Comparison of Methods for Diagnosing Bacterial Vaginosis among Pregnant Women

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1 JOURNAL OF CLINICAL MICROBIOLOGY, June 1989, p /89/ $02.00/0 Copyright C 1989, American Society for Microbiology Vol. 27, No. 6 Comparison of Methods for Diagnosing Bacterial Vaginosis among Pregnant Women MARIJANE A. KROHN,1* SHARON L. HILLIER,2 AND DAVID A. ESCHENBACH2 Departments of Epideiniology' and Obstetrics and Gynec ology,2 University of Washington, Seattle, Washington Received 21 December 1988/Accepted 2 March 1989 The diagnosis of bacterial vaginosis is usually based on clinical criteria including homogeneous vaginal discharge, an elevated vaginal ph, the presence of clue cells, and an amine odor. We have evaluated the vaginal flora and clinical signs for 593 pregnant women. Gardnerella vaginalis, Bacteroides spp., and Mycoplasma hominis were isolated more frequently among women with clinical signs than among those without clinical signs of bacterial vaginosis in multivariable analyses that controlled for other bacteria. To determine the laboratory method that best predicted bacterial vaginosis, we calculated the sensitivity, specificity, and predictive value of positive and negative tests for Gram-stained vaginal smears, gas-liquid chromatography of vaginal fluid, and G. vaginalis cultures compared with clinical signs. G. vaginalis culture was sensitive (92%) and both gas-liquid chromatography (78%) and Gram-stained vaginal smears (62%) were moderately sensitive in identifying women 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 predictive value 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 recognized as a vaginal syndrome over 30 years ago by Gardner and Duke (9). They associated bacterial vaginosis with the isolation of Haemophilus vaginalis, later briefly named Corvnebacteriumr vaginale and currently named Gardnerella iaginalis (10). However, the microbiology of bacterial vaginosis is complex and involves organisms other than G. vaginalis. G. vaginalis can be isolated from the vaginas of 20 to 40% of women without bacterial vaginosis (1, 4, 6), and large quantities of not only G. vaginalis but also anaerobic bacteria (16, 18) and Mycoplasmna hominis (13, 14) can be recovered from women with bacterial vaginosis. The diagnosis of bacterial vaginosis has usually been based on three or more of the following clinical signs of the vaginal discharge: a thin homogeneous appearance, an elevated ph, an amine odor after the addition of 10% KOH, and the presence of clue cells (vaginal epithelial cells studded with bacteria) (1, 6, 9). Four other laboratory methods have been used to diagnose bacterial vaginosis: culture of vaginal fluid for G. vaginalis (9), gas-liquid chromatographic analysis of vaginal fluid for short-chain fatty acids believed to be products of anaerobic bacterial growth (16, 18), Gramstained vaginal smears read microscopically for bacterial morphotypes (5, 19), and an assay for proline aminopeptidase (21). Each laboratory method is based on the assumption that the vaginal flora of women with bacterial 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 laboratory methods have not been compared within a single cohort of women. In the present study, we determined the vaginal flora of patients with bacterial vaginosis diagnosed by clinical signs, gas-liquid chromatography, and Gram-stained vaginal smears to document that each diagnostic method was associated with similar vaginal flora. The sensitivity, specificity, * Corresponding author and predictive value of positive and negative tests for three laboratory methods (Gram-stained smear, G. vaginalis culture, and gas-liquid chromatography) were compared with clinical signs for diagnosing bacterial vaginosis. The importance of an accurate, reproducible, and inexpensive laboratory method to diagnose bacterial vaginosis has increased with the recent association of placental infection (12) and premature delivery (13) with this vaginal syndrome. MATERIALS AND METHODS Women enrolled into the study were part of a 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 of gestation as part of a National Institutes of Health-sponsored collaborative multicenter 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 hypertension, kidney disease, heart disease, or diabetes mellitus requiring therapy. Of 716 women enrolled, Il had unsatisfactory vaginal specimens, and 112 had at least one missing result, 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 the vaginal discharge were determined, a vaginal smear for subsequent Gram staining was obtained, a vaginal smear for saline and potassium hydroxide wet mount was made, and a vaginal wash was taken for culture and gas-liquid chromatographic analysis as previously described (13). The vaginal vault was examined for discharge, which was described as normal (mucoid and floccular), purulent, curdy, or thin 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 examined microscopically for motile trichomonads and clue cells. The 10% potassium hydroxide wet mount was exam-

