Judith Zeh

The acquisition of herpes simplex virus during pregnancy.

BACKGROUND The acquisition of genital herpes during pregnancy has been associated with spontaneous abortion, prematurity, and congenital and neonatal herpes. The frequency of seroconversion, maternal symptoms of the disease, and the timing of its greatest effect on the outcome of pregnancy have not been systematically studied. METHODS We studied 7046 pregnant women whom serologic tests showed to be at risk for herpes simplex virus (HSV) infection. Serum samples obtained at the first prenatal visit, at approximately 16 and 24 weeks, and during labor were tested for antibodies to HSV types 1 and 2 (HSV-1 and HSV-2) by the Western blot assay, and the results were correlated with the occurrence of antenatal genital infections. RESULTS Ninety-four of the women became seropositive for HSV; 34 of the 94 women (36 percent) had symptoms consistent with herpes infection. Women who were initially seronegative for both HSV-1 and HSV-2 had an estimated chance of seroconversion for either virus of 3.7 percent; those who were initially seropositive only for HSV-1 had an estimated chance of HSV-2 seroconversion of 1.7 percent; and those who were initially HSV-2-seropositive had an estimated chance of zero for acquiring HSV-1 infection. Among the 60 of the 94 pregnancies for which the time of acquisition of HSV infection was known, 30 percent of the infections occurred in the first trimester, 30 percent in the second, and 40 percent in the third. HSV seroconversion completed by the time of labor was not associated with an increase in neonatal morbidity or with any cases of congenital herpes infection. However, among the infants born to nine women who acquired genital HSV infection shortly before labor, neonatal HSV infection occurred in four infants, of whom one died. CONCLUSIONS Two percent or more of susceptible women acquire HSV infection during pregnancy. Acquisition of infection with seroconversion completed before labor does not appear to affect the outcome of pregnancy, but infection acquired near the time of labor is associated with neonatal herpes and perinatal morbidity.

Reactivation of Genital Herpes Simplex Virus Type 2 Infection in Asymptomatic Seropositive Persons

BACKGROUND Most persons who have serologic evidence of infection with herpes simplex virus (HSV) type 2 (HSV-2) are asymptomatic. Historically, it has been assumed that these persons have less frequent viral reactivation than those with symptomatic infection. METHODS We conducted a prospective study to investigate genital shedding of HSV among 53 subjects who had antibodies to HSV-2 but who reported having no history of genital herpes, and we compared their patterns of viral shedding with those in a similar cohort of 90 subjects with symptomatic HSV-2 infection. Genital secretions of the subjects in both groups were sampled daily and cultured for HSV for a median of 94 days. RESULTS HSV was isolated from the genital mucosa in 38 of the 53 HSV-2-seropositive subjects (72 percent) who reported no history of genital herpes, and HSV DNA was detected by the polymerase-chain-reaction assay in cultures prepared from genital mucosal swabs in 6 additional subjects. The rate of subclinical shedding of HSV in the subjects with no reported history of genital herpes was similar to that in the subjects with such a history (3.0 percent vs. 2.7 percent). Of the 53 subjects who had no reported history of genital herpes, 33 (62 percent) subsequently reported having typical herpetic lesions; the duration of their recurrences in these subjects was shorter (median, three days vs. five days; P<0.001) and the frequency lower (median, 3.0 per year vs. 8.2 per year; P<0.001) than in the 90 subjects with previously diagnosed symptomatic infection. Only 1 of these 53 subjects had no clinical or virologic evidence of HSV infection. CONCLUSIONS Seropositivity for HSV-2 is associated with viral shedding in the genital tract, even in subjects with no reported history of genital herpes.

Virologic Characteristics of Subclinical and Symptomatic Genital Herpes Infections

