora and clinical signs for 593 pregnant women. Gardnerella vaginalis, Bacteroides spp., and Mycoplasmahominis were isolated more frequently among women with clinical signs than among those without clinical signsof bacterial vaginosis in multivariable analyses that controlled for other bacteria. To determine the laboratorymethod that best predicted bacterial vaginosis, we calculated the sensitivity, specificity, and predictive value ofpositive and negative tests for Gram-stained vaginal smears, gas-liquid chromatography of vaginal fluid, andG. vaginalis cultures compared with clinical signs. G. vaginalis culture was sensitive (92%) and both gas-liquidchromatography (78%) and Gram-stained vaginal smears (62%) were moderately sensitive in identifyingwomen with three of the four clinical signs of bacterial vaginosis. However, the Gram-stained vaginal smear

(95%) was more specific than G. vaginalis culture (69%) or gas-liquid chromatography (81%). The predictivevalue of a positive test was also higher for the Gram staining (76%) than for G. vaginalis culture (41%) or

gas-liquid chromatography (48%).

Bacterial vaginosis (nonspecific vaginitis) was recognizedas a vaginal syndrome over 30 years ago by Gardner andDuke (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 vaginosisis complex and involves organisms other than G. vaginalis.G. vaginalis can be isolated from the vaginas of 20 to 40% ofwomen without bacterial vaginosis (1, 4, 6), and largequantities of not only G. vaginalis but also anaerobic bacte-ria (16, 18) and Mycoplasmna hominis (13, 14) can be recov-ered from women with bacterial vaginosis.The diagnosis of bacterial vaginosis has usually been

based on three or more of the following clinical signs of thevaginal discharge: a thin homogeneous appearance, an ele-vated pH, an amine odor after the addition of 10% KOH, andthe presence of clue cells (vaginal epithelial cells studdedwith bacteria) (1, 6, 9). Four other laboratory methods havebeen used to diagnose bacterial vaginosis: culture of vaginalfluid for G. vaginalis (9), gas-liquid chromatographic analy-sis of vaginal fluid for short-chain fatty acids believed to beproducts of anaerobic bacterial growth (16, 18), Gram-stained vaginal smears read microscopically for bacterialmorphotypes (5, 19), and an assay for proline aminopepti-dase (21). Each laboratory method is based on the assump-tion that the vaginal flora of women with bacterial vaginosisdiffers in some quantifiable way from the flora of normalwomen. In prior reports, individual laboratory methods ofdiagnosing bacterial vaginosis have been compared withclinical signs (1, 4, 6, 11, 18M 21). However, multiple labora-tory methods have not been compared within a single cohortof women.

In the present study, we determined the vaginal flora ofpatients with bacterial vaginosis diagnosed by clinical signs,gas-liquid chromatography, and Gram-stained vaginalsmears to document that each diagnostic method was asso-ciated with similar vaginal flora. The sensitivity, specificity,

* Corresponding author.

and predictive value of positive and negative tests for threelaboratory methods (Gram-stained smear, G. vaginalis cul-ture, and gas-liquid chromatography) were compared withclinical signs for diagnosing bacterial vaginosis. The impor-tance of an accurate, reproducible, and inexpensive labora-tory method to diagnose bacterial vaginosis has increasedwith the recent association of placental infection (12) andpremature delivery (13) with this vaginal syndrome.

MATERIALS AND METHODSWomen enrolled into the study were part of a cohort of

pregnant women attending the University of Washingtonprenatal clinics. Patients were enrolled between July 1984and June 1986 at 23 to 26 weeks of gestation as part of a

National Institutes of Health-sponsored collaborative multi-center study concerning vaginal infection in pregnancy.Women were excluded for the following reasons: maternalage less than 16 years; antibiotic use within the previous 2weeks; twins; cervical cerclage; erythroblastosis; and hyper-tension, kidney disease, heart disease, or diabetes mellitusrequiring therapy. Of 716 women enrolled, Il had unsatis-factory vaginal specimens, and 112 had at least one missingresult, leaving 593 women with complete data who were

used for analysis.At the enrollment visit, women had a vaginal speculum

inserted without lubrication. The appearance and pH of thevaginal discharge were determined, a vaginal smear forsubsequent Gram staining was obtained, a vaginal smear forsaline and potassium hydroxide wet mount was made, and a

vaginal wash was taken for culture and gas-liquid chromato-graphic analysis as previously described (13). The vaginalvault was examined for discharge, which was described as

normal (mucoid and floccular), purulent, curdy, or thin andhomogeneous. The vaginal pH was determined by placingthe vaginal discharge on pH paper (Color pHast; MCBReagents, Gibbstown, N.J.), which had six comparisoncolors for pH 4.0 through 7.0. The saline wet mount was

examined microscopically for motile trichomonads and cluecells. The 10% potassium hydroxide wet mount was exam-

