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ORIGINAL RESEARCH |
From the *Department of Obstetrics and Gynecology, Drexel University College of Medicine, Philadelphia, Pennsylvania; Department of Obstetrics and Gynecology, University of Alabama at Birmingham, Birmingham, Alabama; Department of Obstetrics and Gynecology, Thomas Jefferson University, Philadelphia, Pennsylvania; and Istituto di Ricovero e Cura a Carattere Scientifico Burlo Garofolo, University of Trieste, Trieste, Italy.
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ABSTRACT |
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METHODS: Fifty pregnant women at less than 20 weeks of gestation were consecutively recruited at the time of their first prenatal visit. Three vaginal smears were collected from each woman, 1 from the posterior fornix, 1 from the mid-lateral wall, and 1 from the introitus. Smears were Gram stained and evaluated for bacterial vaginosis using Nugent's criteria. Each smear was classified as positive for bacterial vaginosis if the Nugent score was 7 or greater, intermediate if the score was between 4 and 6, and negative if the Nugent score was 3 or less. A mean leukocyte value was obtained for each slide by evaluating 5 fields under oil immersion. Repeated-measures analysis of variance was used to compare mean Nugent scores and leukocyte counts across sites.
RESULTS: The sample consisted of mostly African-American, young, low-income women in their first trimester of pregnancy. Forty-seven percent were bacterial vaginosis–positive (Nugent score 7 or greater). Mean Nugent scores (± standard deviation) across the vaginal sample collection sites were similar: posterior fornix = 4.2 (4.4); mid-lateral wall = 4.2 (4.5); introitus = 4.2 (4.6). In contrast, the mean leukocyte count varied significantly across vaginal sample collection sites: 2.5 (4.2); mid-lateral wall = 2.8 (4.4); introitus = 6.5 (8.6) (F = 11.26 1, 47.5; P = .002).
CONCLUSION: Leukocyte counts vary according to the site from which vaginal secretions were obtained, with the highest leukocyte count in samples obtained from the introitus. Bacterial vaginosis diagnosis by Nugent score does not vary by site of sample collection.
LEVEL OF EVIDENCE: II-3
In both nonpregnant and pregnant women, use of the Nugent score is the most widespread and well-accepted method for diagnosing bacterial vaginosis.5,10–14 The Nugent score consists of a standardized scoring system for assessing the vaginal flora by Gram stain and is based on the recognition of the most reliably defined bacterial morphotypes: a decrease in large gram-positive rods (lactobacilli), and increases in small gram-negative to gram-variable rods (Gardnerella, Bacteroides), and curved gram-negative to gram-variable rods (Mobiluncus).13,15,16 A score of 7–10 is used by most investigators to define bacterial vaginosis.
In addition to changes in vaginal bacterial morphotypes, bacterial vaginosis is also characterized by limited numbers of leukocytes. Thus the term "vaginosis" and not "vaginitis" has been adopted to identify this syndrome.3 Why bacterial vaginosis is associated with a decrease in the number of leukocytes in vaginal secretions is unknown. Advancing gestational age,17 amniotic fluid infection, chorioamnionitis, and preterm labor18,19,20 have all been associated with an increase in leukocyte count. Recent research has established an association between vaginal leukocytes and increased risk of adverse reproductive outcomes.19,20,21–25 In addition, recent evidence suggests that there is an association between vaginal leukocytes and elevated levels of proinflammatory cytokines.17,19,26 Because of these associations, research interest in the association between leukocyte count and risk of adverse birth outcomes has grown. Thus, refinement in reliably quantifying these cells is needed.
Therefore, because both bacterial vaginosis Nugent scores and vaginal leukocyte scores have been linked to adverse pregnancy outcomes, are markers of vaginal inflammation or infection, are read from the same slide, and are likely to be the focus for future interventions, we thought it important to determine whether scores and counts differed depending on the vaginal site sampled. To answer this question, we examined the variation in bacterial vaginosis Nugent scores and leukocyte counts in vaginal smears collected from 3 different vaginal sites during routine pelvic examination at the time of the first antenatal visit.
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PATIENTS AND METHODS |
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Smears were obtained from all 50 women at 3 vaginal locations (3 women had only 2 samples that were readable; a total of 49 samples were analyzed for each of the 3 sites): the first slide (introitus) was obtained by inserting a sterile dry Dacron swab 1 cm into the vagina, taking the specimen from the wall after carefully avoiding any contact with labia majora and minora. The next 2 slides were collected after the insertion of a nonlubricated sterile speculum. Using the same type of sterile Dacron swabs, these specimens were collected from the mid-lateral vaginal wall and the posterior fornix.
