2 in those with prevalent infection [18, 19]. Because clinical and subclinical reactivation of HSV may result in the sexual transmission of HSV to a partner, suppressing viral shedding is important at both the individual and population level [12]. The current study was conducted to provide additional natural history data in individuals who have been given a new diagnosis of genital herpes, and to provide the ﬁrst evaluation of the eﬃcacy of valacyclovir 1 g once daily in reducing HSV- 2 shedding in this population. 2. Materials and Methods 2.1. Design. This was a double-blind, randomized, multi- center, two-way crossover study conducted at 14 centers in the United States (US) between February and November 2006. Subjects were randomized to receive valacyclovir 1 g or matching placebo once daily for 60 days in each of two treatment periods in a two-way crossover design. In our study, the term VAL-PBO is used to describe the treatment order for subjects randomized to receive valacyclovir in the ﬁrst treatment period followed by placebo in the second treatment period; the term PBO-VAL is used to describe the treatment order for subjects randomized to receive placebo in the ﬁrst treatment period followed by valacyclovir in the second treatment period. There was a washout period of seven days between treatment periods. The 1 g dose of valacyclovir was selected to optimize treatment in this population, 20% of whom would be expected to have 10 or more outbreaks per year based on previous studies [7]. The primary endpoint was the percent of all days with any HSV-2 viral shedding over 60 days deﬁned, for each subject as the percent of all days with PCR data for which HSV-2 shedding was detected by a positive PCR result. Secondary endpoints included percent of all days with subclinical shedding (without a genital lesion), percent of all days with clinical shedding (with a genital lesion), proportion of subjects with no shedding, proportion of subjects with at least one symptomatic recurrence and time to ﬁrst symptomatic herpes recurrence. 2.2. Study Population. Eligible subjects were diagnosed with a ﬁrst recognized episode of herpes at the screening visit or within 4 months of randomization. Subjects were required to have documented signs and symptoms of genital HSV- 2 infection, as well as laboratory conﬁrmation (positive culture, PCR, or type-speciﬁc serology) of HSV-2 infection to be eligible for the study. Subjects that were enrolled on the basis of a positive culture/PCR, but in the absence of HSV- 2 antibodies, represented true incident HSV-2 infection. It was accepted that a subject’s ﬁrst recognized episode of genital herpes might not represent new infection as prior episodes may have escaped detection. All subjects signed an informed consent document approved by an appropriate review committee prior to any study procedures. 2.3. Procedures. A subject’s participation in the study included a screening evaluation, a randomization visit, and eight study visits (four visits per treatment period, Infectious Diseases in Obstetrics and Gynecology with a 15-day interval between each visit). HSV-1 and HSV-2 type-speciﬁc serologic testing was conducted at the screening visit. Immunocompromised subjects, and subjects seropositive for HIV, were excluded. Subjects were not tested for HIV infection at baseline. Once randomized, subjects attended the clinic for additional visits if they suspected they had a genital herpes recurrence. A genital examination was conducted at the screening visit for subjects who presented with genital herpes signs or symptoms. Genital examinations also were conducted at the randomization and genital herpes recurrence visits for all subjects. During each 60-day treatment period and during washout, subjects collected a single daily swab from their genital/anal-rectal area for HSV-2 detection by PCR. On Day 1 of a genital herpes recurrence, swabs of the lesion also were collected in the clinic for HSV-2 detection by culture and PCR. During a documented recurrence, a subject temporar- ily discontinued blinded study medication and received open-label episodic treatment with valacyclovir 500 mg twice daily for 3 days, after which double-blind therapy resumed. Subjects used a telephonic, interactive voice response system daily to capture information on genital lesions, genital symptoms, and oral outbreaks. Adverse events, concomitant medications, and treatment compliance information were collected at each clinic visit, based on recall and discussion with the subject as well as pill counts. Blood and urine samples were collected for hematology, clinical chemistry, and urinalysis at the screening visit and at the end of each treatment period. Pregnancy testing was performed at baseline and at the end of each treatment period. 2.4. Laboratory Methods. The HerpeSelect ELISA glycopro- tein G-based assay (Focus Diagnostics, Cypress, Calif, USA) was used to detect the presence of HSV-1 and HSV-2 antibodies at screen (Quest Diagnostics, Van Nuys, Calif, USA). Samples demonstrating an index value >1.1 were considered positive. No conﬁrmatory testing was performed. Viral isolates from cultures were conﬁrmed and typed with monoclonal antibodies at Quest Diagnostics. HSV-2 DNA was assessed by qualitative ﬂorescent-based real time PCR assay at the University of Washington Virology Research Laboratory, Seattle, Wash, USA [20]. 2.5. Statistical Analyses. In this study, it was estimated that 47 completed subjects provided at least 90% power to detect a reduction in the mean percent days of HSV-2 shedding from 9.6% in the placebo treatment phase to 2.8% in the valacyclovir treatment phase, assuming standard deviations of 9.8% and 5.6%, respectively, and a correlation of 0.4. This study also was powered for the secondary endpoint of subclinical shedding. Forty-seven completed subjects provided 90% power to detect a reduction in the mean percent days of subclinical HSV-2 shedding from 7.0% in the placebo treatment phase to 2.8% in the valacyclovir treatment phase, assuming standard deviations of 9.3% and 5.4%, respectively, and a correlation of 0.4. To allow for dropouts, a sample size of 66 subjects was chosen.