Quantitative Polymerase Chain Reaction to Assess Response to Treatment of Bacterial Vaginosis and Risk of Preterm Birth

Abstract

Objective The aim of this study was to determine whether quantitative polymerase chain reaction (qPCR) bacterial load measurement is a valid method to assess response to treatment of bacterial vaginosis and risk of preterm birth in pregnant women.

Study Design Secondary analysis by utilizing stored vaginal samples obtained during a previous randomized controlled trial studying the effect of antibiotics on preterm birth (PTB). All women had risk factors for PTB: (1) positive fetal fibronectin (n = 146), (2) bacterial vaginosis (BV) and a prior PTB (n = 43), or (3) BV and a prepregnancy weight < 50 kg (n = 54). Total and several individual BV-related bacteria loads were measured using qPCR for 16S rRNA. Loads were correlated with Nugent scores (Spearman correlation coefficients). Loads were compared pre- and posttreatment with Wilcoxon rank-sum test. Individual patient differences were examined with Wilcoxon signed-rank test.

Results A total of 243 paired vaginal samples were available for analysis: 123 antibiotics and 120 placebo. Groups did not differ by risk factors for PTB. For all samples, bacterial loads were correlated with Nugent score and each of its specific bacterial components (all p < 0.01). Baseline total bacterial load did not differ by treatment group (p = 0.87). Posttreatment total bacterial load was significantly lower in the antibiotics group than the placebo group (p < 0.01). Individual patient total bacterial load decreased significantly posttreatment in the antibiotics group (p < 0.01), but not in the placebo group (p = 0.12). The rate of PTB did not differ between groups (p = 0.24). PTB relative risks calculated for BV positive versus BV negative women and women with the highest quartile total and individual bacterial loads were not statistically significant.

Conclusion qPCR correlates with Nugent score and demonstrates decreased bacterial load after antibiotic treatment. Therefore, it is a valid method of vaginal flora assessment in pregnant women who are at high risk for PTB.

