Suppression of Subclinical Shedding of Herpes Simplex Virus Type 2 with Acyclovir

Genital herpes continues to spread in the United States. A recent nationwide survey conducted by the Centers for Disease Control and Prevention indicates that more than one in five adults older than 15 years of age have been infected with herpes simplex virus type 2 (HSV-2) (Johnson R. Personal communication). The virus is often transmitted to sexual partners and infants through contact with infected secretions during periods of subclinical shedding [1-4]. Daily administration of the antiviral drug acyclovir has been shown to decrease the frequency and duration of symptomatic shedding of genital herpes [5-10]. However, no systematic study of the effects of acyclovir on subclinical HSV shedding, the predominant means by which transmission occurs, has been done [11-13]. To determine whether acyclovir suppresses subclinical shedding of HSV-2 in the genital tract, we conducted a placebo-controlled, crossover trial of suppressive acyclovir therapy in women with recently acquired genital HSV-2 infection. Methods Study Participants and Design Because natural history studies of subclinical HSV shedding have indicated that recent acquisition of genital herpes and infection with HSV-2 alone are risk factors for subclinical shedding [14, 15], only women with these clinical characteristics were considered for enrollment in our study. Other entry criteria included general good health without clinical or laboratory evidence of immunosuppression and without the use of immunosuppressive medication. Potential study participants were recruited by newspaper advertisements and through information distributed to local health care providers. We screened 103 women with the above-mentioned clinical characteristics and enrolled 34 (33%). Among the 69 women who were not enrolled, 29 (42%) were not eligible because serologic testing indicated that they had infection other than that with HSV-2 alone [16]. Of the 40 eligible women, 19 (28%) decided not to participate in the study because of the time that it required. Women were also not enrolled if they were planning to move from the area during the study period (6%) or were planning pregnancy (3%). The study was a double-blind, placebo-controlled, crossover trial that was done at the University of Washington Virology Research Clinic and approved by the Human Subjects Review Committee. Participants gave written informed consent. The study was designed as two 70-day treatments with an intervening 14-day washout period. The randomization list was generated by a computer. Participants were randomly assigned to receive either acyclovir, 400 mg twice daily, or placebo for the first 70 days, followed by a 14-day washout period during which all patients received placebo capsules. Participants then received the alternate regimen for 70 days. The randomization scheme was stratified by the duration of genital herpes: One stratum consisted of women who had had herpes for less than 6 months, and the other consisted of women who had had herpes for more than 6 months but for less than 2 years. The reason for the stratification was the expected difference in shedding rates between these two groups [14, 15]. Both clinicians and participants remained blinded to the results of the cultures until the conclusion of the study. The virology laboratory technologists were blinded to the clinical status of the participants. Study medication was dispensed in 2-week cycles (80 capsules per cycle). At enrollment, a medical history was taken, and a physical examination and a pregnancy test were done. Collection of Genital Swabs for Herpes Simplex Virus The clinical staff instructed women in the collection of cervicovaginal, vulvar, and anal samples for viral cultures [17]. A separate Dacron swab was used to sample each site daily. For vulvar cultures, the swab was passed over the entire labia majora and minora. Cervicovaginal swabs were obtained by touching the exocervix and posterior vaginal fornix. Perianal samples were obtained by inserting the swab into the opening of the anal canal and rotating it. Swabs were placed into individual vials of viral transport medium and refrigerated until transport. Participants were instructed to collect the samples on awakening, if possible. Samples were either delivered to the laboratory by the participants themselves or were collected by courier at least three times per week. Previous studies have shown that specimens obtained by patients in this manner are as sensitive for detecting subclinical HSV shedding as those obtained by clinicians [15]. Each woman received a diary card for recording genital lesions, genital symptoms such as localized pain and irritation, and the number of pills taken daily. The use of open-label acyclovir or other topical or systemic antiviral agents was not permitted during the study. Participants were instructed to visit the clinic within 24 hours of the onset of recurrent genital lesions for genital