Diagnosis of Bacterial Vaginosis Using a Novel Molecular Real-Time PCR Test

Background: Bacterial vaginosis is commonly diagnosed using either Amsel’s or Nugent’s criteria. These diagnostic tests are somewhat subjective and there is a need for more objective and reliable tests for the diagnosis of bacterial vaginosis. Methods: A longitudinal study was conducted at a hospital in Sweden during 2012-2013 involving 300 pregnant women seeking legal abortion was conducted at a hospital in Sweden. Bacterial vaginosis was determined to be absent or present on the basis of a modified Hay/Ison criteria assessment and compared with a molecular test analyzing six different bacteria associated with bacterial vaginosis (A. vaginae, BVAB2, G. vaginalis, Leptotrichia/Sneathia spp., Megasphaera spp., and Mobiluncus spp.) in relation to Lactobacillus spp. using real-time PCR. The Cohen kappa coefficient test was used to determine the measure of agreement between the two diagnostic tests. Results: This study showed that there is an excellent agreement between the compared methods, with a kappa coefficient value of 0.87 (0.76–0.99). As compared to the modified Hay/Ison critera, the molecular test achieved a sensitivity of 91%, specificity of 97%, positive predictive value of 91% and negative predictive value of 97%. Conclusions: The molecular test, using six different pathogens in an algorithm comparing their presence with that of lactobacilli, can accurately diagnose bacterial vaginosisin a clinical setting. The molecular test performed comparably to wet mount microscopy of vaginal swab samples. If the molecular test will be equally as effective when used as a “test of cure” needs to be investigated. Trial registration: Clinical trial registration # NCT04067557, retrospectively registered.


Background
Bacterial vaginosis (BV) is a disturbance in the bacterial flora of the vagina. It is prevalent in fertile women, occurring only rarely in post-menopausal women. BV is the most common cause of abnormal vaginal discharge and diffuse vaginal problems. The etiology is still unknown but is most certainly multifactorial [1]. BV decreases the presence of lactobacilli, consequently raising the pH in the vagina, often to a pH level between 5.5-6.0, beyond the normal healthy vaginal pH level range of 3.8-4.5.
There is no simple and easy test to diagnose BV as there is no single bacterial strain attributable to BV infection. The most commonly used method for diagnosis of BV are the Amsel criteria [2], where it is necessary to demonstrate three out of four of the following clinical criteria in order to accurately diagnose BV, including: typical homogeneous vaginal discharge, elevated pH level of the vaginal secretion (above pH 4.5), positive amine test (whiff test) and the presence of clue cells. The most notable disadvantages of Amsel's criteria is the requirement of a clinical examination of the woman and the unavoidable degree of subjectivity in the assessment. The results are, thus, not easy to replicate. Therefore, other diagnostic tests have been introduced, including another commonly used method, the Nugent score [3], in which a Gram stain is performed on the vaginal sample and is subsequently investigated under a microscope at a magnification of 1000x.The final score for the Nugent scoring system for Gram stained vaginal smears is acquired by adding the Lactobacillus content score to the Gardnerella content score. Lactobacilli are evaluated and scored 0, 1, 2, 3, or 4 based on the average number of lactobacilli observed across multiple fields, where by the presence of more than 30 lactobacilli gives a score of 0,5-30 lactobacilli gives a score of 1, 1-4 lactobacilli gives a score of 2, an average score of less than 1 lactobacilli gives a score of 3, and no lactobacilli observed per visual field gives a score of 4. Gardnerella-like bacteria are scored in a similar manner but in the reverse order, where the absence of Gardnerella-like bacteria gives a score of 0 and more than 30 Gardnerella-like bacteria gives a score of 4. If curved rods -Mobiluncus -are present the score is increased by a score of 1-2 depending on the number of Mobiluncus observed. The scores are then added together to obtain a final score. Thus, a score of 0-3 indicates normal lactobacilli flora, a score of 4-6 indicates intermediate levels of bacterial flora, and a score greater than 7 indicates a bacterial flora consistent with a BV diagnosis.
Nugent's method of BV diagnosis also suffers from some disadvantages. Firstly, the vaginal smears are scored by quantification of the different vaginal morphotypes, which requires an experienced laboratory technician and microscopist as well as considerable time and skill [4,5]. Secondly, the microscope area can differ by as much as 300% between different microscopes [6], eg 30 lactobacilli can be viewed in the field of view of a narrow-angle microscope compared to 90 in the field of view of a wide-angle microscope.
A simpler method was described by Hay et al. [7,8].

Participants
Women were recruited to this study from a cohort com-

Molecular test
The second vaginal sample obtained from the women for the purpose of BV diagnosis was collected using FLOQSwabs™

Statistical analyses
To assess the reliability of the molecular testin comparison to the modified Ison/Hay diagnostic test a kappa value (κ) and the associated 95% confidence intervals were calculated. Cohen's kappa value shows correlation between two methods and considers the possibility of coincidence. The calculations give a value between <0,00-1,00. Values of kappa were categorized based on the amount of agreement they suggest as follows: κ> 0.75 represents excellent agreement, 0.40 ≤ κ ≤ 0.75 represents fair to good agreement, and κ< 0.40 represents poor agreement [18].The statistics were calculated using the Open Epi Software.

Ethics approval and consent to participate
Ethical approval for the study was obtained from the regional ethics committee (EPN) for Gothenburg (EPN reference number 658-09 entitled "Molecular biological methods to verify STD-agents") on 21 January 2010. Participants consented to participate through providing verbal informed consent before their inclusion in the study.

Results
Vaginal swabs were collected from 300 women who had consented to participate in the study. Out of the twelve incomplete sets four lacked sufficient biological material in the wet smears, one contained too many red blood cells to be evaluated by wet smear, and seven lacked either the wet smear or PCR sample. This mean that the study was based on 288 samples that both had a wet smear and a PCR test.
In the wet smear analysis, the majority of the samples were scored as grade 1 (n=199), six were grade 0, three were   smears of these twenty sample sets were re-evaluated by microscope. This second evaluation was made simultaneously by two gynecologists, of which one is a highly experienced diagnostician. The re-evaluation showed that of the twelve sample sets  (Table 2). One interesting observation made during the re-evaluation of the slides was that of the twelve that originally tested positive for BV on wet smear but negative in PCR, seven contained sperm.

Discussion
Vaginal discomfort is a common issue experienced during a woman's menses. BV is an aggravating condition that doesn't necessarily require treatment to clear an infection in non-pregnant healthy women, treatment serves only to relieve discomfort. In pregnant women or women undergoing gynecological procedures, however, treatment for BV is likely to decrease the risk of premature delivery [19] or postoperative complications [20].
When examining the cause of vaginal discomfort the current recommendation is to use microscopy in conjunction with the Hay/Ison criteria for clinical diagnosis of BV [21].This method for diagnosis of BV, as with the Amsel's criteria and the Nugent's score, suffers from reliability on subjective judgments [4], questionable reproducibility, high economic costs and are heavily time-consuming methods requiring highly skilled technicians and physician consultation for sampling and, more importantly, these methods are seldom used to their full extent, except in research. There is therefore a great need for an easy and objective method to assess BV in out-patient care.

Declarations
Ethical approval for the study was obtained from the Clinical trail registration # NCT04067557, retrospectively registered.

Availability of data and materials
The datasets generated and/or analyzed during the current study are not publicly available due individual privacy could be compromised but are available from the corresponding author on reasonable request.

Funding
This study was partly funded by the Skaraborgs Hospital FoU fund.