2 VOL. 27, 1989 DIAGNOSIS OF BACTERIAL VAGINOSIS 1267 TABLE 1. Vaginal flora present in pregnant women with and without bacterial vaginosis diagnosed by 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 bacteria G. vaginalis 83 43' 97 45"' 71 41' Lactobacillus spp b 59 94' 76 97' Viridans group streptococci ' Group B streptococci il 16 Enterococcus spp. il Diphtheroids Coagulase-negative staphylococci Anaerobic bacteria' Bacteroides spp. Peptostreptococcus spp ' 23b ' 25' '> 26'> Genital mycoplasmas U. 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 < 'Growth in the third and fourth streak zones on an agar plate. ined for odor (15) (normal, foul, or amine) and microscopically for hyphae. A vaginal smear was obtained by rolling 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 Spiegel et al. (19). Briefly, if Lactobacillus spp. morphotypes were fewer than five per oil immersion field and if there were five or more G. vaginalis morphotypes together with five or more other morphotypes (gram-positive cocci, small gramnegative rods, curved gram-variable rods, or fusiforms) per oil immersion field, the Gram stain was interpreted as indicating bacterial vaginosis. If five or more Lactobacillus spp. and fewer than five other morphotypes were present per ou immersion field, 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 swab on a plastic shaft and transported in chlamydia medium until it was inoculated onto cycloheximide-treated McCoy cells (20). Vaginal specimens were taken with sterile cotton swabs and inoculated into the following media: modified Diamond medium for the isolation of Trichomonas vaginalis (8), Sabouraud agar for the identification of Candida albicans, T (2) and M broth and A7B (17) agar for the isolation of Ureaplasma urealyticum and M. hominis. Specimens for facultative and anaerobic bacteria were obtained by instilling a prereduced sterile balanced salt solution (13) into the vagina; after the vaginal fluid mixed with the solution, it was removed with a sterile syringe. The vaginal fluid was injected into a sterile stoppered Hungate tube filled with 85% N2, 10% H2, and 5% C02 for transport to the microbiology laboratory and was inoculated onto prereduced medium in an anaerobic glove box. A sample of 100,ul of vaginal wash was inoculated onto each agar plate, which was then streaked for isolation into four zones with decontamination 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 fatty acids, the API Anident and 20A systems (Analytab Products, Plainview, N.Y.), and biochemical tests. G. vaginalis and Lactobacillus spp. were identified by Gram stain, catalase reaction, beta-hemolysis on human bilayer Tween agar (22), and typical Gram stain morphology. Beta-hemolytic streptococci were identified by colony morphology on 5% sheep blood agar and were placed into Lancefield groups by the StrepTex (Welicome Diagnostics, Research Triangle Park, N.C.) typing system. Enterococci and viridans group streptococci were differentiated by growth in a broth containing 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 chromatograph fitted with a chromasorb column (Supelco, lnc., Bellefonte, Pa.). A gas chromatographic pattern was considered abnormal 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 in height. Univariate relationships were tested for statistical significance by chi-square or Fisher exact tests. The odds ratio, also called a cross-product ratio, was used as a measure of association for cross-classified categorical data (7). Multivariable logistic regression analyses were performed to estimate adjusted relationships (3). Confidence intervals of 95% were reported for the odds ratios from multivariable analyses and verified by likelihood ratio tests for significance (3). RESULTS The frequency of recovery of microorganisms from women with bacterial vaginosis diagnosed by clinical criteria was compared with that of women without bacterial vaginosis by univariate analysis (Table 1). The vaginal floras of women with and without bacterial vaginosis were compared by two other methods of diagnosing bacterial vaginosis:

