Diagnosis of bacterial vaginosis: need for validation of microscopic image area used for scoring bacterial morphotypes

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Abstract
Background: The diagnosis of bacterial vaginosis (BV) is often made according to Nugent’s classification, a scoring system based on bacterial counting of Gram stained slides of vaginal secretion. However as the image area of the microscope field will influence the number of morphotypes seen there is a need to standardise the area. Methods: A graph intended for recalculation of number of bacterial morphotypes seen by the observer using 1000× magnification from various microscope set-ups was constructed and applied to data sets typical for scoring BV. The graph was used in recalculation of Nugent scores, which were also compared with the Ison/Hay scores to evaluate the consequences for the diagnosis of BV. Results: The observed image area differed by 300% among the investigated microscope set-ups. In two different data sets, one treatment study and one screening study, a considerable change in the number of women classified as intermediate was seen when the graph was used to standardise the image area. The recalculated numbers were also compared to the Ison/Hay classification. Weighted kappa indexes between the different methods were 0.84, 0.88, and 0.90, indicating that the methods are comparable. Conclusion: Because of the considerable differences among image areas covered by different microscope set-ups used in Nugent and Ison/Hay scoring, there is a need to standardise the area in order to reach comparable scores reflecting the diagnosis of BV in different laboratories. The differences in the intermediate group will have a considerable effect on the results from both treatment and prevalence studies, even though the kappa indexes indicate very good agreement between the methods used.