Cervical and vaginal flora is highly concordant with respect to bacterial vaginosis-associated

Transcription

1 JCM Accepts, published online ahead of print on June 0 J. Clin. Microbiol. doi:0.8/jcm Copyright 0, American Society for Microbiology. All Rights Reserved Cervical and vaginal flora is highly concordant with respect to bacterial vaginosis-associated organisms and commensal lactobacillus species in reproductive-aged women William L. Smith B.S. a, Spencer R. Hedges Ph.D. a, Eli Mordechai Ph.D. a,b, Martin E. Adelson Ph.D. a,b Jason P. Trama Ph.D. b, Scott E. Gygax Ph.D. a, Andrew M. Kaunitz M.D. c and David W. Hilbert Ph.D. a # a Femeris Women s Health Research Center and b Oncoveda Cancer Research Center, Medical Diagnostic Laboratories, A Member of Genesis Biotechnology Group, 9 Kuser Rd., Hamilton, New Jersey, USA c Department of Obstetrics and Gynecology, University of Florida College of Medicine, Jacksonville, Florida, USA Running head: Cervical and vaginal microbial flora #Address correspondence to: dhilbert@mdlab.com

2 ABSTRACT Matched vaginal and cervical specimens from 96 subjects were analyzed by quantitative polymerase chain reaction for the presence and concentration of bacterial vaginosis-associated microbes and commensal Lactobacillus spp. Detection of these microbes was 9% concordant, indicating that microbial flora at these body sites is generally similar.

3 The vaginal microbiome plays an important role in female reproductive tract health. The vaginal microbiome of healthy women generally falls into five categories, four of which are dominated by a single lactobacillus species (L. crispatus, L. gasseri, L. iners or L. jensenii), and a fifth that is characterized by diverse anaerobic and facultative species (). This final group has traditionally been associated with bacterial vaginosis (BV), a disease characterized by vaginal discharge and odor (), as well as Human Immunodeficiency Virus (HIV) transmission (), Trichomonas vaginalis infection () and pre-term labor (). Although less well-studied, the cervical microbiome may arguably be of greater relevance, as the cervix is the site of infection by numerous pathogens such as HIV, human papilloma virus (HPV), Chlamydia trachomatis and Neisseria gonorrhea. A recent study found that a lactobacillus-dominated cervical microbiome is inversely associated with the prevalence of HIV, herpes simplex virus type and high-risk human papilloma virus (6). Studies evaluating the cervico-vaginal flora have variously sampled the cervix, vagina or both sties simultaneously. Therefore, it is of general interest to comparatively analyze the flora at these sites to determine if results generated from sampling the vagina can be generalized to the cervix and vice-versa. For example, the cervcal transformation zone is enriched in T-cells and antigen presenting cells compared to the vagina (7), and thus differential immune at these sites could impact the composition of their respective microbiomes. To our knowledge only two prior studies have addressed this issue (8, 9), and although informative, these studies analyzed small subject cohorts and utilized semiquantitative methodology. Our goal in this study was to quantitatively analyze the cervical and vaginal flora with respect to lactobacillus species as well as microbes associated with abnormal flora, such as BV, from a substantial subject cohort.