3 1268 KROHN ET AL. J. CLIN. MICROBIOL. 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 ( ) 13.3 ( ) 2.2 ( ) Facultative Lactobacillus spp. 0.4 ( ) 0.4 ( ) 0.2 ( ) Viridans group streptococci 0.5 ( ) 0.9 ( ) 0.7 ( ) Bacteriodes spp.b 4.1 ( ) 4.4 ( ) 2.6 ( ) Peptostreptococcus spp.b 1.8 ( ) 2.4 ( ) 0.9 ( ) U. urealyticurm 2.5 ( ) 2.4 ( ) 1.0 ( ) M. hominis 3.7 ( ) 3.7 ( ) 2.8 ( ) 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 chromatography. Bacterial isolates that occurred in fewer than 10% of the women in the cohort (Escherichia coli, Micrococcus spp., Moraxella spp., Actinomyces spp., Eubacteriwn spp., Propionibacterium spp., and Mobilhncus spp.) were not tabulated 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 had increased frequencies of G. 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, Enterococcus spp., diphtheroids, and coagulase-negative staphylococci were not associated with bacterial vaginosis as diagnosed by any of the methods. Bacterial morphotypes such as G. vaginalis and Lactobacillus spp. were identified as part of the Gram stain interpretation, so it would be expected that these species would be associated with the diagnosis of bacterial 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 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 the other microorganisms varied slightly for the three diagnostic methods, a similar flora was statistically significantly associated with each of the three diagnostic methods. TABLE 3. Logistic régression analysis was used to determine whether bacterial isolates associated with bacterial vaginosis by univariate analyses would still be associated after adjustment for (i) coinfections such as C. trachomatis, T. vaginalis, or C. albicans or (ii) the other bacterial isolates also associated with bacterial vaginosis. G. vaginalis, Bacteroides spp., and M. honinis remained positively associated with bacterial vaginosis, and Lactobacillus spp. remained negatively associated with bacterial vaginosis after adjustments in the multivariable analysis (Table 2). These four bacterial isolates were interpreted to be independently related to bacterial vaginosis. Peptostreptococcus spp. and U. urealyticum were no longer statistically significantly related to bacterial vaginosis after these adjustments. Except for G. v'aginalis, these microorganisms had similar relative risks for bacterial vaginosis identified either by three of four clinical signs or Gram-stained vaginal smears. G. vaginalis was strongly independently related to bacterial vaginosis diagnosed by Gram-stained smears because its presence is part of the Gram stain interpretation. In general, the relative risks of the microorganisms were lower when bacterial vaginosis was diagnosed by gas-liquid chromatographic criteria than when it was diagnosed by the other two methods. Bacterial vaginosis was diagnosed by finding three of four clinical criteria in 122 (21%) of 593 pregnant women. In comparison, bacterial vaginosis was diagnosed by Gramstained smears in 12%, by gas-liquid chromatography in 28%, and by G. vaginalis culture (third and fourth streak zones on an agar plate) in 41% of the women. The frequency of each individual clinical sign was compared for the three Frequency of clinical signs of bacterial vaginosis among pregnant women identified with bacterial vaginosis by Gram-stained vaginal 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 ph "' 55 15' 50 14" Amine odor after KOH 74 22' ' Clue cells on wet mount 80 24"' 54 22' 48 18" Three of the above four clinical signs ` Statistical significance indicates that the individual clinical sign was independently related to bacterial vaginosis by logistic regression analysis after adjusting for 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 < P < J P < 0.05.

4 VOL. 27, 1989 DIAGNOSIS OF BACTERIAL VAGINOSIS 1269 TABLE 4. Method Correlation of the diagnosis of bacterial vaginosis made by laboratory tests with clinical criteria for diagnosis of bacterial vaginosis" Clinical criteria for diagnosis of bacterial vaginosis, no./total (%) Sensitivity Specificity predictive Positive value predictive Negative 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 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) 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 (Table 3). Each of the four clinical signs was present among 71 to 84% of patients with bacterial vaginosis and 21 to 24% of those without bacterial vaginosis diagnosed by Gram stain (P < 10-' for each determination). Individual clinical signs were less frequent (44 to 55%) when bacterial vaginosis was diagnosed by gas-liquid chromatography or G. vaginalis culture than when diagnosis was made by Gram stain. Based upon the presence of three of four clinical criteria, bacterial vaginosis was present among 77% of women diagnosed by Gram-stained smear compared with only 47% of women diagnosed by gas-liquid chromatography and 40% of women diagnosed by G. vaginalis