MAJOR ARTICLE. Thomas L. Cherpes, 1,4 Melissa A. Melan, 2 Jeffrey A. Kant, 2 Lisa A. Cosentino, 4 Leslie A. Meyn, 4 and Sharon L.

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1 MAJOR ARTICLE Genital Tract Shedding of Herpes Simplex Virus Type 2 in Women: Effects of Hormonal Contraception, Bacterial Vaginosis, and Vaginal Group B Streptococcus Colonization Thomas L. Cherpes, 1,4 Melissa A. Melan, 2 Jeffrey A. Kant, 2 Lisa A. Cosentino, 4 Leslie A. Meyn, 4 and Sharon L. Hillier 3,4 1 Department of Medicine, Division of Infectious Disease, 2 Division of Molecular Diagnostics, 3 Department of Obstetrics, Gynecology, and Reproductive Sciences, and 4 Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania Background. Genital infections due to herpes simplex virus type 2 (HSV-2) are characterized by frequent reactivation and shedding of the virus and by the attendant risk of transmission to sexual partners. We investigated the effects of vaginal coinfections and hormonal contraceptive use on genital tract shedding of HSV-2 in women. Methods. A total of 330 HSV-2 seropositive women were followed every 4 months for a year. At each visit, one vaginal swab specimen was obtained for detection of HSV-2 by polymerase chain reaction, a second vaginal swab specimen was obtained for detection of group B Streptococcus (GBS) organisms and yeast by culture, and a vaginal smear was obtained for the diagnosis of bacterial vaginosis by Gram staining. Results. HSV-2 DNA was detected in 88 (9%) of 956 vaginal swab specimens. Independent predictors of genital tract shedding of HSV-2 were HSV-2 seroconversion during the previous 4 months (adjusted odds ratio [aor], 3.0; 95% confidence interval [CI], ), bacterial vaginosis (aor, 2.3; 95% CI, ), high-density vaginal GBS colonization (aor, 2.2; 95% CI, ), and use of hormonal contraceptives (aor, 1.8; 95% CI, ). Conclusions. The present study identifies hormonal contraceptive use, bacterial vaginosis, and high-density vaginal GBS colonization as risk factors for genital tract shedding of HSV-2 in women. Because hormonal contraceptives are used by millions of women worldwide and because bacterial vaginosis and vaginal GBS colonization are common vaginal conditions, even modest associations with HSV-2 shedding would result in substantial attributable risks for transmission of the virus. There are well-established correlates of genital tract shedding of herpes simplex virus type (HSV-2) in women, including recent acquisition of infection, young age ( 25 years), pregnancy, and coinfection with HIV [1 4]. The association of genital tract shedding of HSV-2 with hormonal contraceptive use is, however, less clear. In 1990, an investigation of 27 women who had recurrent genital herpes infection and from whom genital Received 29 September 2004; accepted 6 January 2005; electronically published 29 March Presented in part: Annual scientific meeting and symposium of the Infectious Disease Society of Obstetrics and Gynecology, San Diego, 5 August 2004 (oral presentations, session II). Reprints or correspondence: Dr. Thomas L. Cherpes, Magee-Womens Research Institute, 204 Craft Ave., Rm. 540, Pittsburgh, PA (rsitc@mwri.magee.edu). Clinical Infectious Diseases 2005; 40: by the Infectious Diseases Society of America. All rights reserved /2005/ $15.00 tract specimens were obtained daily did not detect an increase in the frequency of HSV-2 shedding in association with the use of hormonal contraceptives [5]. Ten years later, differing results were reported in a crosssectional study of 273 women who were seropositive for herpes simplex virus type 1 (HSV-1), HSV-2, and HIV-1, because genital shedding of HSV-2 was significantly associated with the use of either oral or injectable hormonal contraceptives [6]. A more recent longitudinal study, which compared the frequency of genital tract shedding of HSV-2 among 200 HSV-2 and HIV-1 seropositive women before and 60 days after initiation of hormonal contraceptive therapy found no significant differences [7]. However, extension of these results to an HIV-seronegative population of women or to women using hormonal contraceptives for more-extended periods may not be warranted. Even less completely delineated than the effect of 1422 CID 2005:40 (15 May) Cherpes et al.