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TABLE 1. Vaginal flora present in pregnant women with and without bacterial vaginosis diagnosedby three methods in a univariate analysis

% of women diagnosed as follows:

Organism Three of four clinical signs Gram stain Gas-liquid chromatography"Vaginosis No vaginosis Vaginosis No vaginosis Vaginosis No vaginosis(n = 122) (ti = 471) (n = 73) (n = 520) (n = 117) (n = 307)

Facultative bacteriaG. vaginalis 83 43' 97 45"' 71 41'Lactobacillus spp. 72 94b 59 94' 76 97'Viridans group streptococci 38 31 51 30' 40 31Group B streptococci 12 15 7 16 il 16Enterococcus spp. il 16 10 16 9 17Diphtheroids 71 67 74 67 74 71Coagulase-negative staphylococci 53 63 53 62 56 66

Anaerobic bacteria'Bacteroides spp. 62 15' 70 18' 45 14'>Peptostreptococcus spp. 59 23b 70 25' 45 26'>

Genital mycoplasmasU. urealyticum 94 73b 97 74"' 84 74'>M. hominis 65 17b 74 20' 48 16'>

'A total of 169 women were not included because the chromatographic results were not interpretable.P < 0.01.

'Growth in the third and fourth streak zones on an agar plate.

ined for odor (15) (normal, foul, or amine) and microscopi-cally for hyphae.A vaginal smear was obtained by rolling a swab across the

vaginal wall and then onto a glass slide. The Gram stain wascounterstained with safranin and evaluated by the method ofSpiegel et al. (19). Briefly, if Lactobacillus spp. morphotypeswere fewer than five per oil immersion field and if there werefive or more G. vaginalis morphotypes together with five ormore other morphotypes (gram-positive cocci, small gram-negative rods, curved gram-variable rods, or fusiforms) peroil immersion field, the Gram stain was interpreted asindicating bacterial vaginosis. If five or more Lactobacillusspp. and fewer than five other morphotypes were present perou immersion field, the Gram stain was considered normal.The smears were interpreted by microbiologists withoutknowledge of the clinical examination or of the bacterialisolation.

After the cervix was wiped free of vaginal discharge,specimens for Chlamydia trachomatis were taken with aDacron swab on a plastic shaft and transported in chlamydiamedium until it was inoculated onto cycloheximide-treatedMcCoy cells (20). Vaginal specimens were taken with sterilecotton swabs and inoculated into the following media: mod-ified Diamond medium for the isolation of Trichomonasvaginalis (8), Sabouraud agar for the identification of Can-dida albicans, T (2) and M broth and A7B (17) agar for theisolation of Ureaplasma urealyticum and M. hominis. Spec-imens for facultative and anaerobic bacteria were obtainedby instilling a prereduced sterile balanced salt solution (13)into the vagina; after the vaginal fluid mixed with thesolution, it was removed with a sterile syringe. The vaginalfluid was injected into a sterile stoppered Hungate tube filledwith 85% N2, 10% H2, and 5% C02 for transport to themicrobiology laboratory and was inoculated onto prere-duced medium in an anaerobic glove box. A sample of 100 ,ulof vaginal wash was inoculated onto each agar plate, whichwas then streaked for isolation into four zones with decon-tamination of the loop after inoculating each streak zone.Anaerobic rods and cocci were identified on the basis of

Gram stain, production of volatile and nonvolatile fattyacids, the API Anident and 20A systems (Analytab Prod-ucts, Plainview, N.Y.), and biochemical tests. G. vaginalisand Lactobacillus spp. were identified by Gram stain, cata-lase reaction, beta-hemolysis on human bilayer Tween agar(22), and typical Gram stain morphology. Beta-hemolyticstreptococci were identified by colony morphology on 5%sheep blood agar and were placed into Lancefield groups bythe StrepTex (Welicome Diagnostics, Research TrianglePark, N.C.) typing system. Enterococci and viridans groupstreptococci were differentiated by growth in a broth con-taining 6.5% salt and by bile-esculin hydrolysis.

