GENITAL HERPES REMAINS A highly prevalent sexually transmitted disease. In addition to the physical discomfort and emotional distress caused by symptomatic genital herpes, the association of herpes simplex virus (HSV) 2 antibody positivity with a 2-fold increased risk of HIV 1 transmission1,2 further enhances the importance of this infection. Nearly 22% of the US population over the age of 12 had serologic evidence of HSV 2 infection,3 according to the National Health and Nutrition Examination Survey III (NHANES III) conducted between 1988 and 1994. This represented a 30% increase in HSV 2 seroprevalence since the previous study (NHANES II) conducted between 1976 and 1980. This increase suggests that any changes in sexual behavior in the 1980s in response to the AIDS epidemic did not result in reduced transmission of HSV 2. Although the seroprevalence of HSV 2 may have declined somewhat in recent years,4 it remains a highly prevalent infection.
Less than 6% of the population between the ages of 12 and 19 was HSV 2 antibody positive in the NHANES III study.3,5 The largest prospective study that examined HSV 2 infection in an adolescent female population (median age 16) found a prevalence of HSV 2 antibody on entry of 13.8% and an annualized incidence of 8.2 per 100 person years.6 HSV 2 was second only to Chlamydia trachomatis among the pathogens studied in incident and prevalent infections. The study did not include human papillomavirus infections. There were no symptomatic genital herpes cases among the incident infections. A study of STDs in a homeless adolescent population (female mean age 17.7) found a prevalence of HSV 2 antibody of 11.9% among females and an incidence of 25.4 per 100 person-years.7 Both of these studies examined risk factors for STDs in general but not for HSV 2 individually. Other studies of adolescent women found prevalences of HSV 2 antibody ranging from 10.5% to 19%, with different behavioral risk factors.8,9 A more recent prospective study of 174 adolescent females (mean age 14.5) found an HSV 2 seroprevalence of 7% and an incidence of 4.4 cases per 100 person-years among those who were sexually active.10 This study found that a longer period of sexual activity and a history of more sexually transmitted diseases in the previous 6 months were associated with HSV 2 positivity (combined incident and prevalent infection). These studies indicate that the incidence and prevalence of HSV 2 among adolescent women is significant, but little is known about the factors that are associated with HSV 2 acquisition in this population.
Several studies have indicated that the frequency with which genital herpes is caused by HSV 1 is increasing.11–15 Most of these reports have been cross-sectional or laboratory-based studies that could not evaluate risk factors for acquisition. We conducted a prospective study of HSV 1 and HSV 2 infection in adolescent females in an effort to determine the incidence, prevalence, and risk factors for HSV 1 and HSV 2 infection.
Patients and Methods
Subjects participating in this study have been previously reported.16,17 Briefly, adolescent girls (age 14–17) who were attending an inner-city health clinic were invited to participate in this study of sexual behaviors and sexually transmitted diseases. Subjects (and their parents) who agreed to the study were followed for 27 months with physical examinations, questionnaires, diaries, and self-collected genital swab specimens on a specified schedule. The subjects were seen in the clinic every 3 months. At these quarterly visits, serum was obtained and the subjects had pelvic examinations at which swabs were obtained for nucleic acid amplification tests for C trachomatis, N gonorrhoeae, and T vaginalis. Between the first and second quarterly visit and the third and fourth quarterly visit of each year, subjects collected weekly vaginal swabs that were also tested for C trachomatis, N gonorrhoeae, and T vaginalis by nucleic acid amplification. The HSV component of the study was added after initiation of the project. Subjects already enrolled were asked to sign a supplementary consent form allowing previously collected specimens to be tested for HSV, while newly enrolled subjects signed a consent that included HSV testing. For that reason, over half of the specimens tested for this study were retrieved from a specimen archive. The specimens reported here were collected between April 1999 and April 2004. Only subjects who had at least 2 sera obtained at different time points were included in this analysis. For the purposes of this analysis, the date of study entry was considered to be the date of the first available serum specimen, and the date of termination was the date of the last available serum specimen. In some cases, these dates did not correspond with the entry and termination dates for the overall study, because of missing or expended serum specimens. For this reason, the median age of our subjects was slightly higher than that reported for the overall cohort.16,17 The subjects in this report were otherwise similar to the overall cohort in demographic and behavioral characteristics.
