Raina Tzvetanova GERGOVA, Ivan Gergov MITOV, Konstantsa TOSHEVA-DASKALOVA, Tanya Vasileva STRATEVA

Multiplex PCR detection of problematic pathogens of clinically heterogeneous bacterial vaginosis in Bulgarian women

Background/aim: This study aimed to investigate the correlation between the prevalence of problematic pathogens and the clinical status of women with bacterial vaginosis (BV). Materials and methods: Gardnerella vaginalis, Atopobium vaginae, and Mobiluncus spp. were detected using a multiplex PCR assay, and their role in the infection of Bulgarian women with clinically heterogeneous BV was evaluated. Results: The predominant BV-associated pathogen identified was G. vaginalis with an incidence of 98.39%, followed by A. vaginae (68.05%) and Mobiluncus spp. at 17.01%. The coexistence of A. vaginae and G. vaginalis was more common in women with discharge (in 72.04%) and in patients with chronic recurrent BV than among asymptomatic or newly diagnosed BV cases (P < 0.05). Mobiluncus spp. was detected mostly in coinfections, in association with Trichomonas vaginalis. The coinfections were predominantly related to recurrent BV and with complications (P < 0.05). Conclusion: This is the first study about the correlation between problematic pathogens and clinically heterogeneous BV in Bulgarian women. High frequency of infection with key BV-related pathogens was observed in childbearing women. The incidence was shown to often correlate with coexistent T. vaginalis, with severity of infection, and with complicated and recurrent BV after unsuccessful treatments. Screening should be considered in reproductive health programs.

