Genital herpes simplex virus (HSV) infection is one of the most common viral sexually transmitted diseases in the United States, now affecting an estimated 50 million adolescents and adults.1,2 Although only 5% to 10% of women of reproductive age have a history of genital herpes infection, 25% to 30% have HSV-2 antibodies.1,3–5 The majority of these seropositive women are unaware of their HSV infection. As the incidence of this sexually transmitted infection continues to rise, and because the greatest incidence of HSV infections occur in women of reproductive age, the risk of maternal transmission of the virus to the fetus or neonate has become a major health concern.6–8
Almost 100% of women with HSV-2 experience clinical or subclinical recurrences throughout their lifetime,9 especially in the 8–10 years after primary infection.2 Five to 10% of pregnant women have symptomatic recurrent genital herpes during pregnancy,2,3 and up to 25% of these women will have an HSV outbreak the last month of pregnancy.10 Because 85% of neonatal herpes results from viral transmission near delivery,7,11 the pursuit of strategies to prevent transmission has focused on this period. Currently, it is recommended that a cesarean delivery be offered to all women with an active genital herpes lesion or prodromal symptoms at the time of presentation in labor.8 However, the observation that 70% of neonatal herpes cases occur in infants of women who asymptomatically shed virus near delivery4,11–14 has refocused attention to therapeutic approaches that decrease HSV viral shedding, regardless of clinical lesion status.
In a recent meta-analysis we found that prophylactic acyclovir beginning at 36 weeks of gestation reduced the risk of clinical HSV recurrence at delivery, cesarean delivery for recurrent genital herpes, and viral shedding at delivery.15 Valacyclovir hydrochloride, a prodrug of acyclovir, was developed to improve the bioavailability of acyclovir by enhancing absorption from the gastrointestinal tract. This allows for less frequent dosing requirements.16 In clinical trials that did not include pregnant women, it has been shown to be equally effective in suppressing episodes of recurrent genital herpes.17–20
The purpose of this study was to estimate the efficacy of valacyclovir prophylaxis given from 36 weeks gestation until delivery in reducing clinical and virologic evidence of HSV infection at the time of delivery. We also sought to determine any adverse effects of valacyclovir in the neonate.
MATERIALS AND METHODS
This study was a prospective randomized, double-blind comparison of the efficacy of valacyclovir compared with placebo administered orally from 36 weeks gestation until delivery. An independent party was responsible for the randomization code and kept the study code records. It was conducted at Parkland Health and Hospital System in Dallas, Texas, which serves a predominantly indigent population, over a 6-year period from April 1, 1998, through November 2004. The study protocol was approved by the Institutional Review Board of the University of Texas Southwestern Medical Center at Dallas.
All pregnant women at 36 weeks of gestation or less who had a history of genital HSV antedating the pregnancy or who experienced a first outbreak of genital herpes during the pregnancy were approached regarding the study. An information pamphlet was provided in either English or Spanish. All eligible women had serologic confirmation of prior HSV infection by antibody testing using an HSV-1 and HSV-2 IgG enzyme-linked immunosorbent assay II kit (Wampole Laboratories, Cranburg, NJ). First episode primary genital herpes was diagnosed in women with a new genital lesion positive for HSV-1 or HSV-2 and seronegative for both. First episode nonprimary genital herpes was diagnosed when a genital lesion was positive for HSV-2 and seropositive for HSV-1 but seronegative for HSV-2. Women were excluded from the study if they were allergic to valacyclovir or acyclovir, had an immunosuppressive disorder such as human immunodeficiency virus, were known to require a cesarean section or were greater than 36 weeks of gestation at the time of identification. Women with a known fetal anomaly, intrauterine growth restriction, or oligohydramnios were also excluded.
Informed consent was obtained from eligible patients by 36 completed weeks of gestation. Assignment at 36 weeks was made using a computer-generated random number table. They were then dispensed either valacyclovir 500 mg or an identical-appearing placebo, to be taken 2 times a day until delivery. Patients, physicians, and the research coordinator were blinded to the assigned study regimen. An individual not involved with patient care maintained the randomization list and assigned the study drug groups.
Women were scheduled to return weekly to a specific sexually transmitted disease prenatal clinic staffed by the study investigators. At each visit, a detailed questionnaire was given regarding medication side effects, symptoms of a clinical recurrence or prodromal symptoms and medication adherence (by pill count). A pelvic examination for HSV lesions was performed and an HSV culture was obtained to evaluate any suspicious lesions or if prodromal symptoms were present. The study patients were then dispensed another week's supply of study medication.
