Sexually-transmitted infection (STI) - June 15, 2022

Diagnostic methods for Bacterial Vaginosis and the benefits of molecular testing

Vulvovaginal infections are common in prepubertal and adolescent women and include symptoms such as itching, irritation, burning, and an abnormal vaginal discharge.1 The most common cause of vulvovaginitis is bacterial vaginosis (BV) but other infectious causes are candidiasis, trichomoniasis, and herpes simplex virus (HSV).2 BV prevalence is estimated to be 22.8% among women of reproductive age in Europe and central Asia resulting in an estimated annual cost of $2.4 billion.3

Multiple adverse outcomes are associated with BV, including preterm birth, pelvic inflammatory disease, endometritis, and transmission and acquisition of human immunodeficiency virus type 1 (HIV-1) and sexually transmitted infections (STIs).3 Reinfections are also common with annual recurrence rates being as high as 58% and the likelihood increasing following a previous infection.4 Diagnosis of BV can be difficult as the microflora consists of several pathogens that can be present in healthy patients, with the distinguishing factor being the amounts of each pathogen.5 This can potentially lead to missed or misdiagnosis.

Diagnostic techniques must effectively identify and quantify associated pathogens. As such, molecular diagnostic tests are a promising alternative to traditional diagnostic testing for BV as it allows for the identification and quantification of pathogens with higher sensitivity and specificity. Multiplex PCR also allows for the testing of many pathogens simultaneously, which can test for several types of vaginitis infections that share similar symptoms.


The Vaginal Microflora

The vaginal microflora is an intricate and dynamic microecosystem that constantly undergoes fluctuations.5 It is dominated by single or multiple Lactobacillus species and can be classified into 5 community state types (CTSs). CSTs I, II, III, and V are dominated by L. crispatus, L. gasseri, L. iners, and L. jensenii, respectively.5 CST IV has a highly diverse microbial community characterised by obligate anaerobic bacteria, a composition that resembles BV.5 Lactobacillus species produce various antimicrobial compounds such as lactic acid, hydrogen peroxide, and bacteriocins, which all help to establish a defence against invading pathogens, thus maintaining a healthy vaginal microbiome.5 BV is characterised by the loss or sharp decline in the total number of Lactobacillus and a corresponding 100–1000 fold increase in the concentration of facultative or obligate anaerobic microbes, such as Gardnerella, Atopobium and Bacterial Vaginosis Associated Bacteria-2 (BVAB-2).5 Other types of vulvovaginal infections can also be caused by Candidiasis (17%—39%) and Trichomoniasis (4%—35%).6 The microflora of these vulvovaginal infections can include some of the same pathogens as BV, and coinfections resulting in mixed vaginitis can occur at an estimated rate of 4.44% to 35.06%.7 The complexity of vulvovaginal infections and microflora composition can make accurate diagnosis difficult.


Traditional diagnostic testing of BV

Traditional methods for BV testing are the Amsel’s criteria and the Nugent score.8 Amsel’s criteria include homogenous thin vaginal discharge, a vaginal pH of >4.5, a positive whiff amine test result, and the detection of clue cells in vaginal fluid using wet mount microscopy; at least 3 of these 4 criteria are necessary for a diagnosis of BV.9 Determining the Nugent score of Gram stains from vaginal secretions is a more objective way of diagnosing BV. Scoring ranges from 0–10 and is calculated by assessing the quantity of morphotypes per oil immersion field from 1 to 4+ with regard to the number of Lactobacillus spp., G. vaginalis morphotypes, and Mobiluncus spp. morphotypes (Table 1).10,11 A total score of 7 to 10 is classed as BV. 10,12

Score Lactobacillus morphotypes G. vaginalis morphotypes Mobiluncus morphotypes
0 4+ 0 0
1 3+ 1+ 1+ or 2+
2 2+ 2+ 3+ or 4+
3 1+ 3+
4 0 4+

Table 1. Nugent Scoring system; 0-3 = normal microflora, 4-6 = intermediate microflora, 7-10 = BV.10


The Gram stain Nugent score has a higher sensitivity than the Amsel’s criteria (89% vs 70%), with the sensitivity of Amsel’s criteria ranging from 37% to 70%.11,13 However, Amsel’s criteria has a higher specificity (94% vs 83%).13 Some clinicians prefer using these methods because they are relatively fast, and a diagnosis can be made during the office visit.12 However, data suggest that clinicians do not routinely assess all criteria or may incorrectly diagnose BV due to a lack of time or skills.12

In one study, 25 out of 163 flora samples (15%) from pregnant women were confirmed to have BV by either or both the Amsel criteria and Nugent score.14 Ten of these samples had conflicting results: five samples categorised as BV by the Nugent score were normal according to the Amsel criteria and five samples categorized as BV by the Amsel criteria were rated intermediate by the Nugent score.14 This clearly demonstrates that neither test is sufficient on its own to accurately diagnose BV.


Molecular testing of BV and multiplex PCR testing

Molecular diagnostic techniques have high performance and allow the identification of various pathogens by detection of their nucleic acids. These techniques have advantages over microscopy-based tests as they are objective, can detect fastidious bacteria, enable quantitation and are ideal for self-collected vaginal swabs.12

Nucleic acid amplification tests (NAATs), such as PCR, are able to detect as little as one organism in a sample. PCR assays have both a high sensitivity (100%) and specificity (93%).14 Despite its value in diagnosing samples as either normal or BV, the Nugent scoring system is not as reliable when diagnosing intermediate flora.15,16 PCR assays show that intermediate flora can have a molecular profile more similar to that of BV than to normal samples.17 PCR quantification of BV-associated pathogens clearly defines a reproducible and standardised molecularly defined BV, irrespective of the clinical and microscopic characteristics of vaginal flora.14

The polymicrobial composition of BV highlights the need to amplify more than one target sequence for proper diagnosis. It is important that multiple pathogens found in the microflora of BV and their quantity are detected. In women with BV, an average of 11.1 species were detected, while women without BV had an average of 3.6 species (P=0.0001).18 The detection of either BVAB-2 or Megasphaera-1 had a sensitivity of 95.9% and specificity of 93.7% using Nugent score as the point of reference.18

Quantitative multiplex PCR assays makes this possible.12 If targeted alone for diagnosis, different bacterial species that are associated with BV have varying positive predictive values (PPV).12 However, the combined detection of several different bacterial species can improve the test characteristics for BV diagnostics. Multiplex PCR assays also make it possible to use a targeted syndromic approach. As bacterial vaginosis shares symptoms with other vaginal infections caused by other microorganisms, multiplex assays allow for the testing of multiple pathogens that could be causing shared symptoms.



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