PDF

___

1. Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Delanghe J, Van Simaey L, De Ganck C, Temmerman M, Vaneechoutte M. Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis. BMC Microbiol 2004; 4: 16.
2. Marrazzo JM. Interpreting the epidemiology and natural history of bacterial vaginosis: are we still confused? Anaerobe 2011; 17: 186-190.
3. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol 1991; 29: 297-301.
4. Schwebke J R, Lawing LF. Prevalence of Mobiluncus spp. among women with and without bacterial vaginosis as detected by polymerase chain reaction. Sex Transm Dis 2001; 28: 195-199.
5. Aroutcheva AA, Simoes JA, Behbakht K, Faro S. Gardnerella vaginalis isolated from patients with bacterial vaginosis and from patients with healthy vaginal ecosystems. Clin Infect Dis 2001; 33: 1022-1027.
6. Patterson JL, Girerd PH, Karjane NW, Jefferson KK. Effect of biofilm phenotype on resistance of Gardnerella vaginalis to hydrogen peroxide and lactic acid. Am J Obstet Gynecol 2007; 197: 170-177.
7. Bretelle F, Rozenberg P, Pascal A, Favre R, Bohec C, Loundou A, Senat MV, Aissi G, Lesavre N, Brunet J et al. High Atopobium vaginae and Gardnerella vaginalis vaginal loads are associated with preterm birth. Clin Infect Dis. 2015; 60: 860-867.
8. Gergova RT, Strateva TV, Mitov IG. Gardnerella vaginalisassociated bacterial vaginosis in Bulgarian women. Braz J Infect Dis 2013; 17: 313-318.
9. Goodfellow M, Peter HA. The Actinobacteria Part A. Genus IV. Mobiluncus. In: Whitman W, Goodfellow M, Kämpfer P, Busse HJ, Trujillo M, Ludwig W, Suzuki KI, Parte A, editors. Bergeys Manual of Systematic Bacteriology, Vol. 5. 2nd ed. New York, NY, USA: Springer; 2012. pp. 126-138.
10. Hardy L, Jespers V, Dahchour N, Mwambarangwe L, Musengamana V, Vaneechoutte M, Crucitti T. Unravelling the bacterial vaginosis-associated biofilm: a multiplex Gardnerella vaginalis and Atopobium vaginae fluorescence in situ hybridization assay using peptide nucleic acid probes. PLoS One 2015; 10: e0136658.
11. Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SK, McCulle SL, Karlebach S, Gorle R, Russell J, Tacket CO et al. Vaginal microbiome of reproductive-age women. P Natl Acad Sci USA 2011; 108: 4680-4687.
12. Polatti F. Bacterial vaginosis, Atopobium vaginae and nifuratel. Curr Clin Pharmacol 2012; 7: 36-40.
13. Swidsinski A, Doerffel Y, Loening-Baucke V, Swidsinski S, Verstraelen H, Vaneechoutte M, Lemm V, Schilling J, Mendling W. Gardnerella biofilm involves females and males and is transmitted sexually. Gynecol Obstet Invest 2010; 70: 256-263.
14. Catlin BW. Gardnerella vaginalis: characteristics, clinical considerations, and controversies. Clin Microbiol Rev 1992; 5: 213-37.
15. Gilbert NM, Lewis WG, Lewis AL. Clinical features of bacterial vaginosis in a murine model of vaginal infection with Gardnerella vaginalis. PLoS One 2013; 8: e59539.
16. Josey WE, Schwebke JR. The polymicrobial hypothesis of bacterial vaginosis causation: a reassessment. Int J STD AIDS 2008; 19: 152-154.
17. Machado A, Castro J, Cereija T, Almeida C, Cerca N. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method. Peer J 2015; 3: e780.
18. Menard JP, Fenollar F, Henry M, Bretelle F, Raoult D. Molecular quantification of Gardnerella vaginalis and Atopobium vaginae loads to predict bacterial vaginosis. Clin Infect Dis 2008; 47: 33-43.
19. Patterson JL, Stull-Lane A, Girerd PH, Jefferson KK. Analysis of adherence, biofilm formation and cytotoxicity suggests a greater virulence potential of Gardnerella vaginalis relative to other bacterial-vaginosis-associated anaerobes. Microbiology 2010; 156: 392-399.
20. Zariffard MR, Saifuddin M, Sha BE, Spear GT. Detection of bacterial vaginosis-related organisms by real-time PCR for Lactobacilli, Gardnerella vaginalis and Mycoplasma hominis. FEMS Immunol Med Microbiol 2002; 34: 277-281.
21. Ferris MJ, Muyzer G, Ward DM. Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl Environ Microbiol 1996; 62: 340-346.
22. Madico G, Quinn TC, Rompalo A, McKee KT Jr, Gaydos CA. Diagnosis of Trichomonas vaginalis infection by PCR using vaginal swab samples. J Clin Microbiol 1998; 36: 3205-3210.
23. Udayalaxmi J, Bhat GK, Kotigadde S. Biotypes and virulence factors of Gardnerella vaginalis isolated from cases of bacterial vaginosis. Indian J Med Microbiol 2011; 29: 165-168.
24. Gergova R, Sirakov I, Mitov I, Strateva T. Detection and phylogenetic characterization of Gardnerella vaginalis exotoxin in samples from Bulgarian women with bacterial vaginosis. C R Acad Bulg Sci 2016; 69: 1085-94.
25. Harwich MD Jr, Alves JM, Buck GA, Strauss JF 3rd, Patterson JL, Oki AT, Girerd PH, Jefferson KK. Drawing the line between commensal and pathogenic Gardnerella vaginalis through genome analysis and virulence studies BMC Genomics 2010; 11: 375.
26. Kusters JG, Reuland EA, Bouter S, Koenig P, Dorigo-Zetsma JW. A multiplex real-time PCR assay for routine diagnosis of bacterial vaginosis. Eur J Clin Microbiol Infect Dis 2015; 34: 1779-1785.
27. Bradshaw CS, Tabrizi SN, Fairley CK, Morton AN, Rudland E, Garland SM. The association of Atopobium vaginae and Gardnerella vaginalis with bacterial vaginosis and recurrence after oral metronidazole therapy. J Infect Dis 2006; 194: 828- 836.
28. Ling Z, Kong J, Liu F, Zhu H, Chen X, Wang Y, Li L, Nelson KE, Xia Y, Xiang C. Molecular analysis of the diversity of vaginal microbiota associated with bacterial vaginosis. BMC Genomics 2010; 11: 488.
29. Lamont RF, Sobel JD, Akins RA, Hassan SS, Chaiworapongsa T, Kusanovic JP, Romero R. The vaginal microbiome: new information about genital tract flora using molecular based techniques. BJOG 2011; 118: 533-549.
30. Spear GT, Gilbert D, Sikaroodi M, Doyle L, Green L, Gillevet PM, Landay AL, Veazey RS. Identification of rhesus macaque genital microbiota by 16s pyrosequencing shows similarities to human bacterial vaginosis: implications for use as an animal model for HIV vaginal infection. AIDS Res Hum Retroviruses 2010; 26: 193-200.
31. Bahar H, Torun MM, Oçer F, Kocazeybek B. Mobiluncus species in gynaecological and obstetric infections: antimicrobial resistance and prevalence in a Turkish population. Int J Antimicrob Agents 2005; 25: 268-271.
32. Schuyler JA, Mordechai E, Adelson ME, Sobel JD, Gygax SE, Hilbert DW. Identification of intrinsically metronidazoleresistant clades of Gardnerella vaginalis. Diagnostic Mcrob Inf Dis 2016; 84: 1-3.
33. Togni G, Battini V, Bulgheroni A, Mailland F, Caserini M, Mendling W. In vitro activity of nifuratel on vaginal bacteria: could it be a good candidate for the treatment of bacterial vaginosis? Antimicrob Agents Chemother 2011; 55: 2490- 2492.
34. Cruciani F, Biagi E, Severgnini M, Consolandi C, Calanni F, Donders G, Brigidi P, Vitali B. Development of a microarraybased tool to characterize vaginal bacterial fluctuations and application to a novel antibiotic treatment for bacterial vaginosis. Antimicrob Agents Chemother 2015; 59: 2825-2834.
35. Decena DC, Co JT, Manalastas RM Jr, Palaypayon EP, Padolina CS, Sison JM, Dancel LA, Lelis MA. Metronidazole with Lactacyd vaginal gel in bacterial vaginosis. J Obstet Gynaecol Res 2006; 32: 243-251.
36. Recine N, Palma E, Domenici L, Giorgini M, Imperiale L, Sassu C, Musella A, Marchetti C, Muzii L, Benedetti Panici P. Restoring vaginal microbiota: biological control of bacterial vaginosis. A prospective case-control study using Lactobacillus rhamnosus BMX 54 as adjuvant treatment against bacterial vaginosis. Arch Gynecol Obstet 2016; 1: 101-107.
37. Swidsinski A, Loening-Baucke V, Swidsinski S, Verstraelen H. Polymicrobial Gardnerella biofilm resists repeated intravaginal antiseptic treatment in a subset of women with bacterial vaginosis: a preliminary report. Arch Gynecol Obstet 2015; 291: 605-609.
38. Machado D, Castro J, Palmeira-de-Oliveira A, Martinez-deOliveira J, Cerca N. Bacterial vaginosis biofilms: challenges to current therapies and emerging solutions. Front Microbiol 2016; 6: 1528.