On admission to the labor and delivery unit, each patient was examined for evidence of genital lesions by visual examination of external genitalia, vagina, and cervix using a speculum. They were questioned regarding prodromal symptoms by trained nurse-practitioners and physicians. An HSV culture of the usual lesion site and the cervix was performed to detect asymptomatic shedding. Visible genital or cervical lesions were also cultured. Another sample was collected using a polyester fiber swab from the above sites, air dried and stored at 4°C for DNA isolation and PCR analysis (described below) at a later date. Results of PCR analysis were not available to the managing clinicians. Women underwent cesarean delivery if they presented in active labor or with ruptured membranes with either a lesion suspicious for HSV or prodromal symptoms.
Each neonate was evaluated at 12–36 hours of age by a pediatrician aware of the mother's participation in the study but blinded to the study allocation. Under protocol, neonates did not receive prophylactic antiviral treatment for possible exposure at delivery. A physical examination was performed; HSV cultures of the newborn's conjunctiva, oropharynx, rectum, scalp electrode site, and any suspicious lesions were obtained. A polyester fiber swab was also obtained from these sites and stored at 4°C for DNA isolation and PCR analysis (described below). Due to reports of neutropenia in newborns exposed to high dose intravenously acyclovir,21,22 we included a nested substudy of neonatal safety in a group of 126 consecutive neonates to examine neonatal safety after in utero acyclovir exposure. Complete blood count with differential, liver function tests, serum creatinine, and urinalysis were performed. Mothers were instructed to contact investigators for any problems in the first month after discharge, and the infants were followed up at 1 month of age to assess any readmissions or adverse events.
Maternal and neonatal samples obtained for DNA analysis were air dried and stored at 4°C. They were then placed in 0.4 mL phosphate-buffered saline and incubated at room temperature for 30 minutes. After removal of the swab, the resulting patient sample (approximately 0.2 mL) was processed for DNA isolation using the QIAmp DNA Blood Mini Kit (QIAGEN Inc., Valencia, CA) according to the manufacturers' protocol for isolation of genomic DNA from buccal swabs. Final DNA sample volume was 0.15 mL. Quantification of HSV-2 by PCR was performed using the HHVQ-1 internal calibration standard (ICS), essentially as described.23–25 Briefly, PCR reactions contained 10-μL patient sample DNA, 20 molecules of HHVQ-1, 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer, Wellesley, MA), 60 mM Tris-HCl pH 9.0, 15 mM ammonium sulfate, 2 mM MgCl2, 20 mmol of each HSV-2 oligonucleotide primer (5′primer-cgtcctggagtttgacagcg; 3′ primer – cagcagcgagtcctgcacacaa), and 100 μM each of dATP, dCRP, dGTP, and dUTP in 50 μL final volume. Amplification reactions were initially heated to 95°C for 2 minutes and then subjected to 36 cycles of 94°C for 0.5 minutes, 60°C for 0.5 minutes, and 72°C for 1 minute, followed by a final extension at 72°C for 9 minutes in a GeneAmp 9600 thermocycler (Perkin-Elmer). PCR products were separated by 2% agarose gel electrophoresis and quantified following staining with SyberGold (Molecular Probes, Eugene, OR) on a fluorimager (Typhon 8600, Amersham Biosciences, Piscataway, NJ) using ImageQuant software. Quantities of HSV-2 were determined by comparison of band intensities derived from the ICS and HSV-2, and are expressed as HSV-2 viral genomes (or viral targets)/patient sample. For patient samples with high levels of HSV-2, DNA samples were diluted in ddH2O before amplification such that roughly equivalent ICS and HSV-2 PCR product band intensities were obtained.
To verify the identity of any positive HSV-2-derived PCR products, DNA from the agarose gels was transferred to Zeta-Probe Blotting Membranes (Bio-Rad Laboratories, Hercules, CA) using a vacuum blot apparatus and an alkaline transfer buffer (0.5 M NaOH, 0.6 M NaCl). The blots were air dried and then prehybridized for 1 hour at room temperature in 10 mL of Hybridization Buffer (1% SDS, 1.5X SSPE, and 100 mg of nonfat dry milk). An HSV-2–specific oligonucleotide probe (ggcatcatcaccgacaaggtcaaactctccagc) was end-labeled with 1 μCi of [γ32P]ATP using T4 polynucleotide kinase. The probe was denatured for 10 minutes at 95°C and then incubated with the filter for 4 hours at room temperature in 10 mL of fresh Hybridization Buffer. After hybridization, the filter was washed four times shaking for 15 minutes as follows: 2X SSC at room temperature (RT), 2X SSC and 0.1% SDS at RT, ol5 X SSC and 0.1% SDS at RT, 0.5 X SSC and 1% SDS at 37°C. The wet filter was placed in plastic wrap and exposed to a phosphorimager screen overnight. The screen was scanned and analyzed using a phosphorimager (Typhoon 8600). All positive PCR samples were confirmed using this procedure.