Keywords

bacterial vaginosis - bacterial load - quantitative PCR - preterm birth

• References

• 1 Lewit EM, Baker LS, Corman H, Shiono PH. The direct cost of low birth weight. Future Child 1995; 5 (1) 35-56
• 2 Institute of Medicine (US) Committee on Understanding Premature Birth and Assuring Healthy Outcomes. Behrman RE, Butler AS , editors. Preterm Birth: Causes, Consequences, and Prevention. Washington (DC): National Academies Press (US); 2007. . 12, Societal Costs of Preterm Birth. Available from: http://www.ncbi.nlm.nih.gov/books/NBK11358/
• 3 Slattery MM, Morrison JJ. Preterm delivery. Lancet 2002; 360 (9344) 1489-1497
• 4 Hamilton BE, Martin JA, Ventura SJ. Births: Preliminary Data for 2005, Health E-Stats. Center for Disease Control and Prevention National Center for Health Statistics: Hyattsville, MD: 2006
• 5 Martin JA, Hamilton BE, Ventura SJ, Menacker F, Park MM, Sutton PD. Births: final data for 2001. Natl Vital Stat Rep 2002; 51 (2) 1-102
• 6 Martin JA, Kochanek KD, Strobino DM, Guyer B, MacDorman MF. Annual summary of vital statistics—2003. Pediatrics 2005; 115 (3) 619-634
• 7 Martin JA, Hamilton BE, Sutton PD , et al. Births: final data for 2007. Natl Vital Stat Rep 2010; 58 (24) 1-85
• 8 Goldenberg RL, Culhane JF, Iams JD, Romero R. Epidemiology and causes of preterm birth. Lancet 2008; 371 (9606) 75-84
• 9 Gravett MG, Nelson HP, DeRouen T, Critchlow C, Eschenbach DA, Holmes KK. Independent associations of bacterial vaginosis and Chlamydia trachomatis infection with adverse pregnancy outcome. JAMA 1986; 256 (14) 1899-1903
• 10 McDonald HM, O'Loughlin JA, Jolley P, Vigneswaran R, McDonald PJ. Prenatal microbiological risk factors associated with preterm birth. Br J Obstet Gynaecol 1992; 99 (3) 190-196
• 11 Meis PJ, Goldenberg RL, Mercer B , et al; National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. The preterm prediction study: significance of vaginal infections. Am J Obstet Gynecol 1995; 173 (4) 1231-1235
• 12 Hillier SL, Nugent RP, Eschenbach DA , et al; The Vaginal Infections and Prematurity Study Group. Association between bacterial vaginosis and preterm delivery of a low-birth-weight infant. N Engl J Med 1995; 333 (26) 1737-1742
• 13 Edwards RK, Ferguson RJ, Reyes L, Brown M, Theriaque DW, Duff P. Assessing the relationship between preterm delivery and various microorganisms recovered from the lower genital tract. J Matern Fetal Neonatal Med 2006; 19 (6) 357-363
• 14 Ralph SG, Rutherford AJ, Wilson JD. Influence of bacterial vaginosis on conception and miscarriage in the first trimester: cohort study. BMJ 1999; 319 (7204) 220-223
• 15 Silver HM, Sperling RS, St Clair PJ, Gibbs RS. Evidence relating bacterial vaginosis to intraamniotic infection. Am J Obstet Gynecol 1989; 161 (3) 808-812
• 16 Hauth JC, Goldenberg RL, Andrews WW, DuBard MB, Copper RL. Reduced incidence of preterm delivery with metronidazole and erythromycin in women with bacterial vaginosis. N Engl J Med 1995; 333 (26) 1732-1736
• 17 McDonald HM, O'Loughlin JA, Vigneswaran R , et al. Impact of metronidazole therapy on preterm birth in women with bacterial vaginosis flora (Gardnerella vaginalis): a randomised, placebo controlled trial. Br J Obstet Gynaecol 1997; 104 (12) 1391-1397
• 18 Carey JC, Klebanoff MA, Hauth JC , et al; National Institute of Child Health and Human Development Network of Maternal-Fetal Medicine Units. Metronidazole to prevent preterm delivery in pregnant women with asymptomatic bacterial vaginosis. N Engl J Med 2000; 342 (8) 534-540
• 19 McDonald HM, Brocklehurst P, Gordon A. Antibiotics for treating bacterial vaginosis in pregnancy. Cochrane Database Syst Rev 2007; (1) CD000262
• 20 Lamont RF, Duncan SLB, Mandal D, Bassett P. Intravaginal clindamycin to reduce preterm birth in women with abnormal genital tract flora. Obstet Gynecol 2003; 101 (3) 516-522
• 21 Kekki M, Kurki T, Pelkonen J, Kurkinen-Räty M, Cacciatore B, Paavonen J. Vaginal clindamycin in preventing preterm birth and peripartal infections in asymptomatic women with bacterial vaginosis: a randomized, controlled trial. Obstet Gynecol 2001; 97 (5 Pt 1) 643-648
• 22 Ugwumadu A, Manyonda I, Reid F, Hay P. Effect of early oral clindamycin on late miscarriage and preterm delivery in asymptomatic women with abnormal vaginal flora and bacterial vaginosis: a randomised controlled trial. Lancet 2003; 361 (9362) 983-988
• 23 Vermeulen GM, Bruinse HW. Prophylactic administration of clindamycin 2% vaginal cream to reduce the incidence of spontaneous preterm birth in women with an increased recurrence risk: a randomised placebo-controlled double-blind trial. Br J Obstet Gynaecol 1999; 106 (7) 652-657
• 24 Kurkinen-Räty M, Vuopala S, Koskela M , et al. A randomised controlled trial of vaginal clindamycin for early pregnancy bacterial vaginosis. BJOG 2000; 107 (11) 1427-1432
• 25 Newton ER, Prihoda TJ, Gibbs RS. A clinical and microbiologic analysis of risk factors for puerperal endometritis. Obstet Gynecol 1990; 75 (3 Pt 1) 402-406
• 26 Srinivasan S, Hoffman NG, Morgan MT , et al. Bacterial communities in women with bacterial vaginosis: high resolution phylogenetic analyses reveal relationships of microbiota to clinical criteria. PLoS ONE 2012; 7 (6) e37818
• 27 Marrazzo JM, Fiedler TL, Srinivasan S , et al. Extravaginal reservoirs of vaginal bacteria as risk factors for incident bacterial vaginosis. J Infect Dis 2012; 205 (10) 1580-1588
• 28 Fredricks DN, Fiedler TL, Thomas KK, Mitchell CM, Marrazzo JM. Changes in vaginal bacterial concentrations with intravaginal metronidazole therapy for bacterial vaginosis as assessed by quantitative PCR. J Clin Microbiol 2009; 47 (3) 721-726
• 29 Sha BE, Chen HY, Wang QJ, Zariffard MR, Cohen MH, Spear GT. 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 2005; 43 (9) 4607-4612
• 30 Schwebke JR, Lawing LF. Prevalence of Mobiluncus spp among women with and without bacterial vaginosis as detected by polymerase chain reaction. Sex Transm Dis 2001; 28 (4) 195-199
• 31 Cartwright CP, Lembke BD, Ramachandran K , et al. Development and validation of a semiquantitative, multitarget PCR assay for diagnosis of bacterial vaginosis. J Clin Microbiol 2012; 50 (7) 2321-2329
• 32 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 (4) 277-281
• 33 Amsel R, Totten PA, Spiegel CA, Chen KC, Eschenbach D, Holmes KK. Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations. Am J Med 1983; 74 (1) 14-22
• 34 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 (2) 297-301