2 hormonal contraceptive use on the frequency of genital tract shedding of HSV-2 in women is the effect of other genital tract coinfections. The primary objective of the present investigation, therefore, was to determine the effects of hormonal contraceptive use, vaginal group B Streptococcus (GBS) colonization, vaginal yeast colonization, or bacterial vaginosis on genital tract shedding of HSV-2 in women. PATIENTS AND METHODS Study population From 1998 through 2000, a longitudinal cohort investigation of the risk factors associated with the acquisition of vaginal GBS was conducted [8]. Asymptomatic women aged years who were willing to return for an additional 3 visits (at 4-month intervals) were enrolled from 3 health clinics in the Pittsburgh, Pennsylvania, area. Women were excluded from the study if they were pregnant, were currently experiencing genital tract symptoms or bleeding, or were currently using systemic antimicrobials. Only women (1) who were not using antiviral agents and who were identified as having an HSV-2 seropositive status at enrollment, or (2) who acquired serum antibodies to HSV-2 during follow-up were included in the current analysis. Although no women who were included in the present analysis were using any antiviral agents, the HIV antibody status of the study participants was not determined. The use of this subset of the original cohort for an assessment of the risk factors associated with genital tract shedding of HSV-2 was approved by the institutional review board of the Magee-Womens Hospital (Pittsburgh, PA). After written, informed consent was obtained, interview data were obtained via a standardized questionnaire administered by trained research personnel. The interview consisted of closed-format questions that were related to demographic characteristics, medical history, use of intravaginal products, and sexual behaviors and contraceptive practices. Laboratory Methods Detection of vaginal GBS and yeast. At each study visit, data on demographic and behavioral characteristics, as determined by patient interviews, were collected; in addition, 2 vaginal swab specimens, a vaginal smear, and a serum sample were obtained from each patient at each visit. For the collection of vaginal swab specimens, a sterile polyester fiber (Dacron; Invista) swab was inserted at least 3 cm past the vaginal introitus and was rotated 10 times around the vaginal wall. One vaginal swab specimen was placed in Amies transport gel (MML Diagnostics Packaging) for detection of GBS and yeast by culture. This vaginal swab specimen was inoculated onto a Columbia agar plate by use of a 4-zone semiquantitative streaking method and was then inoculated into selective broth medium. Both the plates and the broth cultures were incubated overnight in 5% carbon dioxide at 37 C. Growth of GBS was classified as light if it occurred only in broth culture, in the first streak zone on the plate, or in the first and second streak zones. These 3 lightgrowth patterns denote the presence of 10 5 cfu of GBS per ml of vaginal fluid [9]. Growth of GBS was classified as heavy (110 5 cfu/ml) when it was also detected in the third or fourth streak zones. GBS was presumptively identified on the basis of Gram stain morphologic findings and a negative catalase reaction, and detection was confirmed by means of the PathoDX agglutination system (Diagnostic Products). After 48 h of incubation, samples from the broth cultures were subcultured onto Sabouraud dextrose agar plates (Gibson Laboratories) for the detection of yeast. Detection of genital tract HSV-2. The second vaginal swab specimen that was obtained was placed in a cryovial that contained 200 ml of PBS and was stored at 70 C prior to its subsequent utilization for HSV-2 DNA detection with real-time PCR. Detection of HSV-2 DNA in this specimen was performed using real-time PCR that amplified a portion of the HSV-2 DNA polymerase gene (forward primer, 5 -ATGGTGAACATC- GACATGTACGG-3 ; reverse primer, 5 -CCTCCTTGTCGAG- GCCCCGAAAC-3 ). In brief, DNA was extracted using the IT R.A.P.I.D. DNA Purification Kit (Idaho Technologies) according to the manufacturer s directions. The final elution volume from these extractions was reduced from 400 ml to 200 ml, and samples were amplified using the ABI Prism 7000 Sequence Detection System (Applied Biosystems). A total of 22 ml of extracted DNA was added to a master mix that consisted of 0.2 mmol of each of the 2 HSV-2 DNA polymerase gene specific primers per liter, 2% dimethyl sulfoxide, and Sybr Green PCR Master Mix containing ROX passive dye and Amp- Erase Enzyme (Applied Biosystems). After incubation at 50 C for 2 min and incubation at 95 C for 10 min, the samples were amplified for 40 cycles at 95 C for 15 s and at 60 C for 1 min. Fluorescence