Sample size estimates were performed before the study onset using a power of 0.9 (type II error 0.10) and type I error of 0.05. We estimated a recurrence risk at delivery in the placebo group to be 17.9% (pooled estimate), and a recurrence risk at delivery in the valacyclovir group to be 4.9% (pooled estimate based on available acyclovir data). This yielded a minimum sample size of 139 women in each group. Anticipating a lost to follow-up rate of 5% and with the addition of the nested cohort study for neonatal safety, a total of 350 women were enrolled in the study.
Normally distributed continuous data were analyzed using a Student t test; a nonparametric Mann–Whitney U test was used for continuous data. A χ2 test was used to analyze categoric data, and odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Data were analyzed as intent to treat.
Three hundred fifty women were enrolled in the study, 178 in the valacyclovir group and 172 in the placebo group. A total of 12 women (3%) were lost to follow-up during the study period. The final study cohort consisted of 338 women, 170 in the valacyclovir group and 168 in the placebo group. Table 1 details the demographic and social characteristics of the two groups. There were no significant differences with regard to age, race, parity, and marital status. There were also no differences in tobacco, alcohol, or injection drug use.
The majority of women had a history of a sexually transmitted disease or infection, excluding HSV (Table 2). The mean age at first HSV outbreak for the cohort was 21.7 ± 5.1 years (21.2 ± 5.1 years and 22.2 ± 5.1 years in the valacyclovir and placebo groups, respectively). Thirty-nine percent and 34% of the women in the valacyclovir and placebo groups, respectively, reported more than one HSV outbreak per year. All women had a clinical history and serologic confirmation of HSV infection. The majority of women were diagnosed with recurrent disease (79% and 84%, valacyclovir compared with placebo). Overall, 82% of the women had recurrent genital herpes; 12% had first episode, nonprimary genital herpes; 6% had first episode, primary genital herpes. There were no significant differences between the 2 groups with regard to HSV history (Table 2). Finally, medication compliance in the week before delivery was similar between the 2 groups (91% and 88% in the valacyclovir and placebo groups, respectively).
The valacyclovir and placebo cohorts reported a similar number of HSV recurrences before the onset of study medication at 36 weeks (Table 3). Seventy-six women in the study had at least 1 HSV recurrence before randomization. Upon initiation of medication at 36 weeks, a significant decrease was noted in the median number of HSV recurrences per patient and in any HSV recurrence in those women receiving valacyclovir (P = .04 and P = .005, respectively).
Valacyclovir significantly reduced the number of women with clinical genital HSV at delivery by 70%, from 13% in the placebo group to 4% in the valacyclovir group (P = .009; OR .30, 95% CI .12–.73) (Table 4). Two hundred fifty-five women had an HSV culture performed at delivery, and 164 women had PCR data available for analysis. Valacyclovir significantly decreased the likelihood of a positive culture or PCR result (P = .02 and 0.01, respectively; OR .18, 95% CI .04–.82 and OR .28, 95% CI .11–.72, respectively). Finally, valacyclovir decreased the number of cesarean deliveries performed for HSV, from 13% in the placebo group to 4% in the valacyclovir group (P = .009; OR .3 95% CI .12–.73).
There were no significant differences between the 2 groups with regard to estimated gestational age at delivery or mean birthweight (Table 5). There were also no differences between the 2 groups with regard to obstetric and neonatal complications, such as meconium-stained amniotic fluid, neonatal sepsis, and ventilation requirements, and admission to the neonatal intensive care unit. No infant had a positive HSV culture at birth.
One hundred twenty-six infants were enrolled in the nested substudy to evaluate the safety of valacyclovir, 66 in the valacyclovir group and 60 in the placebo group. Toxicity assessments were quantitated using the Division of Acquired Immunodeficiency Syndrome (DAIDS) Toxicity Tables for neonatal laboratory results.26 No infant was diagnosed with neutropenia defined by either a total white cell count less than 4,000 cells/mm3 or an absolute neutrophil count less than 1,000/mm3. There was no significant increase in the number of infants diagnosed with anemia. There was only 1 infant in the valacyclovir group with a hemoglobin less than 10 g% (actual value 9.2 g%). There were no adverse hepatic or renal effects noted when the neonate was exposed to valacyclovir (Table 6).
Adverse events were collected during the study period. There was 1 unexplained fetal demise at term in the placebo group. No evidence of HSV was noted in this infant. There were no major congenital malformations noted in either cohort, and only 1 minor malformation in the placebo group (polydactyly). Finally, 1 neonate in each group was readmitted to the hospital to evaluate neonatal sepsis. No infant was diagnosed with neonatal HSV in the first month of life.
