1887

Abstract

is a Gram-variable anaerobic bacterium present in 100 % of women with bacterial vaginosis (BV). BV is a complex polymicrobial condition with no single causative agent. The current laboratory detection method for BV relies on a Gram-stain Nugent score to estimate the quantity of different bacterial morphotypes in the vaginal micro flora. Whilst the Nugent score can distinguish between women with and without BV, a significant proportion are categorized as intermediate, which fails to differentiate a normal from an abnormal vaginal micro flora. A singleplex TaqMan real-time quantitative PCR (qPCR) assay was developed and compared with the ‘gold standard’ Nugent score. Detection and quantification of was performed on vaginal specimens with positive, negative and intermediate Nugent scores. The qPCR assay demonstrated high analytical specificity against a broad microbial panel and analytical sensitivity down to 3.1 × 10 copies ml. There was a significantly higher load in women with BV compared with intermediate and non-BV women ( value = 5.1 × 10). All Nugent scores in keeping with BV had qPCR loads of ≥ 10 copies ml. Among the 24 undefined women (11.8 %) in the study with an intermediate flora, 14 (58.3 %) had a load of ≥ 10 copies ml. In this study a threshold of 10 copies ml had positive and negative predictive values of 57.1 and 100 % for BV; the high qPCR loads among the intermediate Nugent scores suggest the need for a new approach in classifying BV and the potential for qPCR to play a role.

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2015-09-01
2024-04-24
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References

  1. Amsel R., Totten P. A., Spiegel C. A., Chen K. C., Eschenbach D., Holmes K. K. 1983; Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations. Am J Med 74:14–22 [View Article][PubMed]
    [Google Scholar]
  2. Balashov S. V., Mordechai E., Adelson M. E., Gygax S. E. 2014; Identification, quantification and subtyping of Gardnerella vaginalis in noncultured clinical vaginal samples by quantitative PCR. J Med Microbiol 63:162–175[PubMed] [CrossRef]
    [Google Scholar]
  3. Begum N., Muazzam N., Shamsuzzaman S., Chowdhury A., Rashid A., Islam D. 2010; Diagnosis of bacterial vaginosis by acridine orange staining and its comparison to conventional methods and association of Gardnerella vaginalis with bacterial vaginosis. Bangladesh J Med Microbiol 4:37–42
    [Google Scholar]
  4. Benzaken A. S., Galban E. G., Antunes W., Dutra J. C., Peeling R. W., Mabey D., Salama A. 2006; Diagnosis of gonococcal infection in high risk women using a rapid test. Sex Transm Infect 82:(Suppl. 5)v26–v28 [View Article][PubMed]
    [Google Scholar]
  5. Bustin S. A., Benes V., Garson J. A., Hellemans J., Huggett J., Kubista M., Mueller R., Nolan T., Pfaffl M. W., other authors. 2009; The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622 [View Article][PubMed]
    [Google Scholar]
  6. Cartwright C. P., Lembke B. D., Ramachandran K., Body B. A., Nye M. B., Rivers C. A., Schwebke J. R. 2012; Development and validation of a semiquantitative, multitarget PCR assay for diagnosis of bacterial vaginosis. J Clin Microbiol 50:2321–2329 [View Article][PubMed]
    [Google Scholar]
  7. Dover S. E., Aroutcheva A. A., Faro S., Chikindas M. L. 2008; Natural antimicrobials and their role in vaginal health: a short review. Int J Probiotics Prebiotics 3:219–230[PubMed]
    [Google Scholar]