Gas-liquid chromatographic analysis of vaginal fluid forthe diagnosis of bacterial vaginosis was performed by themethod of Spiegel et al. (18) with a Varian 3700 gas chro-matograph fitted with a chromasorb column (Supelco, lnc.,Bellefonte, Pa.). A gas chromatographic pattern was consid-ered abnormal and consistent with bacterial vaginosis if thepeak ratio of succinate to lactate was -0.4, if the acetatepeak was -3 mm, or if the proprionate, isobutyrate, orisovalerate peak was -1 mm in height.

Univariate relationships were tested for statistical signifi-cance by chi-square or Fisher exact tests. The odds ratio,also called a cross-product ratio, was used as a measure ofassociation for cross-classified categorical data (7). Multi-variable logistic regression analyses were performed toestimate adjusted relationships (3). Confidence intervals of95% were reported for the odds ratios from multivariableanalyses and verified by likelihood ratio tests for significance(3).

RESULTS

The frequency of recovery of microorganisms fromwomen with bacterial vaginosis diagnosed by clinical criteriawas compared with that of women without bacterial vagino-sis by univariate analysis (Table 1). The vaginal floras ofwomen with and without bacterial vaginosis were comparedby two other methods of diagnosing bacterial vaginosis:

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TABLE 2. Microorganisms associated with bacterial vaginosis diagnosed by three methods in a logistic regression analysis'

Odds ratio (95% confidence interval) for women diagnosed as follows:Organism Three of four Gas-liquid

clinical signs 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)

a Adjusted for C. trachomnatis, T. vaginalis, C. albicans, and each of the other tabulated bacterial isolates.b Growth in the third and fourth streak zones on an agar plate.

Gram-stained vaginal smears and gas-liquid chromatogra-phy. Bacterial isolates that occurred in fewer than 10% of thewomen 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. albicanswere not excluded from the univariate analyses. Womenwith bacterial vaginosis diagnosed by any of the methodshad increased frequencies of G. vaginalis, Bacteroides spp.,

Peptostreptococcus spp., U. urealyticurn, and M. hominisand decreased frequencies of facultative Lactobacillius spp.compared with women without bacterial vaginosis (for allcomparisons, P < 0.01). Group B streptococci, Enterococ-cus spp., diphtheroids, and coagulase-negative staphylo-cocci were not associated with bacterial vaginosis as diag-nosed by any of the methods. Bacterial morphotypes such asG. vaginalis and Lactobacillus spp. were identified as part ofthe Gram stain interpretation, so it would be expected thatthese species would be associated with the diagnosis ofbacterial vaginosis made by Gram stain. The recovery of G.vaginalis was higher and the recovery of Lactobacillus spp.was lower among women who had the diagnosis of bacterialvaginosis made by Gram stain than by other methods.However, the presence of G. i'aginalis and Lactobacillusspp. was similar among women without bacterial vaginosisidentified by all three methods. Although the frequencies ofthe other microorganisms varied slightly for the three diag-nostic methods, a similar flora was statistically significantlyassociated with each of the three diagnostic methods.

Logistic régression analysis was used to determinewhether bacterial isolates associated with bacterial vaginosisby univariate analyses would still be associated after adjust-ment for (i) coinfections such as C. trachomatis, T. vagina-lis, or C. albicans or (ii) the other bacterial isolates alsoassociated with bacterial vaginosis. G. vaginalis, Bacteroi-des spp., and M. honinis remained positively associatedwith bacterial vaginosis, and Lactobacillus spp. remainednegatively associated with bacterial vaginosis after adjust-ments in the multivariable analysis (Table 2). These fourbacterial isolates were interpreted to be independently re-lated to bacterial vaginosis. Peptostreptococcus spp. and U.

urealyticum were no longer statistically significantly relatedto bacterial vaginosis after these adjustments. Except for G.

v'aginalis, these microorganisms had similar relative risks forbacterial vaginosis identified either by three of four clinicalsigns or Gram-stained vaginal smears. G. vaginalis wasstrongly independently related to bacterial vaginosis diag-nosed by Gram-stained smears because its presence is partof the Gram stain interpretation. In general, the relative risksof the microorganisms were lower when bacterial vaginosiswas diagnosed by gas-liquid chromatographic criteria thanwhen it was diagnosed by the other two methods.

