vaginal wash was taken for culture and gas-liquid chromato- graphic analysis as previously described (13). The vaginal vault was examined for discharge, which ...
JOURNAL OF CLINICAL MICROBIOLOGY, June 1989, 0095-1137/89/061266-06$02.00/0
p.
Vol. 27, No. 6
1266-1271
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 AND DAVID A. ESCHENBACH2
Departments of Epideiniology' and Obstetrics and
Gynec ology,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 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%).
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.
Bacterial vaginosis (nonspecific vaginitis) was recognized 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,
as a
*
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-
Corresponding author. 1266
DIAGNOSIS OF BACTERIAL VAGINOSIS
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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 No vaginosis (ti = 471)
Gram stain
Vaginosis (n = 122)
Gas-liquid chromatography" No vaginosis Vaginosis (n = 307) (n = 117)
Vaginosis (n = 73)
No vaginosis (n = 520)
97 59 51 7 10 74 53
94' 30' 16 16 67 62
71 76 40 il 9 74 56
41' 97' 31 16 17 71 66
Facultative bacteria G. vaginalis Lactobacillus spp. Viridans group streptococci Group B streptococci Enterococcus spp. Diphtheroids Coagulase-negative staphylococci
83 72 38 12 il 71 53
Anaerobic bacteria' Bacteroides spp. Peptostreptococcus spp.
62 59
23b
70 70
18' 25'
45 45
14'> 26'>
Genital mycoplasmas U. urealyticum M. hominis
94 65
73b 17b
97 74
74"'
84 48
74'> 16'>
43'
94b 31 15 16 67 63
15'
45"'
20'
'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 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:
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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: Three of four Gas-liquid clinical signs Gram stain chromatography
Organism
G. vaginalis"' Facultative Lactobacillus spp. Viridans group streptococci Bacteriodes spp.b
2.5 0.4 0.5 4.1 1.8 2.5 3.7
Peptostreptococcus spp.b U. urealyticurm M. hominis a b
(1.4-4.6) (0.2-0.9) (0.3-0.8) (2.2-7.6) (1.0-3.0) (1.0-6.0) (2.3-6.2)
13.3 0.4 0.9 4.4 2.4 2.4 3.7
(3.0-58.9) (0.2-0.7) (0.5-1.7) (1.7-10.8) (1.2-4.7) (0.5-11.6) (1.9-7.2)
2.2 0.2 0.7 2.6 0.9 1.0 2.8
(1.3-3.9) (0.1-0.5) (0.4-1.3) (1.3-5.2) (0.5-1.7) (0.5-1.9) (1.6-4.9)
Adjusted for C. trachomnatis, T. vaginalis, C. albicans, and each of the other tabulated bacterial isolates. 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.
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
TABLE 3. 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 Vaginosis (n = 73)
Homogeneous discharge pH - 4.7 Amine odor after KOH Clue cells on wet mount Three of the above four clinical signs
71 84 74 80 77
No vaginosis (n = 520)
24
21"' 22' 24"' 13
Gas-liquid chromatography
G. î'aginalis culture"'
Vaginosis (n = 117)
No vaginosis (n = 307)
Vaginosis
No vaginosis
(n = 243)
(n = 350)
51 55 50 54 47
21
44 50 47 48 40
20 14"
15' 22 22' 9
16' 18" 7
` 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.