  8. Espy M. J., Uhl J. R., Sloan L. M., Buckwalter S. P., Jones M. F., Vetter E. A., Yao J. D., Wengenack N. L., Rosenblatt J. E., other authors. 2006; Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 19:165–256 [View Article][PubMed]
    [Google Scholar]
  9. Fredricks D. N., Fiedler T. L., Marrazzo J. M. 2005; Molecular identification of bacteria associated with bacterial vaginosis. N Engl J Med 353:1899–1911 [View Article][PubMed]
    [Google Scholar]
  10. Ginzinger D. G. 2002; Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 30:503–512 [View Article][PubMed]
    [Google Scholar]
  11. Harper J., Davis G. 1982; Cell wall analysis of Gardnerella vaginalis (Haemophilus vaginalis). Int J Syst Bacteriol 32:48–50 [View Article]
    [Google Scholar]
  12. Kobayashi N., Bauer T. W., Tuohy M. J., Lieberman I. H., Krebs V., Togawa D., Fujishiro T., Procop G. W. 2006; The comparison of pyrosequencing molecular Gram stain, culture, and conventional Gram stain for diagnosing orthopaedic infections. J Orthop Res 24:1641–1649 [View Article][PubMed]
    [Google Scholar]
  13. Kubista M., Andrade J. M., Bengtsson M., Forootan A., Jonák J., Lind K., Sindelka R., Sjöback R., Sjögreen B., other authors. 2006; The real-time polymerase chain reaction. Mol Aspects Med 27:95–125 [View Article][PubMed]
    [Google Scholar]
  14. Lamont R. F., Sobel J. D., Akins R. A., Hassan S. S., Chaiworapongsa T., Kusanovic J. P., Romero R. 2011; The vaginal microbiome: new information about genital tract flora using molecular based techniques. BJOG 118:533–549 [View Article][PubMed]
    [Google Scholar]
  15. Mackay I. M. 2004; Real-time PCR in the microbiology laboratory. Clin Microbiol Infect 10:190–212 [View Article][PubMed]
    [Google Scholar]
  16. Menard J. P., Fenollar F., Henry M., Bretelle F., Raoult D. 2008; Molecular quantification of Gardnerella vaginalis and Atopobium vaginae loads to predict bacterial vaginosis. Clin Infect Dis 47:33–43 [View Article][PubMed]
    [Google Scholar]
  17. Menard J. P., Mazouni C., Salem-Cherif I., Fenollar F., Raoult D., Boubli L., Gamerre M., Bretelle F. 2010; High vaginal concentrations of Atopobium vaginae and Gardnerella vaginalis in women undergoing preterm labor. Obstet Gynecol 115:134–140 [View Article][PubMed]
    [Google Scholar]
  18. Menard J. P., Fenollar F., Raoult D., Boubli L., Bretelle F. 2012; Self-collected vaginal swabs for the quantitative real-time polymerase chain reaction assay of Atopobium vaginae and Gardnerella vaginalis and the diagnosis of bacterial vaginosis. Eur J Clin Microbiol Infect Dis 31:513–518 [View Article][PubMed]
    [Google Scholar]
  19. Nathan B., Appiah J., Saunders P., Heron D., Nichols T., Brum R., Alexander S., Baraitser P., Ison C. 2015; Microscopy outperformed in a comparison of five methods for detecting Trichomonas vaginalis in symptomatic women. Int J STD AIDS 26:251–256[PubMed] [CrossRef]
    [Google Scholar]
  20. Nugent R. P., Krohn M. A., Hillier S. L. 1991; Reliability of diagnosing bacterial vaginosis is improved by a standardized method of Gram stain interpretation. J Clin Microbiol 29:297–301[PubMed]
    [Google Scholar]
  21. Patterson J. L., Stull-Lane A., Girerd P. H., Jefferson K. K. 2010; Analysis of adherence, biofilm formation and cytotoxicity suggests a greater virulence potential of Gardnerella vaginalis relative to other bacterial-vaginosis-associated anaerobes. Microbiology 156:392–399 [View Article][PubMed]
    [Google Scholar]
