Nov mikroskopski metod za analizu preparata bojenog po Gramu u dijagnozi poremećaja vaginalne mikroflore
Abstract
Background/Aim. The Nugent’s score is still the gold standard in the great majority of studies dealing with the assessment of vaginal flora and the diagnosis of bacterial vaginosis (BV). The aim of this study was to show that the analysis of Gram-stained vaginal samples under microscope at the magnification of ´200 (a novel microscopic method – NMM), as a fast and simple tool, easily applicable in everyday practice, better reflects complexity of vaginal microflora than the Nugent’s methodology (´1000). Methods. Gram-stained vaginal smears from 394 asymptomatic pregnant women (24–28 week of pregnancy) were classified according to the Nugent’s microscopic criteria (immersion, magnification ´1000). The smears were then reexamined under immersion but at magnification ´200. All samples were classified into 6 groups according to semiquanititative assessment of numbers (cellularity) and the ratio of rod (length < 1.5 μm) and small bacterial (< 1.5 µm) forms: hypercellular (normal full – NF), moderately cellular (normal mid – NM), hypocellular (normal empty – NE), bacterial vaginosis full (BVF), bacterial vaginosis mid (BVM), and bacterial vaginosis empty (BVE). Also yeasts, coccae, bifido and lepto bacterial forms as well polymorphonuclear (PMN) leukocytes were identified. Results. According to the Nugent’s scoring, BV was found in 78, intermediate findings in 63, and yeasts in 48 patients. By our criteria BV was confirmed in 88 patients (37 BVF, 24 BVM, and 27 BVN). Generally, both tools proved to be highly concordant for the diagnosis of BV (Lin’s concordance correlation coefficient = 0.9852). In 40% of the women mixed flora was found: yeasts in 126 (32%), coccae in 145 (37%), bifido forms in 32 (8%) and lepto forms in 20 (5%). Almost a half of BV patients had also yeasts (39/88). Elevated PMN numbers were found in 102 (33%) patients with normal and in 36 (41%) women with BV. Conclusion. The newly described methodology is simpler to apply and much better reflects diversity of vaginal microflora. In this way it may be more valuable to molecular biologists and their attempts based on quantitative polymerase chain reaction (PCR) to define formulas for molecular diagnosis of bacterial vaginosis.
References
Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bac-terial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol 1991; 29(2): 297−301.
Ison CA, Hay PE. Validation of a simplified grading of Gram stained vaginal smears for use in genitourinary medicine clinics. Sex Transm Infect 2002; 78(6): 413−5.
Tohill BC, Heilig CM, Klein RS, Rompalo A, Cu-Uvin S, Brown W, et. Vaginal flora morphotypic profiles and assessment of bacterial vaginosis in women at risk for HIV infection. Infect Dis Obstet Gynecol 2004;12(3-4):121-6.
Pereira L, Culhane J, Mccollum K, Agnew K, Nyirjesy P. Variation in microbiologic profiles among pregnant women with bacterial vaginosis. Am J Obstet Gynecol 2005; 193(3): 746−51.
Srinivasan S, Morgan MT, Liu C, Matsen FA, Hoffman NG, Fiedler TL, et al. More than meets the eye: associations of vaginal bacteria with gram stain morphotypes using molecular phylogenetic analysis. PLoS One 2013; 8(10): 78633.
Srinivasan S, Hoffman NG, Morgan MT, Matsen FA, Fiedler TL, Hall RW, 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): 37818.
Brotman RM, Ravel J, Cone RA, Zenilman JM. Rapid fluctuation of the vaginal microbiota measured by Gram stain analysis. Sex Transm Infect 2010; 86(4): 297−302.
Menard JP, Mazouni C, Fenollar F, Raoult D, Boubli L, Bretelle F. Diagnostic accuracy of quantitative real-time PCR assay versus clinical and Gram stain identification of bacterial vaginosis. Eur J Clin Microbiol Infect Dis 2010; 29(12): 1547−52.
Verhelst R, Verstraelen P, Cools P, Lopes dos Santos Santiago G , Temmerman M, Veneechoutte M. Garnderella. In: Liu D, editor. Molecular detection of human bacterial pathogens. Boca Ra-ton: Press Taylor & Fracis Group; 2011. p. 81−95.
de Backer E, Verhelst R, Verstraelen H, Alqumber MA, Burton JP, Tagg JR, et al. Quantitative determination by real-time PCR of four vaginal Lactobacillus species. Gardnerella vaginalis and Atopobium vaginae indicates an inverse relationship between L. gasseri and L. iners. BMC Microbiol 2007; 19(7): 115.
Lamont RF, Sobel JD, Akins RA, Hassan SS, Chaiworapongsa T, Kusanovic JP, et al. The vaginal microbiome: new information about genital tract flora using molecular based techniques. BJOG 2011; 118(5): 533−49.