4 We performed a cross-sectional study from a prospective cohort, enrolling subjects who were receiving ambulatory gynecologic care at either the Care Center for Women at University of Florida Health (a teaching hospital with related ambulatory clinics) or the University of Florida Southside Women s Health Specialists (a freestanding ambulatory care center). The study took place from May 006 to June 009. Written informed consent was obtained from all subjects and the study was carried out with the approval of Western IRB. The purpose of the study was to evaluate the microbiology and immunology of BV. Therefore, subjects were recruited who were either complaining of vaginitis symptoms (e.g. itching, discharge or odor) or were attending for well-visits or routine care. Subject information was collected by completing a questionnaire. Exclusion criteria included pregnancy and menopausal or post-menopausal status, as these conditions are known to influence the composition of the vaginal flora (0, ). In addition, patients were excluded if they were positive for Chlamydia trachomatis, Neisseria gonorrhea or Trichomonas vaginalis as these infections can provoke cervical and/or vaginal inflammation which could confound immunological analysis of BV. In this study we analyzed separate vaginal and cervical specimens from 96 subjects. Subject ages ranged from 8- years, with an average of. years. Race was self-reported; subjects (6%) were Caucasian, (%) were African American, 9 (9.%) were Hispanic, was Asian (.0%) and was Other (.0%). Patients were diagnosed for BV using the following signs and symptoms (i) vaginal discharge, (ii) odor, (iii) elevated vaginal ph and (iv) the presence of clue cells (exfoliated vaginal epithelial cells with attached bacteria) (i.e. Amsel s Criteria) (). Twenty-four subjects (%) were diagnosed as having BV (Amsel s Criteria=-), subjects (%) neither had BV nor were completely healthy (Amsel s Criteria=-) and subjects (%) were healthy 88 (Amsel s Criteria=0). Specimens were collected with OneSwab collection devices (Medical

5 Diagnostic Laboratories). After placing a vaginal speculum and visualizing the cervix, the os was swabbed. Immediately thereafter, a second swab was taken from the left or right mid-vaginal sidewall. These samples were stored at C overnight and shipped to Medical Diagnostic Laboratories where they were stored at -0 C until analyzed. DNA extracted from OneSwab transport medium by the X-tractor Gene automated nucleic acid extraction system (Corbett Robotics, San Francisco, CA) according to the manufacturer s instructions. A panel of quantitative real time- PCR assays was used to quantify DNA from BV-associated microbes and commensal lactobacillus species (Supplemental Table ). The targets of these reactions are as follows: A. vaginae (tuf encoding Elongation Factor Tu), BVAB (6S rdna) (), G. vaginalis (hisc gene encoding Histidinol-Phosphate Aminotransferase), Megasphaera spp. (6S rdna) () and lactobacillus species (tuf encoding Elongation Factor Tu) (). All assays have a limit of detection of 0 copies of target per reaction, do not cross-react with DNA from a diverse panel of microbes commonly found in the female reproductive tract, and exhibit linear amplification from 0 to 0 8 target copies per reaction () (data not shown). All reaction plates included separate no template control wells. The quantity of PCR target copies detected is expressed per reaction, each one using 0. l of DNA as template in a l reaction. The final primer concentrations were nm. All of the reactions were run using the following conditions: minutes at 0 C, minutes at 9 C, followed by 0 cycles of 0 seconds at 9 C (melting) and seconds at 60 C (annealing and extension). The Megasphaera spp. reaction is run as a duplex PCR using both Type and Type probes, and the Lactobacillus spp. PCR is run as a multiplex, using probes for L. crispatus, L. gasseri, L. iners and L. jensenii. All reactions were performed using a CFX8 Real-Time Thermocycler (Bio-Rad). For

6 each reaction, a standard curve was generated using reactions with 0, 0 or 0 6 copies of a plasmid encoding the amplification target. The distribution of PCR target copy concentration in patient specimens was non-normal (D Agonstino and Pearson omnibus normality test). Therefore, to detect correlations we determined Spearman s rank correlation coefficient. Only comparisons that remained significant after controlling for multiple comparisons using the Bonferroni correction are reported. Statistical analysis was performed using Prism.0 (GraphPad). For significance testing, an alpha of 0.0 was used. We compared cervical and vaginal specimens from the same patients to determine concordance for detection for these microbes (Table ). We found high concordance rates: 9% for A. vaginae, Megasphaera spp and L. jensenii; 9% for BVAB and L. crispatus; 9% for L. gasseri; 8% for G. vaginalis and 8% for L. iners. In total, 70 out of 768 test results were concordant between specimens for each patient, for an overall concordance rate of 9%. Of these concordant results, 88 were matched positive specimens and 7 were matched negative specimens. Of the 6 instances of discordant test results, 9 were cervical-positive and vaginal-negative and were the converse. We next analyzed swab pairs where at least one was positive for the quantity of PCR target in each sample (Fig. ). We found that concentrations at the two sites were correlated for G. vaginalis (P=0.006, =0.) and BVAB (P < 0.00, =0.67). No significant correlations were observed for the other microbes detected.. In addition, there were no significant differences in the concentration of genomic copies of individual microbes when cervical and vaginal specimens were compared (data not shown). 6