Sera were tested for the presence of HSV 1 and HSV 2 antibody using the HerpeSelect immunoblot assay (Focus Diagnostics, Cypress, CA). Type-specific antibody was detected by the presence of a visible band on the strip in the presence of appropriate control reactions, as recommended by the manufacturer. All sera that appeared to show seroconversion to HSV 1, HSV 2, or both (as well as a few additional randomly selected sera) were confirmed by Western blot.18–20 If discrepant results were obtained, the result of the HSV Western blot was used for this report. HSV 1 and HSV 2 DNA were detected in genital swab specimens using the LightCycler HSV 1/2 Detection Kit (Roche Diagnostics GmbH, Penzberg, Germany).
Associations of sexual behavior with incident HSV 2 infection were tested using an exact logistic regression model which included an adjustment for age at the time of the test. Using both prevalent and incident HSV 2 infections, 2-sample t tests were used to test for differences in mean sexual behaviors between HSV 2-positive and -negative subjects. Associations were also tested using simple logistic regression models for being HSV 2 positive.
A total of 104 subjects had appropriate sera available and were included in the study. Four subjects were excluded from the analysis because of inconsistent serologic results. All 4 had HSV 1 and HSV 2 antibody detected in their first serum specimen and only HSV 1 antibody detected in subsequent sera. The demographic and historical characteristics of the 100 subjects included in the analysis are shown in Table 1. The subjects were mostly blacks around 16 years of age. At the time of entry, 84 reported being sexually active.
Incident HSV Infections
Of the 100 subjects in the study, 37 had no HSV antibody at entry, 49 had antibody to HSV 1 only, 4 had antibody to HSV 2 only, and 10 had antibody to both HSV 1 and HSV 2 (Table 1). Over a median follow-up period of 15 months, there were 4 subjects who converted to HSV 1 positive and 7 who converted to HSV 2 positive. These totals include 1 subject who was initially HSV negative who converted to HSV 1 positive after 3 months and became HSV 1 and HSV 2 positive at 9 months. Using only the subjects who were susceptible to HSV 1 infection (n = 41) or HSV 2 infection (n = 86), the incidence rates are 8.9 per 100 person-years for HSV 1 (4 cases in 44.8 person-years of follow-up) and 7.3 per 100 person-years for HSV 2 (7 cases in 95.6 person-years of follow-up). Despite the fact that there were 11 cases of serologically confirmed HSV infections in 10 subjects, there were no symptomatic cases of genital herpes or herpes gingivostomatitis diagnosed in the study cohort.
Predictors of Incident HSV Infections
Using demographic and behavioral data collected on the study subjects, we attempted correlate these parameters with acquisition of HSV 1 or HSV 2. Because of the small number of HSV 1 acquisitions, we were unable to correlate any of the parameters with incident HSV 1. Of note, there was no correlation between a history of oral-genital contact in the previous 3 months and HSV 1 acquisition. Only 1 of the 4 subjects who acquired HSV 1 reported recent oral-genital contact. This proportion was slightly lower than the overall cohort, in which 35 of the 84 sexually active subjects reported oral-genital contact.
For the HSV 2 acquisitions, we also examined several demographic and behavioral parameters for associations. These are shown in Table 2. Although several of these parameters were more common in those who acquired HSV 2 infection, only the number of partners in the past 3 months approached statistical significance (P = 0.053). Other parameters included in the analysis but not shown in Table 2 were number of recent unprotected sexual encounters and number of recent STDs. Neither of these parameters approached statistical significance.
Genital Tract Shedding of HSV
During 2 13-week periods each year, subjects collected weekly vaginal swabs that were tested for C trachomatis, N gonorrhoeae, and T vaginalis by nucleic acid amplification. Many vaginal swab specimens were also tested for the presence of human papillomavirus DNA. In addition, swabs were collected by clinicians during quarterly pelvic examinations and tested for the same pathogens. We assayed selected swab specimens from the incident HSV cases for the presence of HSV DNA sequences by PCR. We selected weekly and quarterly swab specimens collected before seroconversion, as close as possible to the time of seroconversion, and after seroconversion. Because of missing or expended serum and swab specimens and because some of the subjects seroconverted at or near the end of the follow-up period, the proximity of the swab specimens to the time of seroconversion was not always optimal. However, at least some swab specimens were available for testing on all 10 individuals who seroconverted. HSV 1 DNA was not identified in any genital swab specimen (68 genital swab specimens from the 4 HSV 1 seroconverters tested). A total of 133 swab specimens from HSV 2 seroconverters were tested, and 8 were positive for HSV 2 DNA. The 8 HSV 2 DNA positive specimens came from 3 of the 7 subjects who seroconverted to HSV 2. Two of the subjects had 2 consecutive weekly swabs positive for HSV 2 DNA. The positive swabs were obtained between the last HSV 2–negative serum and first HSV 2–positive serum. One of those subjects had an additional swab specimen that was HSV 2 DNA positive obtained 5 months later, after the first HSV 2–positive serum. The third subject had 3 consecutive weekly swab specimens positive for HSV 2 DNA. These specimens were obtained immediately after the first serum specimen that was positive for HSV 2 antibody. No vaginal swab specimens from subjects with prevalent HSV infections were tested for HSV DNA.
