Sexually Transmitted Diseases:
Herpes Simplex Virus Type 2 Infection in Young Adult Women: Risk Factors for Infection and Frequency of Viral Shedding
Fife, Kenneth H. MD, PHD*†; Williams, James A. BS*; Thomas, Allyson L. MD*; Ofner, Susan MS*; Katz, Barry P. PHD*; Fortenberry, J. Dennis MD, MS‡
From the Departments of *Medicine, †Microbiology and Immunology, and ‡Pediatrics, Indiana University School of Medicine, Indianapolis, IN
The authors thank Patricia Brooks and Mabel Peterson for their assistance with this study, Dr. Barbara Van Der Pol for helpful comments on the manuscript, and Dr. Donald Orr who initiated the cohort study. We would also like to thank the study participants whose dedication to this project was essential to its completion.
Supported in part by an investigator-initiated grant from GlaxoSmithKline (Val R-137) to KHF and by NIH grant AI 31494.
Presented in part at the 48th ICAAC/46th IDSA Joint Meeting, Washington, DC, October 2008.
Correspondence: Kenneth H. Fife, MD, PhD, Indiana University School of Medicine, 545 Barnhill Dr., Room 435, Indianapolis, IN 46202. E-mail: firstname.lastname@example.org.
Received for publication June 15, 2009, and accepted December 12, 2009.
Background: Population-based studies suggest that acquisition of herpes simplex virus 2 (HSV 2) is most common between ages 20 and 29, especially in minority women. We examined HSV 2 infection and viral shedding in a cohort of young women.
Methods: Women, age 18 to 24 (median 21), who were part of an observational cohort enrolled between ages 14 to 17, had blood obtained for HSV 2 antibody. Intensive diary collections on sexual behavior and genital symptoms and weekly vaginal swabs were obtained at regular intervals.
Results: HSV 2 antibodies were detected in 43 of 127 participants (33.9%), only 4 of whom were previously known to be positive. Factors associated with a positive test included older age, years of sexual activity, and number of lifetime partners. Testing for HSV 2 DNA by polymerase chain reaction on weekly vaginal swabs from a 13-week sampling period for each HSV 2 antibody positive participant showed 32 of 43 (74.4%) were positive at least once. The positive predictive value of pain for viral shedding was poor.
Conclusions: HSV 2 infection is very common among young adult women, but symptomatic genital herpes is not. Shedding of HSV 2 DNA can be detected in most antibody positive persons. Early intervention strategies will be needed to control HSV 2 infection.
Genital herpes remains a highly prevalent sexually transmitted infection. In addition to the physical and emotional problems caused by symptomatic genital herpes, the association of herpes simplex virus (HSV) 2 antibody positivity with a 2- to 5-fold increased risk of HIV 1 acquisition or transmission1–7 further enhances the importance of this infection. HSV 2 infection has also been shown to increase the plasma and genital tract HIV 1 viral load in persons infected with both viruses.8–11
It has been estimated that more than half a billion people are infected with HSV 2 worldwide and an estimated 23.6 million persons were newly infected in 2003.12 A population-based survey conducted in the United States between 1999 and 2004 showed that the prevalence of HSV 2 antibody was about 17%.13 However, the prevalence of HSV 2 antibody in non-Hispanic black females was 46.1% and black females between the ages of 20 and 29 had a prevalence of 35.3%.13
We reported a study of adolescent women in Indianapolis conducted between 1999 and 2004 that found 13.5% were HSV 2 antibody positive at enrollment and the annual incidence of HSV 2 infection was 7.4%.14 Our study participants had a mean age of 16.3 years and were predominantly non-Hispanic blacks (86%). We have continued to follow our cohort of young women as they enter adulthood. Here we report on the current prevalence of HSV 2 infection. In addition, we collected genital swab specimens and prospective information about genital symptoms that allows us to detect viral shedding and correlate shedding episodes with genital symptoms among those with HSV 2 infection. These observations allow us to better characterize HSV 2 infection in this demographically important population.
SUBJECTS AND METHODS
The results reported here are from a cross-sectional sample taken from a cohort of women who were participating in a longitudinal observational study of risk and protective factors associated with sexually transmitted infections. The longitudinal study is described in more detail elsewhere.15,16 Briefly, adolescent girls (age 14–17) who were attending any of 3 inner-city health clinics were invited to participate. Enrollment took place between 1999 and 2005 and follow-up ended in mid 2009. In December 2007, an amendment was added to permit a single blood specimen to be drawn for HSV 2 antibody testing. Only participants who were at least 18 years of age were enrolled in this component of the study, although nearly all of those who were still being followed were 18 or older. All participants signed a supplementary informed consent statement that was approved by the local institutional review board. The original approved consent statement (signed by the participants and their parents) also included permission to test stored specimens for STD pathogens, including HSV 2. Among the specimens available were self-collected vaginal swabs that had been collected throughout the study as well as data from behavioral diaries. In alternating calendar quarters during each year of follow-up, participants completed daily behavioral diaries and submitted weekly self-collected vaginal swabs. The collection periods were followed in the next quarter by a rest period in which no diary or weekly vaginal samples were collected. The weekly swabs were tested for N. gonorrhoeae, C. trachomatis, and T. vaginalis as part of the longitudinal study and the residual material was frozen and was available for additional testing.