Bacterial vaginosis was diagnosed by finding three of fourclinical criteria in 122 (21%) of 593 pregnant women. Incomparison, bacterial vaginosis was diagnosed by Gram-stained smears in 12%, by gas-liquid chromatography in28%, and by G. vaginalis culture (third and fourth streakzones on an agar plate) in 41% of the women. The frequencyof each individual clinical sign was compared for the three

TABLE 3. Frequency of clinical signs of bacterial vaginosis among pregnant women identified with bacterial vaginosis by Gram-stainedvaginal smear. gas-liquid chromatography, and G. i'aginalis culture"

1%r of women diagnosed as follows:

Clinical sign Grarm-stained smear Gas-liquid chromatography G. î'aginalis culture"'Vaginosis No vaginosis Vaginosis No vaginosis Vaginosis No vaginosis(n = 73) (n = 520) (n = 117) (n = 307) (n = 243) (n = 350)

Homogeneous discharge 71 24 51 21 44 20pH - 4.7 84 21"' 55 15' 50 14"Amine odor after KOH 74 22' 50 22 47 16'Clue cells on wet mount 80 24"' 54 22' 48 18"Three of the above four clinical signs 77 13 47 9 40 7

` Statistical significance indicates that the individual clinical sign was independently related to bacterial vaginosis by logistic regression analysis after adjustingfor the other signs."Growth in the third and fourth streak zones only.A total of 169 women were not included because the chronatography results were uninterpretable.

<'P < 0.001.P < 0.01.

J P < 0.05.

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DIAGNOSIS OF BACTERIAL VAGINOSIS 1269

TABLE 4. Correlation of the diagnosis of bacterial vaginosis made by laboratory tests with clinical criteria fordiagnosis of bacterial vaginosis"

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

Sensitivity Specificity Positive Negativepredictive value predictive value

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

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

Single microorganismS identified by cultureG. 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)a Women with T. vaginalis or C. albicans by culture, Gram stain, or wet mount were excluded. For gas-liquid chromatography, n = 201; for all other variables,

n = 271.b Growth in the third and fourth streak zones on an agar plate.

laboratory methods of identifying bacterial vaginosis (Table3). Each of the four clinical signs was present among 71 to84% of patients with bacterial vaginosis and 21 to 24% ofthose without bacterial vaginosis diagnosed by Gram stain (P< 10-' for each determination). Individual clinical signswere less frequent (44 to 55%) when bacterial vaginosis wasdiagnosed by gas-liquid chromatography or G. vaginalisculture than when diagnosis was made by Gram stain. Basedupon the presence of three of four clinical criteria, bacterialvaginosis was present among 77% of women diagnosed byGram-stained smear compared with only 47% of womendiagnosed by gas-liquid chromatography and 40% of womendiagnosed by G. vaginalis culture.

Logistic regression analysis was performed to determinewhich of the individual clinical signs was independentlyrelated to bacterial vaginosis after adjustment for the otherclinical signs. A pH of -4.7 and clue cells present on wetmount were independently related to bacterial vaginosisidentified by any of the three methods in the multivariableanalysis (Table 3). Homogeneous discharge was not inde-pendently related to bacterial vaginosis as determined byany of the three methods after adjustment for pH, aminecolor, and clue cells. Amine odor after addition of potassiumhydroxide was independently related to bacterial vaginosisdiagnosed by Gram-stained smear or G. vaginalis culture. ApH of .4.7, amine odor, and clue cells provided uniqueinformation that independently contributed to the clinicaldiagnosis of bacterial vaginosis.Because coinfection with T. vaginalis or C. albicans could

influence the clinical signs of bacterial vaginosis, onlywomen negative for these microorganisms by culture, Gramstain, or wet mount were used to evaluate the sensitivity andspecificity of Gram stain, gas-liquid chromatography, and G.vaginalis culture in comparison with clinical criteria fordiagnosing bacterial vaginosis (Table 4). Women with C.trachomatis or Neisseria gonorrhoeae occurred infrequently(3 and 0.5%, respectively) and occurred rarely without T.vaginalis or C. albicans. G. vaginalis culture was moresensitive (92%) than either gas-liquid chromatography (78%)or Gram stain (62%) in predicting the clinical diagnosis ofbacterial vaginosis. However, the Gram-stained vaginalsmear was more specific for diagnosis (95%) than gas-liquidchromatography (81%) or G. vaginalis culture (69%). TheGram-stained vaginal smear had a higher predictive value ofa positive test (76%) than gas-liquid chromatography (48%)or G. vaginalis culture (41%). All three diagnostic methodshad high predictive values of a negative test. In summary,