BACKGROUND The frequency, pattern, and anatomical sites of subclinical shedding of herpes simplex virus (HSV) in the genital tract, along with factors that predict such shedding, have not been well characterized. METHODS We studied prospectively the clinical and virologic course of genital herpes in 110 women. The women kept symptom diaries and provided daily samples from the vulva, cervix, and rectum for viral culture. RESULTS During a median follow-up of 105 days, subclinical shedding of virus was identified in 36 of 65 women (55 percent) with HSV type 2 (HSV-2), in 16 of 31 women (52 percent) with HSV type 1 (HSV-1) and HSV-2, and in 4 of 14 women (29 percent) with only HSV-1. Among women with genital HSV-2 infection, subclinical shedding occurred on a mean of 2 percent of the days. The mean duration of viral shedding during subclinical episodes was 1.5 days, as compared with 1.8 days during symptomatic episodes. HSV was isolated from several sites in the genital tract and rectum in 17 percent of subclinical episodes and 22 percent of symptomatic episodes. Half the episodes of subclinical shedding of HSV occurred within seven days of a symptomatic recurrence. The risk of subclinical shedding increased with the frequency of symptomatic recurrences. Subclinical shedding was more frequent among women with more than 12 recurrences per year than among those with no symptomatic recurrences (odds ratio, 3.3; 95 percent confidence interval, 1.4 to 7.9); it was also more frequent among women who had recently acquired genital herpes (odds ratio for women with HSV acquired in the past year as compared with those who had had the infection for a year or more, 1.85; 95 percent confidence interval, 1.1 to 3.1). CONCLUSIONS Among women with a history of genital herpes infection, subclinical shedding of HSV is common and accounts for nearly one third of the total days of reactivation of HSV infection in the genital tract. Women with frequent symptomatic recurrences also have frequent subclinical shedding and may be at high risk for transmitting HSV.

Human Herpesvirus 6 in Lung Tissue from Patients with Pneumonitis after Bone Marrow Transplantation

BACKGROUND Human herpesvirus 6 (HHV-6) is a recently described herpesvirus that is epidemiologically and biologically similar to cytomegalovirus. It is the cause of exanthem subitum (roseola) in children. METHODS To evaluate the possible role of HHV-6 infection in pneumonitis in immunocompromised patients, we used quantitative HHV-6 polymerase chain reactions to study lung-biopsy specimens from 15 patients with pneumonitis after bone marrow transplantation and lung tissue from 15 immunocompetent subjects without pneumonitis and 6 fetuses. RESULTS HHV-6 DNA was detected in lung tissue from all 15 patients, from 14 seropositive control subjects, and from none of the 7 seronegative control subjects. Six patients had levels of HHV-6 DNA in lung tissue that were 10 to 500 times higher than those in any of the other patients or control subjects. Increased levels of HHV-6 DNA correlated with a decreased risk of death from pneumonitis (P = 0.015), an increased severity of graft-versus-host disease (P = 0.023), and the presence of idiopathic pneumonitis (P = 0.037). Levels of HHV-6 DNA correlated directly with the changes in HHV-6 antibody titers in the interval between the pretransplantation period and the open-lung biopsy (P = 0.002). Low levels of HHV-6 antibody at the time of the open-lung biopsy were also associated with the diagnosis of idiopathic pneumonitis (P = 0.002). CONCLUSIONS The concentrations of HHV-6 genome in lung tissue and their relation to changes in serologic titers support an association between HHV-6 infection and idiopathic pneumonitis in immunocompromised hosts.

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

Frequency of Symptomatic and Asymptomatic Herpes Simplex Virus Type 2 Reactivations among Human Immunodeficiency Virus—Infected Men

Herpes simplex virus (HSV) infection is common in persons coinfected with human immunodeficiency virus (HIV). In a prospective study, daily viral cultures of the mouth, genitals, and rectum were collected from 68 HIV-positive and 13 HIV-negative men who have sex with men. Subjects completed a median of 57 days of follow-up. Anogenital HSV-2 cultures were positive on 405 (9.7%) of 4167 days for HIV-positive men and on 24 (3.1%) of 766 days for HIV-negative men. Most reactivations were perirectal and subclinical. Risk factors for increased HSV-2 shedding among HIV-positive men were low CD4 cell count (odds ratio, 2.5; 95% confidence interval, 1.2-5.4) and antibodies to both HSV-1 and HSV-2 versus HSV-2 only (odds ratio, 1.9; 95% confidence interval, 1.0-3.7). Three isolates obtained from 3 separate subjects were resistant to acyclovir. Thus, subclinical HSV-2 reactivation is an important opportunistic infection in persons with HIV infection. Further studies are necessaryto determine the impact of subclinical HSV-2 reactivation on the natural history of HIV infection.

Changes in Plasma Human Immunodeficiency Virus Type 1 RNA Associated with Herpes Simplex Virus Reactivation and Suppression

In early trials of antiretroviral therapy, acyclovir was associated with increased survival by an unknown mechanism. The hypothesis that subclinical herpes simplex virus (HSV) reactivation was associated, in vivo, with increased plasma human immunodeficiency virus (HIV) RNA and suppression with a reduced plasma HIV RNA load was investigated. HSV cultures were performed daily on HSV-2-positive/HIV-positive patients, and plasma HIV-1 RNA loads were measured at regular intervals. A subset of patients prior to, during, and after HSV suppression with high-dose acyclovir was measured to determine whether HSV suppression was associated with a decrease in HIV replication. Most (25/27 HSV-2-positive/HIV-positive persons) reactivated HSV. Total HSV shedding rate was strongly correlated with plasma HIV-1 RNA load (R=0.54; P=.004), and the plasma HIV-1 RNA level at a given CD4 cell count was 48% lower when treated with acyclovir. These data indicate that frequent mucosal HSV reactivation influences HIV replication in vivo and daily HSV suppression may be important in the management of HSV-positive/HIV-positive persons.