The presence of HSV 2 antibody was detected using the HerpeSelect 2 ELISA assay (Focus Technologies, Cypress, CA). Specimens with ELISA values ≥3.5 were considered positive and those with values between 1.1 and 3.4 underwent confirmatory testing by competitive inhibition assay (Focus Technologies, Cypress, CA).
Throughout this study, participants completed daily diaries and collected weekly vaginal swabs for two 13-week periods each year. Some participants had been in the study for more than 7 years and had 15 or more sets of weekly swabs potentially available for testing. Because of cost considerations, we selected one set of weekly swab specimens from each HSV 2 antibody positive participant for HSV 2 DNA analysis to evaluate viral shedding. We selected a collection period that was after the participant was known to be HSV 2 antibody positive when possible. A few participants were tested for HSV 2 antibody after they completed all swab collections, so the last available set of swabs was selected for those participants. Other criteria for selecting swab sets for testing included completeness of the set (at least 9–10 of 13 weekly swabs available) and collection at a time when questions on genital symptoms were included in the diaries (between 2004 and 2008). Self-collected vaginal swabs were eluted into 1 mL of water and DNA was extracted from a 200 μL aliquot using the High Pure Viral Nucleic Acid Kit (Roche Diagnostics, Indianapolis, IN). HSV DNA was detected using the LightCycler HSV 1/2 assay that amplifies a 215-basepair region in the HSV DNA polymerase gene (Roche Diagnostics, Indianapolis, IN) and can detect and distinguish both HSV 1 and HSV 2 DNA.
Six study participants returned for HSV 2 antibody testing after their formal participation in other study activities (such as behavioral diaries) had ended. Because these participants did not have current behavioral data available, they were excluded from some of the analyses but they were included in the overall prevalence results. Prevalent HSV 2 antibody was tested for association with participant characteristics and measures of sexual experience using univariate logistic regression models. Each model included a term for the participant's age at the time of the current HSV 2 test. Factors found to be significant in the univariate model were included in a multivariate logistic regression model. For the 43 participants with prevalent HSV 2 antibody who also had genital swab specimens collected, shedding of HSV 2 DNA was tested for association with genital symptoms using univariate repeated measures logistic regression models. Variances were estimated using generalized estimating equation methodology with an exchangeable covariance structure to incorporate the correlation induced by repeated sampling of the same participant. All data analyses were performed using SAS/STAT Software, version 9.1 (SAS Institute, Cary, NC) and Logxact PROCs version 6.0 (Cytel Software Corporation, Cambridge, MA).
Demographic Features and Relationship of the Current Population to That Previously Reported
The characteristics of the study population are shown in Table 1. The participants included in this report constitute all of those who remained in the longitudinal study as of December 2007, who were at least 18 years of age, and who consented to the blood draw and HSV 2 antibody testing. The total number of participants enrolled in the longitudinal study between 1999 and the end of enrollment in July 2005 was 386, including some individuals who were enrolled, but did not stay in the study. The characteristics of those included in this report are similar to all of those enrolled in the study (Table 2). Of the 127 participants in the current study, 39 were included in our previous report14; 30 were previously HSV 2 antibody negative and 9 were positive.
Predictors of HSV 2 Infection
The current HSV 2 antibody test was positive in 43 participants (33.9%, 95% confidence interval [CI] 25.6–42.1) in the overall population and in 41 of those for whom we have current behavioral information (Table 1). Only 4 participants were aware that they were HSV 2 positive (2 with symptomatic genital herpes and 2 with a known positive antibody test but no symptoms) before the current test. All participants were given the results of their HSV 2 antibody test, usually at their next quarterly visit. Univariate logistic regression models were used to identify factors associated with a positive HSV 2 antibody test (Table 1). Women who were older, had an earlier sexual debut, and had a larger number of lifetime sexual partners were more likely to have a positive HSV 2 antibody test. The frequency of genital symptoms (pain or discharge) in the 270 days before the test was not associated with the presence of HSV 2 antibody nor was a history of sexually transmitted infections. In a multivariate analysis, none of the factors identified in the univariate analysis was independently associated with a positive HSV 2 antibody test.
HSV 2 Shedding From the Genital Tract
The 43 participants who were HSV 2 antibody positive had one set of available weekly vaginal swabs tested for HSV 2 DNA to evaluate viral shedding (see Methods for details about how these specimens were selected). A total of 32 of the 43 HSV 2 antibody positive participants (74.4%; 95% CI, 61.4%–87.5%) had HSV 2 DNA detected on at least one swab from the collection period selected. The patterns of HSV 2 shedding are shown in Table 3 for the participants who shed virus at least once. Overall, HSV 2 shedding was documented on 73 of 539 specimens tested (13.6%); among the 32 participants who had at least one positive swab specimen, 73 of 397 specimens (18.4%) were positive.