for predicting the clinical criteria of bacterial vaginosis, theGram-stained vaginal smear had a higher specificity andpredictive value of a positive test compared with gas-liquidchromatography and G. vaginalis culture. The Gram stainwas only moderately sensitive in diagnosing women with theclinical criteria for bacterial vaginosis.To determine whether vaginal bacteria other than G.

vaginalis have greater specificity for the diagnosis of bacte-rial vaginosis, further analyses were performed for singlemicroorganisms associated with bacterial vaginosis byunivariate analysis in this report (M. hominis, Bacteroidesspp., and Peptostreptococcus spp.). The sensitivity of M.hominis (69%), Bacteroides spp. (66%), and Peptostrep-tococcus spp. (64%) in predicting women with three of fourclinical signs was lower than the sensitivity of G. vaginalis(92%), whereas the specificity of diagnosing bacterial vagi-nosis was higher for the isolation of any of these threemicroorganisms (76% to 87%) than for G. vaginalis (69%)(Table 4). The predictive value of a positive or negative testbased on the isolation of these single microorganisms wassimilar to that with G. vaginalis isolation (Table 4).

DISCUSSION

The first goal of this report was to describe the vaginalflora associated with bacterial vaginosis both by clinicalsigns and laboratory methods. Three microorganisms con-sistently occurred more frequently among women with bac-terial vaginosis in multivariable analyses: G. vaginalis, Bac-teroides spp., and M. hominis. Facultative lactobacilli wereisolated consistently less often from women with bacterialvaginosis diagnosed by any method. Peptostreptococci wereisolated more frequently from women with bacterial vagino'sis than from women without bacterial vaginosis identifiedby clinical signs or Gram-stained smears but not by gas-liquid chromatography. The consistency of vaginal flora inbacterial vaginosis identified by different methods supportsthe conclusion that the different diagnostic methods identifya similar microbiologic condition.

In other reports that identified bacterial vaginosis bygas-liquid chromatography (18) or by clinical signs (16), itwas concluded that G. vaginalis, Bacteroides spp., andPeptostreptococcus spp. were more frequently isolated andLactobacillus spp. were less frequently isolated from womenwith bacterial vaginosis compared with women without it. G.vaginalis, black-pigmented Bacteroides spp., and M. homi-nis were also positively associated and facultative Lactoba-

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cillus was negatively associated with bacterial vaginosisamong pregnant women as determined by multivariableanalyses (13). The present report confirmed the relationshipof Bacteroides spp., G. vaginalis, and M. hominis to bacte-rial vaginosis after adjustment for coinfections and con-

firmed the decreased frequency of Lactobacillus spp.,among women with bacterial vaginosis. Another reportstudying nonpregnant women from a sexually transmitteddiseases (STD) clinic related Mobiluncus spp. to bacterialvaginosis (11). The present report failed to verify this findingbecause the culture methods were not adequate for theoptimal detection of Mobiluncus spp., and only 3% of thespecimens yielded this microorganism in the third and fourthstreak zones. However, when more sensitive methods were

performed for 120 of the women included in this study,Mobiluncus spp. were recovered from 10 of 23 (43%) womenwith bacterial vaginosis compared to 4 of 97 (4%) women

without bacterial vaginosis identified by Gram stain (data notshown). In addition, curved rods resembling Mobiluncusspp. were detected by Gram stain in 34 of 122 (28%) womenwith bacterial vaginosis identified by clinical signs and in 14of 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 thesefastidious microorganisms from routine genital cultures.The second goal of this report was to examine the sensi-

tivity, specificity, and predictive value of a positive ornegative laboratory test in comparison with clinical signs forthe diagnosis of bacterial vaginosis. We first examined therelationship of individual clinical signs to the diagnosis madeby Gram-stained vaginal smear, gas-liquid chromatography,and G. vaginalis culture. Individual clinical signs and thepresence of three of the four clinical signs were more closelyrelated to the diagnosis of bacterial vaginosis by Gram stainthan by either gas-liquid chromatography or G. vaginalisculture. The close association between clinical signs andGram stain diagnosis of bacterial vaginosis has been notedpreviously among women attending an STD clinic (6). Pa-tients with bacterial vaginosis diagnosed in the STD clinicpopulation and the pregnant women in this report had similarfrequencies of individual clinical signs, although amongthose without bacterial vaginosis the STD clinic populationhad lower frequencies of homogeneous discharge, amine