was read at the end of the extension step (performed at 60 C) of each cycle, to confirm application. Dissociation-curve analysis was then performed to detect the presence of HSV-2 specific product that had a length of 350 bp and a melting temperature of 88 C. Confirmation of HSV-2 specific product was done by comparing the melting peak of a particular patient sample with the peaks obtained from amplification of purified HSV-2 genomic DNA (Advanced Biotechnologies). Positive control standards that contained 1000, 100, or 10 genomic copies per reaction were run for each assay. In all cases, the 10-copy standard showed positive results of amplification. Patient samples that displayed a melting peak that matched the control peaks when superimposed were considered to have positive results. Detection of bacterial vaginosis and HSV-2 infection. Bacterial vaginosis is characterized by the replacement of the normally predominant lactobacilli in the vagina with anaerobic gram-negative rods, genital mycoplasmas, and Gardnerella vagi- Risk Factors for HSV-2 Shedding in Women CID 2005:40 (15 May) 1423

3 Table 1. Univariate correlates of genital tract shedding of herpes simplex virus type 2 (HSV-2) in 330 women aged years, according to selected demographic characteristics. Characteristic No. of women with characteristic No. (%) of women with HSV-2 shedding OR (95% CI) P Race White (21) 1.0 Black (26) 1.0 ( ).95 Other a 8 2 (25) 1.1 ( ).9 Age, years (16) (28) 2.2 ( ) (27) 2.1 ( ).02 Living arrangement With partner (24) 1.0 Without partner (25) 1.3 ( ).3 Education 112 years (20) years (31) 1.6 ( ).04 a Women of Hispanic, Asian, Native American, or multiracial backgrounds. nalis. Vaginal smears underwent Gram staining, and the vaginal flora was evaluated according to a standardized 0 10-point scoring system in which normal (lactobacilli-dominated) vaginalflora received a score of 0 3 points, intermediate flora received a score of 4 6 points, and bacterial vaginosis received a score of 7 10 points [10]. Serum samples were stored at 70 C until they were tested for type-specific HSV-2 antibodies by use of a commercially available EIA, as described elsewhere [11]. Statistical Analysis All statistical analyses were performed using Stata statistical software, version 8.0 (Stata), and all statistical tests were evaluated with statistical significance denoted by P p.05. Generalized estimating equations (GEEs) were used to identify factors that were associated with the detection of HSV-2 in vaginal swab specimens. GEE is a regression method that is able to model the marginal expectation of repeated, correlated outcomes with both time-dependent and time-independent covariates. An exchangeable working correlation matrix was specified, and modified sandwich estimates of the variance were calculated. Statistical inference was based on the generalized Wald test statistic [12]. Multivariable GEE models were developed using forward stepwise regression. Variables were retained in the model if the Wald test statistic had a P value of.05. The population-averaged ORs that were derived from both the univariate and multivariable GEE models are presented along with the 95% CIs and P values. RESULTS At enrollment, 299 women were identified as HSV-2 seropositive, and another 31 women acquired HSV-2 infection during follow-up. Of the 330 HSV-2 seropositive women who were included in the present analysis, 213 (65%) described themselves as black, 109 (33%) as white, and 8 (2%) as Hispanic, Asian, Native American, or multiracial. The latter 8 women were combined into one racial category, which was referred to as the Other category in the analyses. There were a total of 956 visits and 245 woman-years of follow-up, and HSV-2 DNA was detected in 88 (9%) of the 956 vaginal swab specimens obtained. According to univariate analysis, young age ( 25 years) and less education ( 12 years of education) were the demographic characteristics associated with an increased likelihood of HSV-2 shedding (table 1). Of the behavioral variables examined in the univariate analyses, the frequency of detection of HSV-2 in the genital tract of women who acknowledged having a new sex partner during the 4 months before a study visit was found to be nearly identical to the frequency of detection among women who denied having new sexual contacts during this period (table 2). As shown in table 2, data from only 668 study visits were available for inclusion in this particular analysis, because information about new sex partners was not collected at enrollment. Women who had participated in vaginal intercourse or in receptive oral sex during the 4 months before a study visit occurred were not significantly more likely to shed HSV-2 than were women who 1424 CID 2005:40 (15 May) Cherpes et al.

