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

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.

References

1. Zuckerman A, Romano M. Clinical Recommendation: Vulvovaginitis. Journal of Pediatric and Adolescent Gynecology 2016;29:673-679.
2. Goje O, Munoz JL. Vulvovaginitis: Find the cause to treat it. Cleveland Clinic Journal of Medicine 2017;84:215-224.
3. Peebles K, Velloza J, Balkus JE, et al. High Global Burden and Costs of Bacterial Vaginosis: A Systematic Review and Meta-Analysis. Sexually Transmitted Diseases 2019;46:304-311. Available at: https://journals.lww.com/00007435-201905000-00005.
4. Bradshaw CS, Morton AN, Hocking J, et al. High Recurrence Rates of Bacterial Vaginosis over the Course of 12 Months after Oral Metronidazole Therapy and Factors Associated with Recurrence. The Journal of Infectious Diseases 2006;193:1478-1486. Available at: https://academic.oup.com/jid/article-lookup/doi/10.1086/503780.
5. Chen X, Lu Y, Chen T, et al. The Female Vaginal Microbiome in Health and Bacterial Vaginosis. Frontiers in Cellular and Infection Microbiology 2021;11.
6. Hainer BL, Gibson M v. Vaginitis. American family physician 2011;83:807-15. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21524046.
7. Qi W, Li H, Wang C, et al. Recent Advances in Presentation, Diagnosis and Treatment for Mixed Vaginitis. Frontiers in Cellular and Infection Microbiology 2021;11.
8. Mohammadzadeh F, Dolatian M, Jorjani M, et al. Diagnostic Value of Amsel’s Clinical Criteria for Diagnosis of Bacterial Vaginosis. Global Journal of Health Science 2014;7. Available at: http://www.ccsenet.org/journal/index.php/gjhs/article/view/39836.
9. Amsel R, Totten PA, Spiegel CA, et al. Nonspecific vaginitis. The American Journal of Medicine 1983;74:14-22.
10. Nugent, RP, Krohn MA, Hillier3 SL. Reliability of Diagnosing Bacterial Vaginosis Is Improved by a Standardized Method of Gram Stain Interpretation.; 1991.
11. Sha BE, Chen HY, Wang QJ, et al. Utility of Amsel Criteria, Nugent Score, and Quantitative PCR for Gardnerella vaginalis , Mycoplasma hominis , and Lactobacillus spp. for Diagnosis of Bacterial Vaginosis in Human Immunodeficiency Virus-Infected Women. Journal of Clinical Microbiology 2005;43:4607-4612. Available at: https://journals.asm.org/doi/10.1128/JCM.43.9.4607-4612.2005.
12. Coleman JS, Gaydos CA. Molecular Diagnosis of Bacterial Vaginosis: an Update. Kraft CS, ed. Journal of Clinical Microbiology 2018;56. Available at: https://journals.asm.org/doi/10.1128/JCM.00342-18.
13. Schwebke JR, Hillier SL, Sobel JD, et al. Validity of the Vaginal Gram Stain for the Diagnosis of Bacterial Vaginosis.
14. Menard J-P, Mazouni C, Fenollar F, et al. Diagnostic accuracy of quantitative real-time PCR assay versus clinical and Gram stain identification of bacterial vaginosis. European Journal of Clinical Microbiology & Infectious Diseases 2010;29:1547-1552. Available at: http://link.springer.com/10.1007/s10096-010-1039-3.
15. Larsson P-G, Carlsson B, Få L, et al. Diagnosis of bacterial vaginosis: need for validation of microscopic image area used for scoring bacterial morphotypes. Sex Transm Infect 2004;80:63-67. Available at: http://sti.bmj.com/.
16. Libman MD, Kramer M, Platt R. Comparison of Gram and Kopeloff stains in the diagnosis of bacterial vaginosis in pregnancy. Diagnostic Microbiology and Infectious Disease 2006;54:197-201.
17. Bradshaw CS, Tabrizi SN, Fairley CK, et al. The Association of Atopobium vaginae and Gardnerella vaginalis with Bacterial Vaginosis and Recurrence after Oral Metronidazole Therapy. The Journal of Infectious Diseases 2006;194:828-836. Available at: https://academic.oup.com/jid/article-lookup/doi/10.1086/506621.
18. Fredricks DN, Fiedler TL, Thomas KK, et al. Targeted PCR for Detection of Vaginal Bacteria Associated with Bacterial Vaginosis. Journal of Clinical Microbiology 2007;45:3270-3276. Available at: https://journals.asm.org/journal/jcm.