Famciclovir for the Suppression of Symptomatic and Asymptomatic Herpes Simplex Virus Reactivation in HIV-Infected Persons: A Double-Blind, Placebo-Controlled Trial

Persistent genital herpes simplex virus (HSV) infection was one of the first opportunistic infections described in persons with AIDS [1]. Although 60% to 80% of the general population has serum antibodies to HSV-1, HSV-2, or both [2-4], up to 95% of HIV-positive persons are seropositive for either HSV-1, HSV-2, or both [5-11]. This makes HSV one of the most common viral infections complicating HIV infection. Anecdotal reports indicate that clinical reactivations usually persist for an extended period, may involve many cutaneous and mucosal sites, and seem to increase with progression of HIV disease [12, 13]. In addition, long-term antiviral therapy is commonly used to suppress frequent HSV reactivations. Although the efficacy of such therapy is well established in immunocompetent persons [14-16], no published trials have quantitated the efficacy of suppressive antiviral therapy on HSV-1 or HSV-2 reactivation in HIV-infected persons. Famciclovir is a nucleoside analogue recently licensed for the treatment of herpes zoster and recurrent genital HSV infection [17]. To determine the efficacy of famciclovir for the suppression of HSV reactivation in HIV-infected persons, we did a double-blind, placebo-controlled, crossover trial in 48 persons with HIV infection. Methods Study Participants and Design Persons infected with both HIV and HSV were recruited into our study through advertisements in local newspapers and referrals from private physicians. The study was approved by the University of Washington institutional review board. We screened 123 persons and enrolled 48. Reasons for exclusion were lack of HSV antibodies (14%), inability to complete or lack of interest in completing 4 consecutive months of daily home culture (80%), and unwillingness to go without therapy during a recurrence of HSV (6%). At enrollment, each participant completed a standardized interview designed to record history of previous HSV reactivation, frequency of previous antiherpesvirus therapy, and extent of HIV disease. We excluded persons if they were younger than 18 years of age, were currently infected or had previously been infected with acyclovir-resistant HSV, had known gastrointestinal disorders affecting absorption, or had received suppressive antiviral therapy with acyclovir in the 6 months before enrollment. Patients were paid

Clinical Reactivation of Genital Herpes Simplex Virus Infection Decreases in Frequency over Time

300 for completion of the protocol; those who completed only the first arm of the protocol were paid

Human Herpesvirus 6 Infections after Bone Marrow Transplantation: Clinical and Virologic Manifestations