4 Table 2. Univariate correlates of genital tract shedding of herpes simplex virus type 2 (HSV- 2) in women aged years, according to selected behavioral characteristics and genital tract infections. Variable No. of study visits at which the variable was noted No. (%) of study visits at which HSV-2 shedding was noted OR (95% CI) P Behavioral characteristic New sex partner a No (8) 1.0 Yes (9) 1.1 ( ).8 Vaginal intercourse a No 57 2 (4) 1.0 Yes (10) 2.8 ( ).1 Receptive oral sex a No (9) 1.0 Yes (9) 1.0 ( ).99 Douching a No (9) 1.0 Yes (9) 1.0 ( ).99 Hormonal contraceptive use a No (8) 1.0 Yes (11) 1.5 ( ).08 Genital tract infection HSV-2 seroconversion a No (9) 1.0 Yes 31 7 (23) 3.1 ( ).01 Vaginal Gram stain morphologic score b (6) (11) 1.9 ( ) (11) 2.0 ( ).01 Vaginal yeast No (8) 1.0 Yes (12) 1.5 ( ).1 Vaginal GBS growth on culture c None (8) 1.0 Light (9) 1.3 ( ).4 Heavy (15) 2.3 ( ).003 NOTE. GBS, group B streptococci. a During the past 4 months. b A score of 0 3 denotes normal flora; 4 6, intermediate flora; and 7 10, bacterial vaginosis. c 5 Light colonization was defined as detection of GBS at concentrations! 10 cfu/g of vaginal fluid; heavy 5 colonization was defined as detection of GBS at concentrations 10 cfu/g of vaginal fluid. had been sexually abstinent or who had not received receptive oral sex (table 2). Similarly, vaginal douching did not appear to be associated with an increased likelihood of detection of HSV-2 shed from the genital tract. Women who used either depo-medroxyprogesterone acetate or oral contraceptive pills were included in one category (i.e., hormonal contraceptive use ), because the associated estimates of risk were similar when analyzed separately. Although HSV-2 DNA was detected more frequently in women who used hormonal contraceptives, compared with women who did not, this association did not achieve statistical significance in the univariate analysis (OR, 1.5 vs. 1.0; P p.08). Genital tract shedding of HSV-2 was detected more frequently in women who had acquired HSV-2 antibodies during the previous 4 months (table 2). HSV-2 DNA was also identified more often in women with bacterial vaginosis and in women with high-density vaginal GBS colonization, compared with women without these findings. Women with a Gram stain mor- Risk Factors for HSV-2 Shedding in Women CID 2005:40 (15 May) 1425

5 Table 3. Results of multivariable generalized estimating equation modeling of the independent risk factors for genital tract shedding of herpes simplex virus type 2 (HSV-2) in women aged years. Risk factor Adjusted OR a (95% CI) P HSV-2 seroconversion b 3.0 ( ).01 Vaginal Gram stain morphologic score c ( ) ( ).003 Vaginal GBS growth on culture d None 1.0 Light 1.2 ( ).5 Heavy 2.2 ( ).004 Hormonal contraceptive use 1.8 ( ).02 NOTE. GBS, group B streptococci. a Adjusted for all variables shown. b During the past 4 months. c A score of 0 3 denotes normal flora; 4 6, intermediate flora; and 7 10, bacterial vaginosis. d Light colonization was defined as detection of GBS at concentrations! 10 5 cfu/g of vaginal fluid; heavy colonization was defined as detection of GBS at concentrations 10 5 cfu/g of vaginal fluid. phologic score that was classified as intermediate (i.e., a score of 4 6) were also more likely to have HSV-2 detected in their vaginal swab specimens than were women with a normal score for vaginal flora (table 2). However, the detection of vaginal yeast by culture was not associated with genital tract shedding of HSV-2. Because yeast colonization can be either symptomatic or asymptomatic, secondary analysis was performed to evaluate whether HSV-2 shedding was increased among women who had positive yeast culture results and complaints of vaginal pruritis, but we found that the presence of vaginal symptoms in women with yeast colonization did not increase the frequency of detection of