7 The frequent co-colonization of the cervix and vagina with the same microbes is not surprising in light of the essentially continuous nature of the lower female reproductive tract. Interestingly, only two of the eight microbes assayed G. vaginalis and BVAB were correlated with respect to quantity at the two sites.. Studies of G. vaginalis have found that virulent strains are superior to commensal strains in their ability to adhere to both vaginal () and cervical (6) epithelium, which may contribute to the correlated concentrations we observed at these sites. In addition, G. vaginalis has a highly diverse population structure (7) and co-colonization with multiple strains is common (8); possibly we are observing one strain predominating at the cervix and the other the vagina. BVAB and vaginal Megasphaera spp. have not yet been cultured in the laboratory so their adherence properties are unknown; although A. vaginae has been cultured the relevant studies are also lacking. With respect to lactobacillus, a study of human vaginal isolates found that they varied greatly in their ability to adhere to vaginal epithelial cells (9). In addition, an adhesin has been identified in L. crispatus that specifically mediates adherence to stratified squamous epithelium (including vaginal epithelium) but not to columnar epithelium (which constitutes the cervical epitihelium) and its expression is strain-specific (0). Therefore, we speculate that some strains of lactobacillus may be differentially able to colonize the two sites, resulting in the poor correlation we observed. One major possible confounding factor in this study is contamination. One trivial explanation for similarity between cervical and vaginal flora is that cross-contamination occurred during sampling. Based upon physical anatomy it is highly unlikely that a vaginal specimen would be contaminated with cervical flora; in contrast, it is more plausible than a cervical specimen could come in contact 7

8 with the vaginal wall after sampling, resulting in contamination with vaginal flora. There are several observations that lead us to reject cross-contamination as the most likely explanation for our observations. First, of the 6 instances of discordance, 9 of them (6%) were positive cervical specimens and negative vaginal specimens, which cannot be explained by contamination of cervical specimens with vaginal flora. Second, we chose to analyze specimens which were concordant for multiple organisms and compared the concentration, as we would expect to observe a consistently higher concentration in vaginal specimens if systematic contamination occurred. However, among such subjects, we found that for of them (6%) at least one microbe was present at a higher concentration in the cervical specimen than in the vaginal specimen. Again, this pattern argues against cross-contamination as an explanation for the cervical-vaginal concordance. However, future studies could consider the utilization of sham cervical samples as a control for cross-contamination. With respect to prior studies examining cervical and vaginal flora, Kim et al. analyzed a cohort of eight subjects, sampled from the cervix and several vaginal sites using lavage, swabbing and scraping, and PCR amplified 6S rdna, generated amplicon libraries and sequenced them to evaluate microbial community composition (8). Their results were broadly similar to ours, in that the same microbes, such as Lactobacillus spp., Pseudomonas spp. and G. vaginalis, were detected at the cervix and vagina in the same subjects but their concentration varied greatly. The methodology used in this study is advantageous in that it is unbiased in detection; however, it is only semi-quantiative and failed to resolve the lactobacillus detected to the species level. Another study, using DNA hybridization, analyzed cervical and vaginal specimens from patients and also found that microbes such as G. vaginalis, A. vaginae and L. iners were present in both specimens from the 8