150. At study entry, each participant was randomly assigned to receive either famciclovir tablets, 500 mg twice daily, or placebo tablets (identical in appearance to the famciclovir tablets) twice daily, for 8 weeks. This 8-week period was followed by a 7-day washout period, during which no pills were taken, and then by a second 8-week period during which participants received whichever regimen they had not received during the first phase of the trial. We chose this study design because it controlled for the variability between individual persons in CD4 cell count (which can affect the frequency of HSV reactivation [13]) and because a 7-day washout period had been shown not to influence subsequent HSV reactivation [18]. Famciclovir and placebo were dispensed in 28-day supplies (56 pills). During the entire 119-day study period, patients obtained once-daily cultures of the oropharynx, genitals (urethra and penile shaft in men; cervicovaginal area and labia in women), and rectum. Participants returned to the clinic at 28-day intervals so that we could review their daily symptom diaries, monitor compliance, distribute more study medication and culture supplies, and assess safety. All participants were also asked to return to the clinic during episodes of genital herpes. At these visits, we performed genital examinations to confirm the presence of lesions and obtained additional cultures of the lesions. Both participants and investigators were blinded to culture results until the conclusion of the study. Neither open-label oral acyclovir nor topical anti-HSV products for treatment of an HSV recurrence were allowed. Collection of Daily Cultures The methods used to collect swabs for HSV isolation have been described elsewhere [16, 18]. Each participant collected one specimen daily from each of four anatomic sites, using a separate Dacron swab for each culture. Oral-pharyngeal cultures were obtained by inserting a swab into the mouth and vigorously rubbing it along the gum line and over the palate. Men obtained urethral cultures by rubbing a swab over the urethral opening and obtained penile cultures by rubbing a separate swab along the entire ventral and dorsal shaft of the penis. Women obtained labial cultures by rubbing a swab over the entire labia majora and minora. Cervicovaginal cultures were obtained by rubbing a swab over the exocervix and posterior vaginal fornix. Rectal cultures were obtained by inserting the swab approximately 2 to 4 cm into the anus and gently rotating it. Each swab was placed in a separate vial that contained viral transport media, was labeled with site and date, and was stored in the refrigerator. Participants were instructed to obtain the cultures at the same time each day, preferably upon awakening. Cultures were picked up by a courier within 36 hours of collection and were transported to the virology laboratory of the University of Washington, where they were immediately inoculated into tissue culture. Participants also filled out daily diary cards that recorded the site-specific presence or absence of symptoms (pain, tingling, numbness or itching, presence of lesions) and the number of pills taken each day. The cards were collected monthly and reviewed by the study clinician. Unused cards of medication were collected, and pill counts were done to confirm compliance records. Laboratory Studies Serologic and T-Cell Analysis Seropositivity for HIV was confirmed by using standard enzyme-linked immunoassays and Western blot assays. CD4 cell subset analysis was done by using flow cytometry. Tests for antibodies to HSV were performed with Western blot analysis on serum specimens obtained at study entry [11, 19]. Patients were classified into three groups: those who were seropositive for HSV-1 only, those who were seropositive for HSV-2 only, and those who were seropositive for both HSV-1 and HSV-2. Herpes Simplex Virus Culture and Sensitivity Testing For isolation of HSV, each sample was inoculated in triplicate into 48-well microtiter plates that contained human diploid fibroblasts [20]. Each well was examined three times weekly for evidence of cytopathic changes of HSV infection. Cultures that showed cytopathic effects were confirmed and typed by using HSV-specific monoclonal antibodies. The initial culture supernatant from each clinical isolate that was obtained while patients were receiving famciclovir was inoculated into human diploid fibroblast cells [21]. Cell-associated virus (1:200 dilution in MEM [minimal essential media]-10% fetal calf serum) was inoculated in duplicate onto freshly confluent diploid fibroblast cells in 24-well plates. Plates were rocked at 30 tilts per minute for 60 minutes; penciclovir (Smith-Kline Beecham Pharmaceuticals, Philadelphia, Pennsylvania) containing medium was then added at 11 serial log2 dilutions from 40.96 to 0.04 g/mL. Control wells with no penciclovir were also established. When controls without penciclovir demonstrated 4+ cytopathic effect, the Hybriwick kit (Diagnostic Hybrids, Athens, Ohio) was used to measure HSV DNA [22] according to the manufacturers directions. The IC50 value was estimated as the lowest concentration of drug that caused a 50% or greater reduction of mean cycles per minute hybridization of HSV-specific DNA probe to cell lysates. Values for IC50 were calculated with a computer algorithm that used a Michaelis-Menten model and nonlinear least-squares curve fitting. Definition of Terms Total HSV shedding was defined as the total number of days on which HSV was isolated by culture (regardless of anatomic site) divided by the total number of days on which cultures were obtained. Asymptomatic HSV shedding was defined as the total number of days on which a participant reported no symptoms or lesions at an anatomic site from which HSV was isolated divided by the total number of days on which cultures were obtained. Symptomatic HSV shedding was defined as the total number of days on which a lesion or symptom was reported by the participant in the symptom diary at an anatomic site from which HSV was isolated divided by the total number of culture days. A recurrence of genital herpes was defined as lasting from the onset of lesions to the complete healing of lesions. Statistical Analysis Treatment effect was initially evaluated with intention-to-treat analyses that included all randomly assigned participants before crossover. Survival analysis was used to handle data censoring caused by early withdrawals. Time to first isolation of HSV (either HSV-1 or HSV-2) by culture, time to HSV-1 isolation, and time to HSV-2 isolation were examined. Differences in Kaplan-Meier survival curves between the famciclovir and placebo groups in the first treatment period were assessed by using the log-rank test. The relative risk for shedding was estimated by using the Cox proportional-hazards regression model with first-period treatment as the only predictor variable. To determine the effect of famciclovir on reduction of HSV-1 and HSV-2 shedding rates and days with symptoms, we used crossover analysis methods to further analyze participants who successfully completed both arms of the study. Successful completion was defined as more than 28 days with cultures in each treatment arm. Because HSV shedding and symptom rates were not normally distributed, nonparametric tests were used. Wilcoxon rank-sum tests for carryover (residual) and period effects compared the sums and differences, respectively, of rates during placebo and famciclovir administra