Predictors of a Positive HSV 2 Antibody Test
Using similar demographic and behavioral information to that used for the incident HSV cases noted above, we pooled the incident and prevalent HSV 2 infections to determine traits that predict a positive HSV 2 antibody test. These results are shown in Table 2. Age, duration of sexual activity, and number of lifetime sexual partners were the strongest predictors of a positive HSV 2 antibody test. Another predictor of a positive HSV 2 test was a diagnosed infection with C trachomatis, N gonorrhoeae, or T vaginalis before the positive HSV 2 antibody test (P = 0.02 in a simple logistic regression model).
As previously noted, only a few studies have reported incidence rates for HSV 2 infection in adolescent women,6,7,10 and these have been in very different populations. The incidence of HSV 2 infection in our study, 7.3 per 100 person-years, is similar to the 2 previous studies in comparable populations.6,10 This incidence is only slightly lower than the 11.7 per 100 person-years reported in STD clinic attendees in 5 US cities,21 although the incidence in the subjects in that study between 14 and 19 years of age was 13.4 per 100 person-years. Only 1 other study has examined HSV 2 incidence in adolescents, and that study found a very low incidence (0.4 per 100 person years) in adolescents in Italy.22 That study had no information about sexual activity among study subjects. These results suggest that the incidence of HSV 2 infection in adolescent women is high and approaches that of young adults. Because there were no cases of symptomatic genital herpes diagnosed in our cohort during the follow-up period, the subjects who acquired HSV 2 infection must have had few or no symptoms around the time of acquisition. Seroprevalence studies such as NHANES III3 suggest that only about 10% of persons with HSV 2 antibody have a history of symptomatic genital herpes. Because we had only 7 HSV 2 seroconverters, we cannot determine if asymptomatic infection is more or less common in adolescents compared to adults.
Because the seroprevalence of HSV 1 was relatively high at enrollment (59%), fewer subjects were at risk for acquiring HSV 1 infection during the study. Although the numbers are small, the calculated incidence of HSV 1 infection was higher than the incidence of HSV 2. Because there were no symptomatic cases of oral or genital herpes diagnosed in this study, we cannot determine if the subjects who acquired HSV 1 were infected by the oral or genital route. Because of the high frequency of genital HSV 1 infection among young adults,12,13 it is likely that some, perhaps most, of the incident HSV 1 infections in our study were genital. Only 1 of 4 HSV 1 seroconverters engaged in oral sex, but 3 of 4 engaged in vaginal sex, and other studies23 have shown that both oral and vaginal sex are risk factors for HSV 1 acquisition.
The factors we identified that were associated with HSV 2 infection (age, duration of sexual activity, number of lifetime sex partners, and history of other sexually transmitted diseases) are similar to those found in other studies.10,24 Because of our relatively small numbers, we were not able to determine if each of these factors is an independent predictor of a positive HSV 2 antibody test.
Although we did not perform daily HSV shedding studies that others have used to document asymptomatic shedding of HSV,25,26 we did have access to weekly vaginal swabs that were used to document other sexually transmitted pathogens. Using these specimens, we were able to document that at least 3 of the 7 HSV 2 seroconverters shed virus on multiple occasions. These women would be at risk for transmitting the virus to a sexual partner. This is especially concerning because none of these subjects had symptoms of genital herpes and were unaware of their ability to transmit HSV 2.
This study has a number of limitations. First, the sample size is relatively small and the number of incident infection is also small, making the determination of risk factors for incident infections relatively weak. Second, we studied teens who were followed in public adolescent health clinics. They were inner city, mostly black young women who may not be representative of teens with other demographic characteristics. Finally, because this study was performed using specimens collected for another study and a number of specimens were not available, our ability to determine the time of infection was limited for some subjects, and many subjects in the cohort were not included in the analysis because specimens were not available.
In summary, HSV infections are common in adolescent women. Efforts to reduce these infections through either behavioral interventions or immunization need to target children before adolescence.
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