culture. Logistic regression analysis was performed to determine which of the individual clinical signs was independently related to bacterial vaginosis after adjustment for the other clinical signs. A ph of -4.7 and clue cells present on wet mount were independently related to bacterial vaginosis identified by any of the three methods in the multivariable analysis (Table 3). Homogeneous discharge was not independently related to bacterial vaginosis as determined by any of the three methods after adjustment for ph, amine color, and clue cells. Amine odor after addition of potassium hydroxide was independently related to bacterial vaginosis diagnosed by Gram-stained smear or G. vaginalis culture. A ph of.4.7, amine odor, and clue cells provided unique information that independently contributed to the clinical diagnosis of bacterial vaginosis. Because coinfection with T. vaginalis or C. albicans could influence the clinical signs of bacterial vaginosis, only women negative for these microorganisms by culture, Gram stain, or wet mount were used to evaluate the sensitivity and specificity of Gram stain, gas-liquid chromatography, and G. vaginalis culture in comparison with clinical criteria for diagnosing 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 more sensitive (92%) than either gas-liquid chromatography (78%) or Gram stain (62%) in predicting the clinical diagnosis of bacterial vaginosis. However, the Gram-stained vaginal smear was more specific for diagnosis (95%) than gas-liquid chromatography (81%) or G. vaginalis culture (69%). The Gram-stained vaginal smear had a higher predictive value of a positive test (76%) than gas-liquid chromatography (48%) or G. vaginalis culture (41%). All three diagnostic methods had high predictive values of a negative test. In summary, for predicting the clinical criteria of bacterial vaginosis, the Gram-stained vaginal smear had a higher specificity and predictive value of a positive test compared with gas-liquid chromatography and G. vaginalis culture. The Gram stain was only moderately sensitive in diagnosing women with the clinical criteria for bacterial vaginosis. To determine whether vaginal bacteria other than G. vaginalis have greater specificity for the diagnosis of bacterial 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 of M. hominis (69%), Bacteroides spp. (66%), and Peptostreptococcus spp. (64%) in predicting women with three of four clinical signs was lower than the sensitivity of G. vaginalis (92%), whereas the specificity of diagnosing bacterial vaginosis was higher for the isolation of any of these three microorganisms (76% to 87%) than for G. vaginalis (69%) (Table 4). The predictive value of a positive or negative test based on the isolation of these single microorganisms was similar to that with G. vaginalis isolation (Table 4). 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 consistently occurred more frequently among women with bacterial vaginosis in multivariable analyses: G. vaginalis, Bacteroides spp., and M. hominis. Facultative lactobacilli were isolated consistently less often from women with bacterial vaginosis diagnosed by any method. Peptostreptococci were isolated more frequently from women with bacterial vagino' sis than from women without bacterial vaginosis identified by clinical signs or Gram-stained smears but not by gasliquid chromatography. The consistency of vaginal flora in bacterial vaginosis identified by different methods supports the conclusion that the different diagnostic methods identify 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 more frequently isolated and Lactobacillus spp. were less frequently isolated from women with bacterial vaginosis compared with women without it. G. vaginalis, black-pigmented Bacteroides spp., and M. hominis were also positively associated and facultative Lactoba-

5 1270 KROHN ET AL. J. CLIN. MICROBIOL. cillus was negatively associated with bacterial vaginosis among pregnant women as determined by multivariable analyses (13). The present report confirmed the relationship of Bacteroides spp., G. vaginalis, and M. hominis to bacterial vaginosis after adjustment for coinfections and confirmed 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). The present report failed to verify this finding because the culture methods were not adequate for the optimal detection of Mobiluncus spp., and only 3% of the specimens yielded this microorganism in the third and fourth streak 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%) women with bacterial vaginosis compared to 4 of 97 (4%) women without bacterial vaginosis identified by Gram stain (data not shown). In addition, curved rods resembling Mobiluncus spp. were detected by Gram