odor, and clue cells and higher frequencies of pH .4.7 thandid the pregnant women. These differences may be due topregnancy or the inclusion of women with T. vaginalis in theanalysis done in Table 3 of this report. Among nonpregnantwomen, Bump et al. (4) found frequencies of each clinicalsign among women with or without bacterial vaginosis thatwere lower than the frequencies found in this report.The isolation of G. vaginalis was examined as a predictor

of bacterial vaginosis defined by clinical signs; it had excel-lent sensitivity but low specificity and a poor positivepredictive value. These results are not surprising in view ofthe high frequency of recovery of G. vaginalis amongwomen without clinical signs of bacterial vaginosis in thisand other reports (1, 6, 16, 22). None of the other singlemicroorganisms associated with bacterial vaginosis (Bac-teroides spp., Peptostreptococcus spp., and M. hoininis) hada good positive predictive value for identifying patients withclinical signs ofbacterial vaginosis. The isolation ofonly onemicroorganism does not reliably predict women with bacte-rial vaginosis. Thus, the value of vaginal cultures for any ofthese microorganisms is doubtful for the identification ofbacterial vaginosis in women.

In this report, gas-liquid chromatography was 78% sensi-

tive and 81% specific for diagnosing women with clinicalsigns of bacterial vaginosis. Others have reported higherspecificities (90 to 98%) (1, 4, 16, 18, 21) and both higher (1,18) and lower (4, 21) sensitivities. These differences mayresult from differences in gas-liquid chromatographic tech-niques, differences in the clinical signs used to define bacte-rial vaginosis, and differences in the population or the studydesigns. Reports with cohort designs (4, 18, 21), includingthe present report, have, in general, obtained lower sensi-tivities than reports with a case-control design (1). Womenidentified as having bacterial vaginosis among a cohort ofwomen may have a wider spectrum of disease that is lessclosely associated with gas-liquid chromatographic changesthan among women identified in a case-control study.The Gram-stained smear had moderate sensitivity and

predictive value of a positive test with excellent specificityand predictive value of a negative test. The positive predic-tive value of the Gram stain method was 76%, representingthe highest predictive value of a positive test among themethods evaluated. In a small case-control series, Gram-stained vaginal smears were more closely related to bacterialvaginosis as identified by clinical signs than was found in thisreport (19). The cohort study design used in this reportinvolved examinations performed by several clinicians, andinterobserver differences may have led to less controlledresults than exist in a case-control study performed by asingle clinician.

In this report, pregnant women were evaluated to deter-mine the vaginal flora associated with bacterial vaginosis andthe laboratory test that best identified women with theclinical signs of bacterial vaginosis. Many components of thefindings in this report have been studied among nonpregnantwomen in other reports with similar results. Other reportsbased on the evaluation of nonpregnant women have asso-ciated bacterial vaginosis with an increased frequency ofisolating G. vaginalis, Bacteroides spp., and Peptostrep-tococcus spp. and a decreased frequency of isolating Lacto-bacillus spp. (16, 18). The close association of identifyingbacterial vaginosis by Gram stain smear with the clinicalsigns of bacterial vaginosis has been noted among nonpreg-nant women (16). The high frequency of isolating G. vagi-nalis from nonpregnant women without bacterial vaginosishas been reported (1, 6, 16). Thus substantial evidence existsin this and other studies that the vaginal Gram stain issuperior to vaginal cultures for the diagnosis of bacterialvaginosis in both pregnant and nonpregnant women.

In addition to the scientific considerations in choosing anaccurate laboratory method of diagnosing bacterial vagino-sis, there are considerations of complexity, cost, and thefrequency of uninterpretable specimens. Vaginal culturesand gas-liquid chromatography methods to identify bacterialvaginosis require skilled personnel and costly equipment.Up to 30% of specimens analyzed by gas-liquid chromatog-raphy may yield results that are not interpretable. Further,these methods had a poor predictive value of a positive test.These findings support the conclusion that the Gram-stainedvaginal smear identifies women with the clinical signs ofbacterial vaginosis from a cohort of women better than otherlaboratory tests. Combined with its high frequency of inter-pretable results, low cost, ease of transport, and ease ofstorage, the Gram-stained smear provides a good screeningtest for bacterial vaginosis. In our experience, both clini-cians and clinical microbiologists are able to accuratelyinterpret 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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DIAGNOSIS OF BACTERIAL VAGINOSIS 1271

the same group of vaginal microorganisms as identified byclinical criteria.