  22. Public Health England 2014; Commercial and In-House Diagnostic Tests: Evaluations and Validations. UK Standards for Microbiology Investigations. Q 1 Issue 4.3 http://www.hpa.org.uk/SMI/pdf
  23. Romoren M., Velauthapillai M., Rahman M., Sundby J., Klouman E., Hjortdahl P. 2007; Trichomoniasis and bacterial vaginosis in pregnancy: inadequately managed with the syndromic approach. Bull World Health Organ 85:297–304 [View Article][PubMed]
    [Google Scholar]
  24. Rose W. A. II, McGowin C. L., Spagnuolo R. A., Eaves-Pyles T. D., Popov V. L., Pyles R. B. 2012; Commensal bacteria modulate innate immune responses of vaginal epithelial cell multilayer cultures. PLoS One 7:e32728 [View Article][PubMed]
    [Google Scholar]
  25. Rosenstein I. J., Morgan D. J., Sheehan M., Lamont R. F., Taylor-Robinson D. 1996; Bacterial vaginosis in pregnancy: distribution of bacterial species in different Gram-stain categories of the vaginal flora. J Med Microbiol 45:120–126 [View Article][PubMed]
    [Google Scholar]
  26. Sha B. E., Chen H. Y., Wang Q. J., Zariffard M. R., Cohen M. H., Spear G. T. 2005; 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. J Clin Microbiol 43:4607–4612 [View Article][PubMed]
    [Google Scholar]
  27. Sivaranjini R., Jaisankar T., Thappa D. M., Kumari R., Chandrasekhar L., Malathi M., Parija S., Habeebullah S. 2013; Spectrum of vaginal discharge in a tertiary care setting. Trop Parasitol 3:135–139 [View Article][PubMed]
    [Google Scholar]
  28. Smith R., Copas A. J., Prince M., George B., Walker A. S., Sadiq S. T. 2003; Poor sensitivity and consistency of microscopy in the diagnosis of low grade non-gonococcal urethritis. Sex Transm Infect 79:487–490 [View Article][PubMed]
    [Google Scholar]
  29. Sobel J. D. 2000; Bacterial vaginosis. Annu Rev Med 51:349–356 [View Article][PubMed]
    [Google Scholar]
  30. Srinivasan S., Morgan M. T., Liu C., Matsen F. A., Hoffman N. G., Fiedler T. L., Agnew K. J., Marrazzo J. M., Fredricks D. N. 2013; More than meets the eye: associations of vaginal bacteria with Gram stain morphotypes using molecular phylogenetic analysis. PLoS One 8:e78633 [View Article][PubMed]
    [Google Scholar]
  31. Whiley D. M., Tapsall J. W., Sloots T. P. 2006; Nucleic acid amplification testing for Neisseria gonorrhoeae: an ongoing challenge. J Mol Diagn 8:3–15 [View Article][PubMed]
    [Google Scholar]
  32. WHO 2009 CDC Realtime RTPCR (rRTPCR) Protocol for Detection and Characterization of Swine Influenza Geneva, Switzerland: World Health Organization;
  33. Willett L. L., Centor R. M. 2005; Evaluating vaginitis. The importance of patient factors. J Gen Intern Med 20:871 [View Article][PubMed]
    [Google Scholar]
  34. Witkin S. S., Linhares I. M., Giraldo P., Ledger W. J. 2007; An altered immunity hypothesis for the development of symptomatic bacterial vaginosis. Clin Infect Dis 44:554–557 [View Article][PubMed]
    [Google Scholar]
  35. Yeoman C. J., Yildirim S., Thomas S. M., Durkin A. S., Torralba M., Sutton G., Buhay C. J., Ding Y., Dugan-Rocha S. P., other authors. 2010; Comparative genomics of Gardnerella vaginalis strains reveals substantial differences in metabolic and virulence potential. PLoS One 5:e12411 [View Article] (Electronic Resource). [PubMed]
    [Google Scholar]
  36. Zozaya-Hinchliffe M., Lillis R., Martin D. H., Ferris M. J. 2010; Quantitative PCR assessments of bacterial species in women with and without bacterial vaginosis. J Clin Microbiol 48:1812–1819 [View Article][PubMed]
    [Google Scholar]
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