9 same paients (9). Again, quantitative PCR is a superior method to DNA hybridization with respect to sensitivity and quantitation. In summary, utilizing quantitative PCR methods to analyze a series of BV-associated and commensal lactobacillus species we found a high overall degree of concordance with respect to colonization, although the quantity of a particular microbe detected at both sites varied substantially. With respect to diagnostic testing for the microbes in question, cervical and vaginal specimens are generalizable with respect to qualitative detection; however, quantitative methodology may require independent validation for cervical and vaginal specimens. 9

10 References. Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SS, McCulle SL, Karlebach S, Gorle R, Russell J, Tacket CO, Brotman RM, Davis CC, Ault K, Peralta L, Forney LJ. 0. Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci U S A 08 Suppl : Sobel JD Vaginitis. N Engl J Med 7: Mirmonsef P, Krass L, Landay A, Spear GT. 0. The role of bacterial vaginosis and trichomonas in HIV transmission across the female genital tract. Curr HIV Res 0:0-0.. Martin DH, Zozaya M, Lillis RA, Myers L, Nsuami MJ, Ferris MJ. 0. Unique vaginal microbiota that includes an unknown Mycoplasma-like organism is associated with Trichomonas vaginalis infection. J Infect Dis 07:9-9.. Hillier SL, Nugent RP, Eschenbach DA, Krohn MA, Gibbs RS, Martin DH, Cotch MF, Edelman R, Pastorek JG, nd, Rao AV, McNellis D, Regan JA, Carey JC, Klebanoff MA. 99. Association between bacterial vaginosis and preterm delivery of a low-birthweight infant. The Vaginal Infections and Prematurity Study Group. N Engl J Med : Borgdorff H, Tsivtsivadze E, Verhelst R, Marzorati M, Jurriaans S, Ndayisaba GF, Schuren FH, van de Wijgert JH. 0. Lactobacillus-dominated cervicovaginal microbiota associated with reduced HIV/STI prevalence and genital HIV viral load in African women. The ISME journal. 0

11 Pudney J, Quayle AJ, Anderson DJ. 00. Immunological microenvironments in the human vagina and cervix: mediators of cellular immunity are concentrated in the cervical transformation zone. Biol Reprod 7: Kim TK, Thomas SM, Ho M, Sharma S, Reich CI, Frank JA, Yeater KM, Biggs DR, Nakamura N, Stumpf R, Leigh SR, Tapping RI, Blanke SR, Slauch JM, Gaskins HR, Weisbaum JS, Olsen GJ, Hoyer LL, Wilson BA Heterogeneity of vaginal microbial communities within individuals. J Clin Microbiol 7: Nikolaitchouk N, Andersch B, Falsen E, Strombeck L, Mattsby-Baltzer I The lower genital tract microbiota in relation to cytokine-, SLPI- and endotoxin levels: application of checkerboard DNA-DNA hybridization (CDH). APMIS 6: Romero R, Hassan SS, Gajer P, Tarca AL, Fadrosh DW, Nikita L, Galuppi M, Lamont RF, Chaemsaithong P, Miranda J, Chaiworapongsa T, Ravel J. 0. The composition and stability of the vaginal microbiota of normal pregnant women is different from that of non-pregnant women. Microbiome :.. Brotman RM, Shardell MD, Gajer P, Fadrosh D, Chang K, Silver MI, Viscidi RP, Burke AE, Ravel J, Gravitt PE. 0. Association between the vaginal microbiota, menopause status, and signs of vulvovaginal atrophy. Menopause :0-8.. Amsel R, Totten PA, Spiegel CA, Chen KC, Eschenbach D, Holmes KK. 98. Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations. Am J Med 7:-.