HSV-2 (data not shown). To identify the factors independently associated with the presence of HSV-2 DNA in vaginal fluid, multivariable GEE regression analyses were performed (table 3). The strongest predictor of shedding of HSV-2 from the genital tract was recent HSV-2 seroconversion (OR, 3.0; 95% CI, ; P p.01). Absence of a Lactobacillus-predominant vaginal flora also increased the risk of genital tract shedding of HSV-2. Both an intermediate vaginal Gram stain morphologic score, which reflects reduced numbers of lactobacilli, and bacterial vaginosis were identified as independent risk factors (OR, 1.9 [95% CI, ; P p.04] and 2.3 [95% CI, ; P p.003], respectively). Women with high-density GBS colonization were significantly more likely to shed HSV-2 than were women without vaginal GBS colonization or women with low-density GBS colonization (OR, 2.2; 95% CI, ; P p.004). Finally, the use of hormonal contraception (either depo-medroxyprogesterone acetate or oral contraceptive pills) was identified as an independent risk factor for the detection of HSV-2 in the genital tract (OR, 1.8; 95% CI, ; P p.02). DISCUSSION In the present longitudinal cohort investigation of the risk factors associated with genital tract shedding of HSV-2 in women, we found that recent HSV-2 seroconversion, use of hormonal contraceptives, bacterial vaginosis, and high-density vaginal GBS colonization were among the independent risk factors for increased detection of the virus. These results are in agreement with the results of previous studies that demonstrated genital tract shedding of HSV-2 to be more frequent in women with recent acquisition of HSV-2 [1, 13]. More novel, and more unexpected, are the associations of increased frequency of genital tract shedding of HSV-2 with the presence of bacterial vaginosis and high-density vaginal GBS colonization. A more thorough understanding of how these seemingly disparate vaginal infections independently increase the likelihood of genital tract shedding of HSV-2 will require further research. It is known that when individuals have concurrent primary oropharyngeal and genital HSV-2 infections, the frequency of genital shedding is significantly higher than the frequency of oral shedding [14]. These disparate rates of HSV-2 shedding suggest that local immune or host cellular factors influence viral reactivation, and it is possible that perturbations of genital tract mucosal immunity produced by either bacterial vaginosis associated anaerobic gram-negative rods or high densities of encapsulated gram-positive cocci may be the types of events that can promote reactivation of HSV-2 from latency. GBS is regarded as an organism whose presence in the vagina does not elicit a significant host inflammatory response [15]. In fact, detection of GBS in the vagina is typically considered to denote colonization rather than infection [16], because its isolation does not appear to be causally related to the presence of vulvovaginal symptoms [17, 18]. However, pregnant women with heavy vaginal GBS colonization at weeks gestation have a significantly increased risk of delivery of a preterm, lowbirth-weight infant, compared with pregnant women with either no or light GBS colonization [19]. This finding suggests that, although vaginal GBS colonization does not typically produce overt vulvovaginal symptoms, alterations in the vaginal microenvironment may be induced, and, furthermore, larger quantities of GBS in the vagina may be more likely to create these alterations. Bacterial vaginosis is another noninflammatory condition of the genital tract that is associated with a number of adverse obstetric and gynecologic events, including pelvic inflammatory disease [20] and preterm birth [21]. Although bacterial vaginosis is generally regarded as a disease that does not elicit an 1426 CID 2005:40 (15 May) Cherpes et al.

