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

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 10006 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.

Assessment of vaginal microbiota in Brazilian women with and without bacterial vaginosis and comparison with Nugent score

“…Intermediate status has been described by several investigators as women harboring a mixed vaginal microbiota transitioning among healthy and BV [40,41]. In the present study, even though there were a small number of intermediate subjects, their vaginal microbiota structure was more similar to those found in BV women, especially regarding to A. vaginae, G. vaginalis and Mobiluncus sp.…”

Treatment of Bacterial Vaginosis in Early Pregnancy and its Effect on Spontaneous Preterm Delivery and Preterm Premature Rupture of Membranes

“…The vaginal swab sample was transferred to a microscope slide, air dried, and transported to the gynaecological clinic in Skaraborgs Hospital where it was microscopically examined using a phase contrast microscope with a magnification of 400x, according to the modified Hay/Ison criteria for the diagnosis of BV [9]. The Hay/ Ison criteria were developed using Gram-stained smears by oil immersion at a magnification of 1000x; however, because this criteria assesses the type of flora and does not indicate the number of individual bacteria, which is achieved using Nugent's criteria, it is possible to use these criteria with non-stained smears at lower magnification [5]. The vaginal flora is divided into the following three categories: normal (grade 1), intermediate (grade 2), and BV (grade 3) florae, depending on the relative number of lactobacillus morphotypes as compared to gardnerella morphotypes.…”

“…First, the smears are scored by quantification of the different vaginal morphotypes that requires an experienced slide reader, as well as considerable time and skill [4]. Second, quantification of the morphotypes also depends on the area of the microscope, which can differ by more than 300% between different microscope vision fields [5], particularly in a modern laboratory microscope. A total of 30 gardnerella morphotypes will result in a score of 4, thereby indicating that the slide shows intermediate BV.…”

Utility of Microbiological Profile of Symptomatic Vaginal Discharge in Rural Women of Reproductive Age Group

“…The first swab taken and smears were made on a clean and grease free glass slide, then heat fixed by just passing the glass slide over the flame. Then the smear was stained by Gram's method and observed under oil immersion objective and assessed using the Nugent's score for diagnosis of Bacterial vaginosis and the presence gram positive oval yeast like budding cells (2-4μm) and/or pseudohyphae [5] [ …”