ACKNOWLEDGMENTS

This work was supported by Public Health Service contractHD3-2832 and grant AI 12192 from the National Institutes of Health.

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8. Fouts, A. C., and S. J. Kraus. 1980. Trichomonas vaginalis:reevaluation of its clinical presentation and laboratory diagno-sis. J. Infect. Dis. 141:137-143.

9. Gardner, H. L., and C. D. Dukes. 1955. Haemophilus vaginalisvaginitis. Am. J. Obstet. Gynecol. 69:962-976.

10. Greenwood, J. R., and M. J. Pickett. 1980. Transfer of Hae-mophilus vaginalis Gardner and Dukes to a new genus, Gard-nerella: G. vaginalis (Gardner and Dukes) comb. nov. Int. J.Syst. Bacteriol. 30:170-178.

11. Hallen, A., C. Pahlson, and U. Forsum. 1987. Bacterial vaginosisin women attending STD clinic: diagnostic criteria and preva-lence of Mobiluncus spp. Genitourin. Med. 63:386-389.

12. Hillier, S., J. Martius, M. Krohn, N. Kiviat, K. K. Holmes, andD. A. Eschenbach. 1988. A case-control study of chorioamnionicinfection and histologic chorioamnionitis in prematurity. N.Engl. J. Med. 319:972-978.

13. Martius, J., M. A. Krohn, S. L. Hillier, W. E. Stamm, K. K.Holmes, and D. A. Eschenbach. 1988. Relationships of vaginalLactobacillus species, cervical Chlamydia trachomatis, andbacterial vaginosis to pfeterm birth. Obstet. Gynecol. 71:89-95.

14. Paavonen, J., A. Miettenen, C. E. Stevens, K. C. S. Chen, andK. K. Holmes. 1983. Mycoplasma hominis in nonspecific vagini-tis. Sex. Transm. Dis. 10(Suppl.):271-275.

15. Pheifer, T. A., P. A. Forsyth, M. Durfee, H. Pollock, and K. K.Holmes. 1978. Nonspecific vaginitis: role of Haemophilus vag-inalis and treatment with metronidazole. N. Engl. J. Med.298:1429-1434.

16. Piot, P., E. Van Dyck, P. Godts, and J. Vanderheyden. 1982. Thevaginal microbial flora in non-specific vaginitis. Eur. J. Clin.Microbiol. 1:301-306.

17. Shepard, M. C., and R. S. Comb. 1979. Enhancement ofUreaplasma urealyticum growth on a differential agar medium(A7B) by a polyamine putrescine. J. Clin. Microbiol. 10:931-933.

18. Spiegel, C. A., R. Amsel, D. Eschenbach, F. Schoenknecht, andK. K. Holmes. 1980. Anaerobic bacteria in nonspecific vaginitis.N. Engl. J. Med. 303:601-607.

19. Spiegel, C. A., R. Amsel, and K. K. Holmes. 1983. Diagnosis ofbacterial vaginosis by direct Gram stain of vaginal fluid. J. Clin.Microbiol. 18:170-177.

20. Stamm, W. E., M. Tam, M. Koester, and L. Cles. 1983.Detection of Chlamydia trachomatis inclusions in McCoy cellcultures with fluorescein-conjugated monoclonal antibodies. J.Clin. Microbiol. 17:666-668.

21. Thomason, J. L., S. M. Gelbart, L. M. Wilcoski, A. K. Peterson,R. J. Anderson, B. J. Jilly, and P. R. Hamilton. 1988. Prolineaminopeptidase as a rapid diagnostic test to confirm bacterialvaginosis. Obstet. Gynecol. 71:607-611.

22. Totten, P. A., R. Amsel, J. Hale, P. Piot, and K. K. Holmes.1982. Selective differential human blood bilayer media forisolation of Gardnerella (Haemophilus) vaginalis. J. Clin. Mi-crobiol. 15:141-147.

VOL. 27, 1989

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