6 overt local host inflammatory response, a number of recent studies have demonstrated that it is associated with significant alterations in the concentrations of several immunomodulatory cytokines, compared with the concentrations of these cytokines associated with normal vaginal flora [22 24]. Further research will be necessary to determine what local or systemic changes elicited by vaginal GBS colonization or bacterial vaginosis are responsible for the increased frequency of reactivation of HSV- 2 in the genital tract that was observed in the present investigation. It will be equally important to determine the specific effects of other genital tract pathogens, such as Chlamydia trachomatis or Trichomonas vaginalis, on reactivation and shedding of HSV-2. The present study is the first longitudinal investigation to demonstrate an association of the use of oral and injectable hormonal contraceptives with increased genital tract shedding of HSV-2. The specific pathomechanisms responsible for this association await elucidation, but they may include sex steroid induced changes in either local genital tract immunity or physiology. Alterations of the T cell mediated immune response in particular, those caused by immunosuppression are thought to be of primary importance in the reactivation of HSV-2 from latency [25]. Increasing evidence suggests that CD8 + T lymphocytes are the cell type that is primarily responsible for maintaining herpes simplex virus latency with sensory neurons [26]. Furthermore, viral clearance from recurrent genital HSV-2 lesions is highly correlated with the local infiltration of HSV-specific cytotoxic T lymphocytes [27]. Because increased levels of estrogen and progesterone are thought to suppress cell-mediated immune responses in order to create a more receptive environment for allogeneic sperm and a fetus [28], attenuation or modulation of this response by exogenous sex steroids may act to increase the duration and/or frequency of shedding of HSV-2 from the genital tract of women. On the other hand, women who use oral contraceptive pills are known to have larger areas of cervical ectopy, compared with women who do not use oral contraceptive pills [29, 30], and this extension of the single-layered columnar epithelium onto the ectocervix may facilitate genital tract shedding of HSV-2. We found that oral and injectable hormonal contraceptives are equally as likely to increase genital tract shedding of HSV-2. However, because cervical ectopy is more commonly associated with the use of oral contraceptive pills, cervical ectopy may not be the predominant mechanism responsible for these results. Although the strengths of the present study include its longitudinal design and its capture of 245 woman-years of followup investigation, it does possess several limitations. Daily, rather than intermittent, collection of vaginal swab specimens would probably have resulted in increased detection of HSV-2 [5]. Collection of vulvar and perianal specimens, which would have likewise increased the likelihood of detection of HSV-2, were not obtained [2]. Recent evidence also suggests that cervicovaginal lavage is a more sensitive technique than is use of swab specimens obtained from the genital tract for PCR detection of HSV-2 [31]. Despite these sampling limitations, the frequency of detection of HSV-2 in the present study (9%) is similar to the frequency of detection (11%) observed among HSV-2 infected women who, on a daily basis for 2 months, self-collected swab specimens from the cervicovaginal, vulvar, and perianal regions in a recent study of transmission of genital herpes [32]. Our analyses are also limited by the absence of data concerning the presence of other reproductive