12 Fredricks DN, Fiedler TL, Thomas KK, Oakley BB, Marrazzo JM Targeted PCR for detection of vaginal bacteria associated with bacterial vaginosis. J Clin Microbiol : Balashov SV, Mordechai E, Adelson ME, Sobel JD, Gygax SE. 0. Multiplex quantitative polymerase chain reaction assay for the identification and quantitation of major vaginal lactobacilli. Diagn Microbiol Infect Dis 78:-7.. Jr HM, JM A, GA B, JL P, AT O, PH G, KK J. 00. Drawing the line between commensal and pathogenic Gardnerella vaginalis through genome analysis and virulence studies. BMC Genomics :7. 6. Castro J, Henriques A, Machado A, Henriques M, Jefferson K, Cerca N. 0. Reciprocal interference between Lactobacillus spp. and Gardnerella vaginalis on initial adherence to epithelial cells. Int J Med Sci 0: Ahmed A, Earl J, Retchless A, Hillier SL, Rabe LK, Cherpes TL, Powell E, Janto B, Eutsey R, Hiller NL, Boissy R, Dahlgren ME, Hall BG, Costerton JW, Post JC, Hu FZ, Ehrlich GD. 0. Comparative genomic analyses of 7 clinical isolates of Gardnerella vaginalis provide evidence of multiple genetically isolated clades consistent with subspeciation into genovars. J Bacteriol 9: Balashov SV, Mordechai E, Adelson ME, Gygax SE. 0. Identification, quantification and subtyping of Gardnerella vaginalis in noncultured clinical vaginal samples by quantitative PCR. J Med Microbiol 6:6-7.

13 Andreu A, Stapleton AE, Fennell CL, Hillier SL, Stamm WE. 99. Hemagglutination, adherence, and surface properties of vaginal Lactobacillus species. J Infect Dis 7: Edelman SM, Lehti TA, Kainulainen V, Antikainen J, Kylvaja R, Baumann M, Westerlund-Wikstrom B, Korhonen TK. 0. Identification of a high-molecular-mass Lactobacillus epithelium adhesin (LEA) of Lactobacillus crispatus ST that binds to stratified squamous epithelium. Microbiology 8:7-7.

14 Figure Legends Figure. Quantity of PCR target copies measured in matched cervical and vaginal specimens. P values are displayed for significant correlations. Due to the log scale, discordant samples (where one sample as a value of zero) are not displayed. Downloaded from on October, 08 by guest

15 Figure G. vaginalis BVAB P=0.006 P<0.00 Cervical (log copies) Cervical (log copies) A. vaginae Megasphaera spp. 6 Cervical (log copies) 6 Cervical (log copies) Vaginal ( log copies) Vaginal (log copies) Vaginal (lo g copies) Vaginal (log copies)

16 Vaginal ( log copies) Vaginal (lo og copies/rxn) L. crispatus 6 Cervical (log copies) L. gasseri Cervical (log copies/rxn) ) Vaginal (lo og copies/rxn) Vaginal (log co opies) L. jensenii Cervical (log copies/rxn) L. iners Cervical (log copies)

17 Table. Concordance for detection of microbial genomic DNA in matched specimens from 96 subjects. n (%) Concordant Discordant Organism Total Cer+ Vag+ Cer- Vag- Total Cer+ Vag- Cer- Vag+ G. vaginalis 8 (8) () 8 (0) () 7 (7.) 7 (7.) A. vaginae 9 (9) 8 (9) 6 (66) (.) (.) (.) BVAB 90 (9) 9 (0) 7 (7) 6 (6.) (.) (.) Megasphaera spp. 9 (9) 6 (7) 6 (68) (.) (.) (.0) L. crispatus 90 (9) () 67 (70) 6 (6.) (.) (.) L. gasseri 89 (9) 6 (6.) 8 (86) 7 (7.) (.) (.) L. jensenii 9 (9) 8 (8.) 8 (86) (.) (.) (.) L. iners 78 (8) 0 (9) 8 (0) (6) 6 (6.) 9 (9.) Total 70 (9) 88 () 7 (67) 6 (8.) 9 (.8) (.)