examination and confirmation of an HSV recurrence. Patients also visited the clinic at 2-week intervals, at which time the diary cards were reviewed and collected, the study drug was refilled, and compliance was monitored. Laboratory Studies Each collected sample was inoculated daily into triplicate wells of diploid fibroblasts in 48-well microtiter plates [18]. We examined wells three times during the first week and twice during the second week. All wells that exhibited cytopathic effect were confirmed and typed using HSV-specific monoclonal antibodies as previously described [19]. At the completion of the trial, acyclovir sensitivity testing was done on selected isolates using previously reported methods [20-22]. Definition of Subclinical and Symptomatic Herpes Simplex Virus Shedding We evaluated the effect of acyclovir therapy on the frequency and duration of subclinical shedding of HSV, the primary end point of our study, and on the frequency and duration of symptomatic shedding. We defined subclinical shedding as isolation of HSV in the absence of genital or perianal lesions as noted by patient or clinician. The subclinical shedding rate was calculated as the number of days without genital lesions but with positive viral cultures divided by the total number of days without lesions. The rate of symptomatic HSV shedding was defined as the number of days with reported or observed genital lesions and with positive viral cultures, divided by the total number of days with genital lesions. Statistical Analyses All data collection, entry, analyses, and interpretation were done at the University of Washington, independent of the company sponsor. We evaluated the treatment effect in two ways. The first was an intent-to-treat analysis that included all randomized participants for the initial treatment period and that used Mann-Whitney two-sample test and chi-square test to compare shedding rates between the women who received placebo and those who received acyclovir therapy. The time to subclinical shedding and the risk factors for subclinical shedding were estimated using Kaplan-Meier survival curves and the Cox proportional-hazards model. A second analysis was done on the data from the 26 women who successfully complied with both sequences of the trial (successful compliance was defined as collection of cultures on at least 80% of the study days). This analysis included a paired analysis of treatment, period, and sequence effects using the Wilcoxon signed-rank test and the McNemar test [23]. Thus, we were able to assess the influence of the sequence of treatment (acyclovir followed by placebo compared with placebo followed by acyclovir) on the results of the study. The relation between the days on which women observed lesions while receiving acyclovir therapy and while receiving placebo was examined with the Spearman correlation coefficient. Results Thirty-four women were enrolled in the study between July 1992 and August 1993. Eighteen of these women had had genital herpes for less than 6 months (median time since acquisition, 3.5 months), and 16 had had genital herpes for more than 6 months but for less than 2 years (median time since acquisition, 18 months) (Table 1). The median age of the participants was 26 years, and 29 were white. Nine (50%) of the 18 women who had had genital herpes for less than 6 months and 8 (50%) of the 16 who had had genital herpes for more than 6 months were assigned to receive acyclovir first and placebo second; the 17 remaining women received the study drugs in the reverse order. Table 1. Demographic and Clinical Characteristics of 34 Study Participants Thirty-three of the 34 women successfully completed the first arm of the study, and 26 women completed both the first and the second arm. The median number of days on study was 154 (range, 54 to 177 days), and the median number of days on which cultures were obtained was 146 (range, 53 to 158 days). The median number of days on which cultures were obtained was 65 for the acyclovir arm, 66 for the placebo arm, and 13.5 for the washout period. The median percentage of the prescribed drug dosage taken was 98% during the first arm of the study, 99% during the second arm of the study, and 96% during the washout period. Of the eight women who failed to complete both portions of the study successfully, four were assigned to receive placebo initially and four were assigned to receive acyclovir initially. Six women withdrew from the study for administrative reasons (such as moving from the area), and two women completed the study but obtained cultures on only 47% and 65% of the study days, respectively. No patient withdrew from the study because of side effects or toxicity caused by the study drugs, nor did any participant drop out from either of the study arms to receive antiviral therapy. Intent-to-Treat Analysis Twenty-six of the 34 women had either a culture-confirmed clinical or subclinical episode of genital herpes during the study. Of the 17 women who were assigned to receive p