To perform a wet-mount preparation correctly, dilute the vaginal discharge with 1 or 2 drops of 0.9% saline and place it on a slide. Examine the slide under lowand high-powered fields for vaginal squamous cells, white blood cells (WBCs), lactobacilli, clue cells, and trichomonads. An increased number of WBCs can be defined as >5–10 WBC/HPF or WBCs exceeding the number vaginal epithelial cells.
To prepare the potassium hydroxide (KOH) slide, place a generous amount of vaginal discharge on a slide with 10% KOH solution. Air- or flame-drying before examination under low-power microscopy may improve sensitivity. A positive KOH preparation will have hyphae, mycelial tangles, or spores.
Whiff test. The whiff test is performed by adding drops of 10% potassium hydroxide solution to the vaginal fluid. A positive result is a “fishy” amine odor. In a study16 of 100 women complaining of malodorous discharge, a positive whiff test was predictive of positive culture results for anaerobic flora such as Bacteroides sp. with sensitivity 67%, specificity 94%, and a positive predictive value of 95%. The whiff test was not positive in any of the 5 cases with positive culture results for G vaginalis in the absence of anaerobes. There were also 12 cases positive for anaerobes without G vaginalis.
pH >4.5. Since the abnormal flora of BV is consistently associated with a vaginal pH >4.5, a normal pH excludes a diagnosis of BV.17,18 The determination of pH in the narrow range around 4.5 is not accurate using standard nitrazine paper. Narrower-range test paper is available and more accurate. Examples include pH paper for 4.5 to 5.5 (Micro Essential Laboratory), FemExam pH and Amines Test Card (Litmus Concepts), pHem-ALERT: pH paper on a stick (Imagyn Gynecology). Cervical mucous, semen, and blood are alkaline and can interfere with pH testing. Estrogen production is also necessary to maintain an acidic environment. A pH of 3.8 to 4.5 is consistent with normal vaginal flora in premenopausal women with normal estrogen production.17
Clue cells. Clue cells are vaginal epithelial cells coated with coccobacilli giving an appearance as if coated with ground black pepper. Clue cells on wet mount preparation is considered the most accurate of Amsel’s diagnostic criteria for BV.19 On the other hand, office evaluation of the wet mount is considered by some authors to be unreliable due to dependence on the clinician’s microscopy skills and lack of a durable record of the patient sample.
Gram stain a more objective test. A Gram stain evaluation using Nugent’s criteria has been adopted as the gold standard test for research purposes, including studies of prematurity. The Gram-stained vaginal specimen is scored from 0 to 10 based on semi-quantitative assessment of 3 classes of morphotypes (Table 2): large gram-positive rods (Lactobacilli), small gram-negative rods (Gardnerella and Bacteroides spp.), and small curved gram-variable rods (Mobiluncus spp.).11
Diagnosis of BV is typically made when the Nugent score is 7 or more, which appears qualitatively as dominant morphotypes other than Lactobacilli. Gram staining is more objective and reproducible compared with wet-mount examination, with a sensitivity of 93% and specificity of 70% if Amsel’s criteria are used as the gold standard.14 It is useful for the evaluation of asymptomatic women. It also provides a durable record of the patient specimen. Compared with Gram stain, Amsel’s criteria tend to underdiagnose cases. We can expect that if screening for BV in pregnancy becomes a recommendation, Gram staining in a clinical laboratory will be the recommended method of diagnosis.
Other diagnostic tests for BV. DNA testing for Gardnerella is accurate for detection, but it is not synonymous with a diagnosis of BV, as described.20 DNA testing is further described under “Differential Diagnosis.” Gram staining is more reliable than gas-liquid chromatography21 and an assay for proline aminopeptidase (a metabolic product of some of the bacteria associated with BV).22 Latex agglutination testing for vaginal lactoferrin is a nonspecific marker for leukocytes, and thus inflammation. It is of little clinical utility in the diagnosis of vaginal discharge.23
TABLE 1 Predictive values of Amsel’s criteria (using 3 of 4 positive as diagnostic reference standard)