stain in 34 of 122 (28%) women with bacterial vaginosis identified by clinical signs and in 14 of 471 (3%) women without clinical signs of bacterial vaginosis. Thus the lower frequency of Mobiluncus spp. detected by culture reflects the difficulty in recovering these fastidious microorganisms from routine genital cultures. The second goal of this report was to examine the sensitivity, specificity, and predictive value of a positive or negative laboratory test in comparison with clinical signs for the diagnosis of bacterial vaginosis. We first examined the relationship of individual clinical signs to the diagnosis made by Gram-stained vaginal smear, gas-liquid chromatography, and G. vaginalis culture. Individual clinical signs and the presence of three of the four clinical signs were more closely related to the diagnosis of bacterial vaginosis by Gram stain than by either gas-liquid chromatography or G. vaginalis culture. The close association between clinical signs and Gram stain diagnosis of bacterial vaginosis has been noted previously among women attending an STD clinic (6). Patients with bacterial vaginosis diagnosed in the STD clinic population and the pregnant women in this report had 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 and higher frequencies of ph.4.7 than did the pregnant women. These differences may be due to pregnancy or the inclusion of women with T. vaginalis in the analysis done in Table 3 of this report. Among nonpregnant women, Bump et al. (4) found frequencies of each clinical sign among women with or without bacterial vaginosis that were 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 excellent 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 (Bacteroides spp., Peptostreptococcus spp., and M. hoininis) had a good positive predictive value for identifying patients with clinical signs of bacterial vaginosis. The isolation ofonly one microorganism does not reliably predict women with bacterial vaginosis. Thus, the value of vaginal cultures for any of these microorganisms is doubtful for the identification of bacterial vaginosis in women. In this report, gas-liquid chromatography was 78% sensitive and 81% specific for diagnosing women with clinical signs of bacterial vaginosis. Others have reported higher specificities (90 to 98%) (1, 4, 16, 18, 21) and both higher (1, 18) and lower (4, 21) sensitivities. These differences may result from differences in gas-liquid chromatographic techniques, differences in the clinical signs used to define bacterial vaginosis, and differences in the population or the study designs. Reports with cohort designs (4, 18, 21), including the present report, have, in general, obtained lower sensitivities 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 associated with gas-liquid chromatographic changes than among women identified in a case-control study. The Gram-stained smear had moderate sensitivity and predictive value of a positive test with excellent specificity and predictive value of a negative test. The positive predictive value of 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, Gramstained vaginal smears were more closely related to bacterial vaginosis as identified by clinical signs than was found in this report (19). The cohort study design used in this report involved examinations performed by several clinicians, and interobserver differences may have led to less controlled results than exist in a case-control study performed by a single clinician. In this report, pregnant women were evaluated to determine the vaginal flora associated with bacterial vaginosis and the laboratory test that best identified women with the clinical signs of bacterial vaginosis. Many components of the findings in this report have been studied among nonpregnant women in other reports with similar results. Other reports based on the evaluation of nonpregnant women have associated bacterial vaginosis with an increased frequency of isolating G. vaginalis, Bacteroides spp., and Peptostreptococcus spp. and a decreased frequency of isolating Lactobacillus spp. (16, 18). The close association of identifying bacterial vaginosis by Gram stain smear with the clinical signs of bacterial vaginosis has been noted among nonpregnant women (16). The high frequency of isolating G. vaginalis from nonpregnant women without bacterial vaginosis has been reported (1, 6, 16). Thus substantial evidence exists in this and other studies that the vaginal Gram stain is superior to vaginal cultures for the diagnosis of bacterial vaginosis in both pregnant and nonpregnant women. In addition to the scientific considerations in choosing an accurate laboratory method of diagnosing bacterial vaginosis, there are considerations of complexity, cost, and the frequency of uninterpretable specimens. Vaginal cultures and gas-liquid chromatography methods to identify bacterial vaginosis require skilled personnel and costly equipment. Up to 30% of specimens analyzed by gas-liquid chromatography 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-stained vaginal smear identifies women with the clinical signs of bacterial vaginosis from a cohort of women better than other laboratory tests. Combined with its high frequency of interpretable 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 clinicians 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 interpretation of the Gram-stained smear can identify women with