All smears were air-dried, Gram stained and scored according to Nugent.14 Bacterial vaginosis was diagnosed if the score was 7 or greater.14 Leukocyte count was also evaluated on the Gram-stained smears. A mean count was obtained by evaluating 5 different fields under oil immersion (x 1,000 magnification).
Repeated measures analysis of variance was used to compare mean leukocyte counts and Nugent scores across 3 sites while correcting for the lack of independence among samples collected from the same person. Box's conservative epsilon corrected P values were used with 
(2-tialed) = .05.27 For statistically significant findings, we conducted post hoc pair-wise comparisons using repeated-measures analysis of variance. To take into account multiple comparisons, we considered 2 sites statistically significantly different if the Box's conservative epsilon corrected P values were less than (2-tailed) = 0.05/3.
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RESULTS |
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We first compared Nugent scores by site of sample collection for each of the 50 study participants and found that 39 (78%) had an identical Nugent score at all 3 sites, 9 had identical Nugent scores at 2 of the 3 sites, and 2 had a different value for each of the 3 sites. (Three women had only 2 samples that were readable; a total of 49 samples were analyzed for each of the 3 sites.) When we compared the mean Nugent score (SD) for all study participants, we found that there were no statistically significant differences across sites (Fig. 1): posterior fornix 4.2 (4.4), mid-lateral vaginal wall 4.2 (4.5), introitus 4.2 (4.6) (F = 0.08 1, 47.5; P = .785) (Table 2).
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We then classified women as bacterial vaginosis–positive, intermediate, or negative based on the Nugent score from each location and found that all study participants had the same bacterial vaginosis classification for all 3 sites, despite the small variation in Nugent score for some study participants (data not shown). The percentage of women that was bacterial vaginosis–positive, negative, and intermediate were as follows: 47% were bacterial vaginosis–positive (Nugent score 7–10), 53% were bacterial vaginosis–negative (Nugent score 0–3), and none were bacterial vaginosis–intermediate. Taken together, these findings suggest that although there may be slight variation in Nugent score by site of collection, classification of bacterial vaginosis status does not vary by site.
In contrast, leukocyte count did differ by site of collection such that the introitus had the highest count with an average (SD) of 6.5 (8.6) leukocytes per oil immersion field (Fig. 1). (Three women had only 2 samples that were readable; a total of 49 samples were analyzed for each of the 3 sites.) The mean (SD) leukocyte count for the mid-lateral wall and posterior fornix were 2.8 (4.4) and 2.5 (4.2), respectively (Table 2). The difference between the posterior fornix and introitus was significantly different (F = 12.68 1,47; P = .001) as was the difference between the introitus and the mid-lateral vaginal wall (F = 11.93 1,47; P = .001). However, the difference between the mid-lateral wall and the introitus was not statistically significant (F = 0.32 1,47; P = .575) (Table 2).
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DISCUSSION |
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In this study, the Nugent score did not vary with respect to the vaginal site of collection. Our results are consistent with those of a previous study, where the use of self-collected vaginal samples (site of collection unknown) agreed with the results obtained from the posterior wall of the vaginal fornix at the time of speculum examination,28 suggesting reasonably good reliability and reproducibility of the Nugent method. Because the results of our study were similar regardless of where in the vagina the sample originated, this study provides further evidence that for assessing bacterial vaginosis by Nugent Score, self-sampling should not detract from the validity of the results.
In contrast, because the leukocyte count differed by vaginal location and because the same vaginal location is not likely posted at each sampling, self-sampling for leukocyte assessment is not likely to yield consistent results and should probably not be used without further investigation. Finally, we should state clearly that we do not know why the leukocyte count at the introitus is higher than other areas, but suggest that the introitus may be the site where various pathogens and foreign proteins are first encountered by the host; it is therefore reasonable to believe that at least during pregnancy, the increased leukocyte count at the introitus serves a protective immunologic function. In contrast, it is likely that the distribution of the bacteria that are counted in the Nugent score are distributed equally throughout the vagina.
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Footnotes |
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Received May 25, 2004. Received in revised form August 26, 2004. Accepted September 9, 2004.
doi:10.1097/01.AOG.0000147842.69832.7d
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REFERENCES |
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