tract pathogens, including Neisseria gonorrhoeae, C. trachomatis, and T. vaginalis. Because the present study demonstrates that common genital syndromes and colonizing organisms are associated with increased shedding of HSV-2, it will be important to evaluate the role of these other genital tract coinfections in future studies of HSV-2 shedding. Because the great majority of genital infections due to HSV- 2 in adults are the result of heterosexual transmission of HSV- 2, and because almost all neonatal HSV-2 infections are perinatally acquired through contact with infected genital secretions during delivery, a more complete understanding of the factors that promote shedding of HSV-2 in the genital tract of women is likely to help decrease the risk of both heterosexual and mother-to-child transmission of HSV-2. The present study has identified hormonal contraceptive use, bacterial vaginosis, and vaginal GBS colonization as independent risk factors for genital tract shedding of HSV-2 in women. Because hormonal contraceptives are used by 1100 million women worldwide [33], and because bacterial vaginosis and vaginal GBS colonization are 2 of the most common genital tract conditions present among women of reproductive age, even modest associations between these variables and genital tract shedding of HSV-2 would result in substantial attributable risks for transmission of the virus. Acknowledgments Financial support. National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH [Bethesda, MD]; contract N01- A and grant 5U01-A ) and the AIDS and Molecular Microbiology/Epidemiology Training Program, NIH (grant T32-AI07333 [to T.L.C.]). Potential conflicts of interest. All authors: no conflicts. References 1. Rattray MC, Corey L, Reeves WC, Vontver LA, Holmes KK. Recurrent genital herpes among women: symptomatic v. asymptomatic viral shedding. Br J Vener Dis 1978; 54: Benedetti J, Corey L, Ashley R. Recurrence rates in genital herpes after symptomatic first-episode infection. Ann Intern Med 1994; 121: Augenbraun M, Feldman J, Chirgwin K, et al. Increased genital shed- Risk Factors for HSV-2 Shedding in Women CID 2005:40 (15 May) 1427

7 ding of herpes simplex virus type 2 in HIV-seropositive women. Ann Intern Med 1995; 123: Wald A, Zeh J, Selke S, Ashley RA, Corey L. Virologic characteristics of subclinical and symptomatic genital herpes infections. N Engl J Med 1995; 333: Brock BV, Selke S, Benedetti J, Douglas JM Jr, Corey L. Frequency of asymptomatic shedding of herpes simplex virus in women with genital herpes. JAMA 1990; 263: Mostad SB, Kreiss JK, Ryncarz AJ, et al. Cervical shedding of herpes simplex virus in human immunodeficiency virus infected women: effects of hormonal contraception, pregnancy, and vitamin A deficiency. J Infect Dis 2000; 181: McClelland RS, Wang CC, Richardson BA, et al. A prospective study of hormonal contraceptive use and cervical shedding of herpes simplex virus in human immunodeficiency virus type 1 seropositive women. J Infect Dis 2002; 185: Meyn L, Moore D, Hillier S, Krohn M. Association of sexual activity with colonization and vaginal acquisition of Group B Streptococcus in nonpregnant women. Am J Epidemiol 2002; 155: Gentry YM, Hillier SL, Eschenbach DA. Evaluation of a rapid enzyme immunoassay test for detection of Group B Streptococcus. Obstet Gynecol 1991; 78: 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: Cherpes TL, Ashley R, Meyn LA, Hillier SL. Longitudinal reliability of the Focus glycoprotein G based type-specific enzyme immunoassays for herpes simplex virus types 1 and 2 in women. J Clin Microbiol 2003; 41: Hardin JW, Hilbe JM. Generalized estimating equations. Boca Raton: Chapman & Hall/CRC, Wald A, Corey L, Cone R, Hobson A, Davis G, Zeh J. Frequent genital herpes simplex virus 2 shedding in immunocompetent women: effect of acyclovir treatment. J Clin Invest 1997; 99: Lafferty WE, Coombs RW, Benedetti J, Critchlow C, Corey L. Recurrences after oral and genital herpes simplex virus infection. N Engl J Med 1987; 316: Edwards MS, Baker CJ. Streptococcus agalactiae (group B streptococcus). In: Mandell GL, Douglas RG, Bennett JE, eds. Principles and practice of infectious diseases. 