Association of Symptoms With Viral Shedding
During the diary periods, participants recorded whether they experienced vaginal pain and whether they had a vaginal discharge each day. These symptoms were subjectively defined by the participants; they were not given guidance or definitions for these symptoms. No other symptoms (such as itching or genital lesions) were recorded in the diaries. For participants who had shedding of HSV 2 DNA detected on a genital swab, we evaluated the presence of genital symptoms on the day that shedding was detected or in a window that extended from 1 day before to 1 day after the swab was collected. The odds ratio for shedding of HSV 2 DNA was 2.0 (95% CI, 1.1–3.6, P = 0.02) if there was pain recorded in the 3-day window around the time of the swab. There was no association between the presence of vaginal discharge and detection of HSV 2 DNA. Although there was a statistically significant association between genital pain and HSV 2 shedding, the sensitivity of pain as a predictor of shedding was low (4/73 or 5.5%) and the positive predictive value was only 23.5%. The days when symptoms were associated with HSV 2 DNA shedding are shown in Table 3.
Despite the apparent recent decline in HSV 2 seroprevalence in the United States, infection remains common in some populations. Although not statistically significant, the prevalence of HSV 2 antibody among non-Hispanic blacks between the ages of 20 and 29 increased in the 1999–2004 survey compared with the previous survey conducted in 1988–1994.13 That survey did not break the population down further by gender, but in other comparisons, women had seroprevalence rates that were about twice that of men. Thus, a combined prevalence of 35% would likely represent a prevalence of about 23% in men and 46% in women. Our finding that about 34% of women at the beginning of the 20 to 29 age range were HSV 2 antibody positive is consistent with those projections. Young black women remain at very high risk of acquiring HSV 2 infection.
Black women comprise the group with the most rapid increase in HIV infection in the United States in recent years.17 The epidemiologic association of HSV 2 antibody with increased risk of HIV acquisition and the high prevalence of HSV 2 antibody in black women suggest that the 2 observations could be related. Modeling studies suggest that HSV 2 infection may be an important driver of the heterosexual HIV epidemic in sub-Saharan Africa.18 Studies that have examined the population attributable risk of HSV 2 to HIV spread1,6 show that a quarter to a third of HIV infections in black women in the United States could be related to HSV 2. Other studies have linked clinical genital herpes to HIV in black women.19 The underlying causes of disparities in the prevalence of HSV 2 and HIV in black women are complex and multifactorial.20 Our study suggests that these disparities are present from an early age.
Despite the fact that fewer than 10% of our participants knew they had genital herpes, nearly 75% of those with HSV 2 antibody shed virus from the genital tract at least once in the 13-week period that was sampled. Because we were only able to evaluate shedding once a week, unlike more typical shedding studies that evaluate subjects daily21–24 or more often,25 this must be considered a minimum estimate of the proportion of our participants who shed virus and of the frequency of shedding within individuals. Several of the participants who did not have HSV 2 shedding detected had another swab collection period assayed for various reasons and one of the participants had shedding detected in that other period (data not shown). It is possible that even more participants would have shedding detected if more specimen collection periods were assayed. In addition, vaginal swabs were the only specimens available to us for testing. Most shedding studies include other genital sites, so this may have further underestimated the number of participants who might have had detectable HSV 2 DNA. The proportion of participants shedding virus in our study was similar to that found in a previous study of subjects who were HSV 2 antibody positive but had no symptoms of genital herpes24 but somewhat higher than the placebo group in 2 other studies that evaluated suppressive antiviral therapy in HSV 2 antibody positive subjects with no history of genital herpes.26,27 The reason for these differences is not clear, but it could be because our young subjects are likely to be closer to their time of initial infection when viral shedding is more frequent.28
This study is the only one to our knowledge that prospectively collected information about genital symptoms before the participants were known to be HSV 2 antibody positive. Our finding of an association of HSV 2 shedding with genital pain in participants who did not know they had HSV 2 is unique. However, this observation is limited in at least 2 ways. First, we only collected information on 2 symptoms, pain and vaginal discharge, that were self-defined and do not represent the full spectrum of symptoms that are likely to be experienced. The second limitation is that despite demonstrating a significant association between genital pain and HSV 2 shedding, the observation has little clinical utility because of its poor positive predictive value. However, our observation does reinforce the contention that some persons with “asymptomatic” genital herpes do have subtle symptoms that they do not recognize.24,29
Our observations and those of others emphasize the need for early behavioral intervention or an HSV 2 vaccine to prevent young people, especially young black women, from becoming infected with HSV 2 and the potential downstream consequences of that event such as increased susceptibility to HIV infection. Because suppression of HSV 2 does not appear to mitigate the increased susceptibility to HIV,30,31 primary prevention of HSV 2 infection is the only viable solution. The HSV 2 vaccine that is currently in phase 3 clinical trials is only effective in women who are also HSV 1 antibody negative.32 In our earlier study of this adolescent population, 59% were HSV 1 antibody positive at age 16. Population-based studies in the United States have also shown that black Americans acquire HSV 1 infection at a relatively early age.33 We clearly need either a better HSV 2 vaccine or a strategy that intervenes at an age before most people are exposed to either HSV type. Such a strategy remains to be defined.
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