Valacyclovir was tolerated well in the study cohort. There was no serious morbidity in either group. Seven (4%) women compared with 5 (3%) women in the valacyclovir and placebo groups, respectively, experienced mild nausea and vomiting. Headache or dizziness was reported in 8% of the women, with no difference between valacyclovir and placebo groups.
We report a large randomized placebo-controlled trial comparing valacyclovir to placebo suppression in pregnancy from 36 weeks until delivery. Valacyclovir decreased the rate of clinical HSV at delivery by 69%. It also decreased asymptomatic shedding at delivery, shown using HSV culture and PCR. A decrease in cesarean delivery for HSV was noted. There was no significant increase in adverse obstetric or neonatal events in the valacyclovir group. Neonatal laboratory evaluation showed no neutropenia and no hepatic or renal dysfunction in infants exposed to valacyclovir. Finally, valacyclovir was tolerated well by pregnant women with only minor side effects, comparable to the placebo group.
Valacyclovir suppression has been shown in adults to decrease recurrent genital HSV, asymptomatic shedding, and HSV transmission18–20,27 comparable to previously reported acyclovir data. The benefits of valacyclovir hydrochloride are 2-fold. It is rapidly absorbed from the gastrointestinal tract with improved bioavailability. It is also rapidly converted to acyclovir and achieves higher plasma levels than acyclovir alone.28 This allows for less frequent dosing, potentially improving patient compliance. Valacyclovir is well tolerated and adverse events reported have been mild. The experience was similar in our cohort, with no increase in maternal side effects compared with placebo.
The benefit of treating pregnant women with a history of HSV with suppressive antiviral therapy has been controversial. The American College of Obstetricians and Gynecologists (ACOG) recommendations are based on limited or inconsistent scientific evidence (level B) or on consensus and expert opinion (level C). The ACOG Practice Bulletin of October 1999 states, “For women at or beyond 36 weeks of gestation with a first episode of HSV during the current pregnancy, antiviral therapy should be considered” (level B) and "For women at or beyond 36 weeks of gestation who are at risk for recurrent HSV, antiviral therapy also may be considered, although such therapy may not reduce the likelihood of cesarean delivery (level C). We recently performed a meta-analysis of the 5 randomized controlled trials evaluating acyclovir suppression in pregnant women with a history of genital HSV.15 Acyclovir suppression was found to be effective in reducing the risk of both clinical genital herpes recurrences and HSV shedding at delivery. The results of this study confirm these findings about antiviral suppression and extend them to include valacyclovir.
A concern with the use of antiviral prophylaxis has been that suppression of viral shedding might be incomplete. In other words, a reduction in the rate of cesarean delivery could theoretically increase the risk of neonatal herpes by increasing vaginal deliveries and genital tract exposure to subclinical HSV shedding. The data from this study and the previously described meta-analysis15 indicate that antiviral prophylaxis is effective in reducing the risk of active lesions and in reducing the risk of asymptomatic shedding.
Some investigators have expressed concerns about the effect of valacyclovir on the fetus and neonate. In two studies by Kimberlin et al21,22 neutropenia, defined as an absolute neutrophil count less than 1,000/mm,3 was noted in 21% of infants with neonatal HSV treated with prolonged intravenous acyclovir and in 46% treated with prolonged suppressive oral acyclovir. These safety concerns led us to initiate a nested substudy, evaluating neonatal safety laboratory studies and scheduling a one-month follow-up examination. Although the numbers of exposed infants is limited, no neutropenia was noted in either study group. Furthermore, valacyclovir did not have an adverse effect on liver or renal function. No neonate had anemia that required blood transfusion. We did not observe any association with congenital malformations, but infants were not exposed to the drug until 36 weeks of gestation.
This study was powered sufficiently to address the primary outcomes. However, there are several limitations. The delivery collection of HSV PCR and culture specimens were incomplete. There was also insufficient power to fully address neonatal safety and to determine any effects on neonatal HSV transmission. Finally, the safety of valacyclovir use only addressed maternal and fetal exposure from 36 weeks of gestation until delivery. No statement can be made regarding safety at earlier gestations.
In conclusion, valacyclovir prophylaxis was tolerated well in late pregnancy, had no adverse effects on the neonate and significantly reduced clinical recurrences at delivery, laboratory detection of HSV at delivery, and cesarean delivery for HSV. From a practical point of view, 11 women would need to be treated with valacyclovir to prevent 1 cesarean delivery for HSV. Only 7 women would need to be treated with valacyclovir to prevent one positive HSV PCR at delivery. Given the near epidemic rise in the incidence of genital HSV, antiviral prophylaxis late in pregnancy needs to be considered in managing this increasingly large number of women.
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