5th ed. Philadelphia: Churchill Livingstone, 2000: Bliss SJ, Manning SD, Tallman P, et al. Group B Streptococcus colonization in male and nonpregnant female university students: a crosssectional prevalence study. Clin Infect Dis 2002; 34: Honig E, Mouton JW, van der Meijden WI. The epidemiology of vaginal colonisation with group B streptococci in a sexually transmitted disease clinic. Eur J Obstet Gynecol Reprod Biol 2002; 105: Shaw C, Mason M, Scoular A. Group B streptococcus carriage and vulvovaginal symptoms: causal or casual? A case-control study in a GUM clinic population. Sex Transm Infect 2003; 79: Regan JA, Klebanoff MA, Nugent RP, et al. Colonization with group B streptococci in pregnancy and adverse outcome. Am J Obstet Gynecol 1996; 174: Paavonen J, Teisala K, Heinonen PK, et al. Microbiological and histopathological findings in acute pelvic inflammatory disease. Br J Obstet Gynecol 1987; 94: Gravett MG, Hummel D, Eschenbach D, Holmes KK. Preterm labor associated with subclinical amniotic fluid infection and with bacterial vaginosis. Obstet Gynecol 1986; 67: Cohen CR, Plummer FA, Mugo N, et al. Increased interleukin-10 in the endocervical secretions of women with non-ulcerative sexually transmitted diseases: a mechanism for enhanced HIV-1 transmission? AIDS 1999; 13: Spandorfer SD, Neuer A, Giraldo PC, Rosenwaks Z, Witkin SS. Relationship of abnormal vagina flora, proinflammatory cytokines and idiopathic fertility in women undergoing IVF. J Reprod Med 2001; 46: Yudin MH, Landers DV, Meyn L, Hillier SL. Clinical and cervical cytokine response to treatment with oral or vaginal metronidazole for bacterial vaginosis during pregnancy: a randomized trial. Obstet Gynecol 2003; 102: Theil D, Derfuss T, Paripovic I, et al. Latent herpesvirus infection in trigeminal ganglia causes chronic immune response. Am J Pathol 2003; 163: Khanna KM, Bonneau RH, Kinchington PR, Hendricks RL. Herpes simplex virus-specific memory CD8 + T cells are selectively activated and retained in latently infected sensory ganglia. Immunity 2003; 18: Koelle DM, Posavad CM, Barnum GR, Johnson ML, Frank JM, Corey L. Clearance of HSV-2 from recurrent genital lesions correlates with infiltration of HSV-specific cytotoxic T lymphocytes. J Clin Invest 1998; 101: Grossman CJ. Regulation of the immune system by sex steroids. Endocr Rev 1984; 5: McGregor JA, Hammill HA. Contraception and sexually transmitted diseases: interactions and opportunities. Am J Obstet Gynecol 1993; 168: Critchlow CW, Wolner-Hanssen P, Eschenbach DA, et al. Determinants of cervical ectopia and of cervicitis: age, oral contraception, specific cervical infection, smoking, and douching. Am J Obstet Gynecol 1995; 173: Ndjoyi-Mbiguino A, Ozouaki F, Legoff J, et al. Comparison of washing and swabbing procedures for collecting genital fluids to assess cervicovaginal shedding of herpes simplex virus type 2 DNA. J Clin Microbiol 2003; 41: Corey L, Wald A, Patel R, et al. Once-daily valacyclovir to reduce the risk of transmission of genital herpes. N Engl J Med 2004; 350: United Nations Population Division Department for Economic and Social Information and Policy Analysis. Levels and trends of contraceptive use as assessed in New York: United Nations, 1996: CID 2005:40 (15 May) Cherpes et al.