Thies FL, König W, König B. Rapid characterization of the nor-mal and disturbed vaginal microbiota by application of 16S rRNA gene terminal RFLP fingerprinting. J Med Microbiol 2007; 56(Pt 6): 755−61.
Fredricks DN, Fiedler TL, Marrazzo JM. Molecular identification of bacteria associated with bacterial vaginosis. N Engl J Med 2005; 353(18): 1899−911.
Zhou X, Brotman RM, Gajer P, Abdo Z, Schüette U, Ma S, et al. Recent advances in understanding the microbiology of the fe-male reproductive tract and the causes of premature birth. In-fect Dis Obstet Gynecol 2010; 2010: 737425.
Aagaard K, Riehle K, Ma J, Segata N, Mistretta TA, Coarfa C, et al. A metagenomic approach to characterization of the vaginal microbiome signature in pregnancy. PLoS One 2012; 7(6): e36466.
Zhou X, Bent SJ, Schneider MG, Davis CC, Islam MR, Forney LJ. Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods. Mi-crobiology 2004; 150(Pt 8): 2565−73.
Zhou X, Brown CJ, Abdo Z, Davis CC, Hansmann MA, Joyce P, et al. Differences in the composition of vaginal microbial com-munities found in healthy Caucasian and black women. ISME J 2007; 1(2): 121−33.
Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SS, Mcculle SL, et al.Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci USA 2011; 108(Suppl 1): 4680−7.
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.
Larsson P, Carlsson B, Fåhraeus L, Jakobsson T, Forsum U. Diag-nosis of bacterial vaginosis: need for validation of microscopic image area used for scoring bacterial morphotypes. Sex Transm Infect 2004; 80(1): 63−7.
Verhelst R, Verstraelen H, Claeys G, Verschraegen G, Van Simaey L, de Ganck C, et al. Comparison between Gram stain and culture for the characterization of vaginal microflora: definition of a distinct grade that resembles grade I microflora and revised categorization of grade Imicroflora. BMC Microbiology 2005; 5(1): 61.
Lin LI. A concordance correlation coefficient to evaluate re-producibility. Biometrics 1989; 45(1): 255−68.
Sobel JD, Subramanian C, Foxman B, Fairfax M, Gygax SE. Mixed vaginitis-more than coinfection and with therapeutic implications. Curr Infect Dis Rep 2013; 15(2): 104−8.
Forsum U, Jakobsson T, Larsson PG, Schmidt H, Beverly A, Bjør-nerem A, et al. An international study of the interobserver vari-ation between interpretations of vaginal smear criteria of bac-terial vaginosis. APMIS 2002; 110(11): 811−8.
Menard J, 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(1): 33−43.
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(6): 828−36.
Shipitsyna E, Roos A, Datcu R, Hallén A, Fredlund H, Jensen JS, et al. Composition of the vaginal microbiota in women of repro-ductive age--sensitive and specific molecular diagnosis of bac-terial vaginosis is possible. PloS One 2013; 8(4): 60670.
Menard JP, Fenollar F, Raoult D, Boubli L, Bretelle F. 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 2012; 31(4): 513−8
Ling XZ, Kong MJ, Liu F, Zhu BH, Chen YX, Wang ZY, et al. Molecular analysis of the diversity of vaginal microbiota asso-ciated with bacterial vaginosis. BMC Genomics 2010; 11(1): 488.
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): 721−6.
Biagi E, Vitali B, Pugliese C, Candela M, Donders GGG, Brigidi P. Quantitative variations in the vaginal bacterial population as-sociated with asymptomatic infections: a real-time polymerase chain reaction study. Eur J Clin Microbiol Infect Dis 2009; 28(3): 281−5.
Libman MD, Kramer M, Platt R. Comparison of Gram and Ko-peloff stains in the diagnosis of bacterial vaginosis in pregnan-cy. Diagn Microbiol Infect Dis 2006; 54(3): 197−201.
McDonald HM, Brocklehurst P, Gordon A. Antibiotics for treatin g bacterial vaginosis in pregnancy. Cochrane Database Syst Rev 2007; 2(1): CD00026.
Cartwright CP, Lembke BD, Ramachandran K, Body BA, Nye MB, Rivers CA, et al. Development and validation of a semiquanti-tative, multitarget PCR assay for diagnosis of bacterial vagi-nosis. J Clin Microbiol 2012; 50(7): 2321−9.
Cartwright CP, Lembke BD, Ramachandran K, Body BA, Nye MB, Rivers CA, et al. Comparison of nucleic acid amplification as-says with BD affirm VPIII for diagnosis of vaginitis in symp-tomatic women. J Clin Microbiol 2013; 51(11): 3694−9.