6 VOL. 27, 1989 the same group of vaginal microorganisms as identified by clinical criteria. ACKNOWLEDGMENTS This work was supported by Public Health Service contract HD and grant AI from the National Institutes of Health. LITERATURE CITED 1. Amsel, R., P. A. Totten, C. A Spiegel, K. C. S. Chen, D. Eschenbach, and K. K. Holmes Nonspecific vaginitis. Am. J. Med. 74: Bowie, W. R., S.-P. Wang, E. R. Alexander, J. Floyd, P. S. Forsyth, and H. M. Pollock, J.-S. L. Lin, T. M. Buchanan, and K. K. Holmes Etiology of nongonococcal urethritis: evidence for Chlamydia trachomatis and Ureaplasma urealyticum. J. Clin. Invest. 59: Breslow, N., and N. E. Day Statistical methods in cancer research, the analysis of case-control studies, p In IARC scientific publication no. 32., vol. 1. International Agency for Research on Cancer, Lyons, France. 4. Bump, R. C., F. P. Zuspan, W. J. Buesching, L. W. Ayers, and T. J. Stephens The prevalence, six-month persistence, and predictive values of laboratory indicators of bacterial vaginosis (nonspecific vaginitis) in asymptomatic women. Am. J. Obstet. Gynecol. 150: Dunkelberg, W. E Diagnosis of Hemophilus vaginalis by Gram-stained smears. Am. J. Obstet. Gynecol. 91: Eschenbach, D. A., S. L. Hillier, C. Critchlow, C. Stevens, T. DeRouen, and K. K. Holmes Diagnosis and clinical manifestations of bacterial vaginosis. Am. J. Obstet. Gynecol. 158: Fleiss, J. L Statistical methods for rates and proportions, 2d ed., p. 58. John Wiley & Sons, Inc., New York. 8. Fouts, A. C., and S. J. Kraus Trichomonas vaginalis: reevaluation of its clinical presentation and laboratory diagnosis. J. Infect. Dis. 141: Gardner, H. L., and C. D. Dukes Haemophilus vaginalis vaginitis. Am. J. Obstet. Gynecol. 69: Greenwood, J. R., and M. J. Pickett Transfer of Haemophilus vaginalis Gardner and Dukes to a new genus, Gardnerella: G. vaginalis (Gardner and Dukes) comb. nov. Int. J. Syst. Bacteriol. 30: DIAGNOSIS OF BACTERIAL VAGINOSIS Hallen, A., C. Pahlson, and U. Forsum Bacterial vaginosis in women attending STD clinic: diagnostic criteria and prevalence of Mobiluncus spp. Genitourin. Med. 63: Hillier, S., J. Martius, M. Krohn, N. Kiviat, K. K. Holmes, and D. A. Eschenbach A case-control study of chorioamnionic infection and histologic chorioamnionitis in prematurity. N. Engl. J. Med. 319: Martius, J., M. A. Krohn, S. L. Hillier, W. E. Stamm, K. K. Holmes, and D. A. Eschenbach Relationships of vaginal Lactobacillus species, cervical Chlamydia trachomatis, and bacterial vaginosis to pfeterm birth. Obstet. Gynecol. 71: Paavonen, J., A. Miettenen, C. E. Stevens, K. C. S. Chen, and K. K. Holmes Mycoplasma hominis in nonspecific vaginitis. Sex. Transm. Dis. 10(Suppl.): Pheifer, T. A., P. A. Forsyth, M. Durfee, H. Pollock, and K. K. Holmes Nonspecific vaginitis: role of Haemophilus vaginalis and treatment with metronidazole. N. Engl. J. Med. 298: Piot, P., E. Van Dyck, P. Godts, and J. Vanderheyden The vaginal microbial flora in non-specific vaginitis. Eur. J. Clin. Microbiol. 1: Shepard, M. C., and R. S. Comb Enhancement of Ureaplasma urealyticum growth on a differential agar medium (A7B) by a polyamine putrescine. J. Clin. Microbiol. 10: Spiegel, C. A., R. Amsel, D. Eschenbach, F. Schoenknecht, and K. K. Holmes Anaerobic bacteria in nonspecific vaginitis. N. Engl. J. Med. 303: Spiegel, C. A., R. Amsel, and K. K. Holmes Diagnosis of bacterial vaginosis by direct Gram stain of vaginal fluid. J. Clin. Microbiol. 18: Stamm, W. E., M. Tam, M. Koester, and L. Cles Detection of Chlamydia trachomatis inclusions in McCoy cell cultures with fluorescein-conjugated monoclonal antibodies. J. Clin. Microbiol. 17: Thomason, J. L., S. M. Gelbart, L. M. Wilcoski, A. K. Peterson, R. J. Anderson, B. J. Jilly, and P. R. Hamilton Proline aminopeptidase as a rapid diagnostic test to confirm bacterial vaginosis. Obstet. Gynecol. 71: Totten, P. A., R. Amsel, J. Hale, P. Piot, and K. K. Holmes Selective differential human blood bilayer media for isolation of Gardnerella (Haemophilus) vaginalis. J. Clin. Microbiol. 15: