Herpes simplex virus (HSV) type 2 is one of the most common pathogens found among HIV-1-infected individuals, with prevalence levels above 80% in Africa . HSV-2 infection may facilitate HIV acquisition  and potentially increase HIV-1 genital shedding and infectiousness [3,4], although the latter hypothesis was challenged by recent data from Zimbabwe . The possible mechanisms for facilitation include the local influx of activated CD4 lymphocytes in HSV-infected lesions  and transactivation of the HIV-1 tat  and long terminal repeat genes by HSV proteins [7–9], which could result in a higher replication of HIV-1 at both the systemic and genital levels.
Randomized controlled trials of HSV-2 suppressive strategies are required to demonstrate a causal role of HSV-2 in HIV-1 transmission [1,10]. The impact of such interventions at all stages of HIV disease, including among patients taking HAART, would inform on the possible mechanisms of action, in particular whether compartmentalization of HIV replication occurs as suggested [11,12], and whether this could be specifically targeted. Suppressive antiherpetic therapy currently offers the most attractive means of HSV-2 control, as it has proved successful in reducing the frequency of genital herpes episodes and in increasing the duration of lesion-free periods among HIV-infected patients, including those taking HAART .
We conducted two randomized controlled trials of HSV-2 suppressive therapy among HIV-1 and HSV-2 co-infected women in Bobo-Dioulasso, Burkina Faso, measuring the impact on genital HIV-1 RNA, plasma HIV-1 RNA and genital HSV-2 DNA among women taking HAART (ANRS 1285b study), or not eligible for HAART (ANRS1285a study). Here we report the results of the trial in women taking HAART.
Patients and methods
The ANRS1285b study was a double blind, placebo-controlled, randomized trial of valacyclovir suppressive therapy 500 g administered twice a day for 3 months to women already taking HAART. The study protocol complied with the Ethical Charter of the French Agence Nationale de Recherches sur le SIDA et les Hepatites (ANRS) and was approved by the Centre Muraz Institutional Review Board, the Ethical Committees of the Burkina Faso Ministry of Health and the London School of Hygiene and Tropical Medicine.
Study participants were recruited, between August 2004 and January 2005, either from the Yerelon cohort of high-risk women described elsewhere , from local organizations of individuals living with HIV/AIDS, or from the University Hospital in Bobo-Dioulasso. Women aged 16 years and over, with serum antibodies to HSV-2 and HIV-1, and taking HAART for a minimum of 4 months were eligible for the ANRS1285b trial. Exclusion criteria included: significant renal impairment or hypersensitivity to acyclovir; pregnancy or the desire to become pregnant in the next 6 months; breastfeeding; taking acyclovir or being eligible for HSV-2 suppressive therapy because of a history of frequent (six or more) genital herpes episodes in the preceding 12 months; and predicted poor adherence with study procedures.
Participants had started HAART as part of the medical management provided by the recruiting institution.
HAART initiation and monitoring followed the Burkinabe national guidelines derived from the World Health Organization (WHO) recommendations for developing countries . Women with a stage IV AIDS classification or CD4 lymphocyte count less than 200 cells/μl were eligible for HAART. The first line treatment regimen included a standard combination of zidovudine, or stavudine in case of anaemia, lamivudine and efavirenz.
Potentially eligible and consenting women were informed of the study aims and procedures during a screening visit, and were assessed for eligibility. After one week, eligible women who signed consent forms were enrolled into the baseline phase, which consisted of six biweekly visits over 3 months. At the scheduled sixth visit, eligibility criteria were assessed again, and after the collection of appropriate samples consenting participants were randomly assigned to study drugs following a random allocation list independently generated by the study drug manufacturer (www.randomization.com) with a 1: 1 allocation scheme and block random assignment of 10 individuals. This list was kept by the Data Monitoring Committee. Investigators and participants were blinded to treatment allocation through the use of a prelabelled sequentially numbered list of treatment packs. The treatment phase began 2 weeks after randomization and lasted 3 months with biweekly visits.
Women who became pregnant were withdrawn from study drugs but were invited to carry on with regular follow-up procedures. Menstruating women were deferred for genital sampling until 2 days after bleeding had ceased. Patients with genital ulcers were initially given antibiotics following national guidelines for the syndromic management of sexually transmitted infections. Non-healing ulcers after 7 days were treated with open-label acyclovir 200 mg five times a day for 5 days. The assigned study drug was not discontinued.
At each visit, swabs were collected for the diagnosis of vaginal infections (Trichomonas vaginalis, Candida albicans and bacterial vaginosis), and enriched cervicovaginal lavages for the detection and quantitation of genital HIV-1 RNA and HSV-2 DNA, as described previously . At enrolment, additional cervical swabs were collected for the diagnosis of Neisseria gonorrhoeae and Chlamydia trachomatis for treatment purposes.
Blood samples were drawn on alternate visits for the quantitation of plasma HIV-1 RNA, and for repeat CD4 T-lymphocyte counts at the first visit of the treatment phase only.
Urine samples were collected just before randomization and once a month during the treatment phase for pregnancy testing (Vikia HCG-SJ; Bio-Merieux, France).
Serological assays and CD4 T-lymphocyte analysis
Depending on the source of recruitment, the diagnosis of HIV-1 infection relied on two complementary enzyme-linked immunosorbent assays  or a rapid testing strategy using Determine (Abbott, Tokyo, Japan) followed by Genie II (Bio-Rad, Paris, France) as recommended by the WHO . HSV-2 seropositivity was determined using a specific IgG2 enzyme-linked immunosorbent assay (Kalon; Kalon Biologicals, Surrey, UK) with high sensitivity (92.3%) and specificity (97.7%) in African sera . Serological diagnosis of syphilis was based on positive results for rapid plasma reagin test (BioMérieux, Lyon, France) and Treponema pallidum haemagglutination assay (New Market Laboratory Ltd., Kentford, UK). The CD4 lymphocyte count was performed using a standard fluorocytometry technique (FACSCAN; Becton Dickinson, USA).
Detection and quantitation of HIV-1 RNA and herpes simplex virus 2 DNA
HIV-1 RNA and HSV-2 DNA were quantitated using real-time PCR techniques with the ABI 7000 system and a manual nucleic acid extraction (Qiagen RNA and DNA kits) as described previously [16,20]. Testing was performed in duplicate, and the mean value of the two measures was used in the analyses. The technique proved reliable, with quantitation thresholds of 300 (2.48 log10) , 250 (2.40 log10)  and 500 (2.70 log10) copies/ml for plasma HIV-1 RNA, genital HIV-1 RNA in enriched cervicovaginal lavages, and genital HSV-2 DNA, respectively. The Centre Muraz Laboratory was enrolled into an ANRS-organized external quality control scheme for HIV-1 RNA quantitation, and a commercial panel (HSV 1/2 Clear QC panel; Argen, Varilhes, France) was used as an internal quality control for HSV-2 DNA quantitation.
N. gonorrhoeae was identified by the culture of cervical swabs on modified Thayer–Martin media; C. trachomatis was diagnosed using a commercial PCR assay (Amplicor, Roche Diagnostics Inc., Branchburg, USA); T. vaginalis and C. albicans were identified by culture on specific media, the InPouch TV (Biomed Diagnostics, San Jose, California, USA) and Sabouraud's milieu, respectively; and bacterial vaginosis was diagnosed from Gram-stained vaginal smears using the Nugent method (score of 7–10) .
Study endpoints and definitions
The primary study endpoints were the detection of any genital HIV-1 RNA above the threshold level, the frequency of detection, and mean viral load. Secondary endpoints were the detection and quantity of plasma HIV-1 RNA, the detection, frequency and quantity of genital HSV-2 DNA, and the occurrence of ‘clinical ulcer episodes’, defined as the proportion of women experiencing at least one episode of genital ulceration or vesicles.
The sample size was determined by calculations for the companion ANRS1285a trial. This led to the recruitment of 60 women eligible for HAART and the ANRS1285b trial, which provided 80% power to detect a reduction of 0.7 log10 copies/ml of genital HIV-1 RNA between the two arms (with variance of 0.4, 95% significance level, allowing for 70% of participants with detectable genital HIV-1 RNA at least once over the study period and 10% loss to follow-up).
Analyses were conducted using Stata version 9.0 (Stata Corporation, College Station, Texas, USA), using a modified intention-to-treat approach whereby pregnant women were censored at the time of the first positive urine test. Data from the prerandomization baseline phase were used to adjust for the variability in genital HIV-1 RNA and HSV-2 DNA, as appropriate.
The impact of valacyclovir on the presence and frequency of plasma HIV-1 RNA, genital HIV-1 RNA and genital HSV-2 DNA was assessed using ‘per-woman’ summary measures. The impact on the proportion of women with a detectable genital or plasma viral load at least once was estimated using binomial regression, adjusting for the presence of detectable virus during the baseline phase. The impact on the frequency of a detectable genital or plasma viral load (proportion of visits during which the virus was detected, per woman) was estimated by an ordered logistic regression model adjusting for baseline frequency.
The impact of valacyclovir on the quantity of HIV-1 RNA and HSV-2 DNA was assessed on a ‘per-visit’ basis, using random effects linear regression among episodes with detectable HIV-1 RNA or detectable HSV2 DNA (as appropriate), as the distributions of HIV-1-RNA and HSV-2-DNA viral load were highly skewed when including non-detectable visits.
Secondary analyses of detection and frequency were carried out on a ‘per-visit’ basis, using random effects logistic regression. An interaction term for phase and arm was used to estimate the impact on the presence of genital HIV-1 RNA and HSV-2 DNA, respectively. The trend of the treatment effect with time was assessed by including an interaction term of arm and time (time equals 0 for the baseline phase, 1–6 for visits in the treatment phase).
Finally, predefined subgroup analyses were performed by restricting the analyses to women who shed HIV-1 at least once during the baseline phase.
Eighty-two women were screened for trial eligibility, 61 were eligible, and 60 were randomly assigned to receive valacyclovir (n = 30) or placebo (n = 30). None of the participants was lost to follow-up, but two women (in the valacyclovir arm) were withdrawn from study treatment because of pregnancy and were therefore censored (Fig. 1).
The mean age of the participants was 33.3 years (range 23–47; Table 1). There were no major imbalances between the treatment arms at enrolment in terms of demographic, behavioural or biological factors. The median time since HAART initiation was 19.3 weeks (range 15.9–217).
The mean treatment compliance assessed by pill count was 98.9% and 99.1% in the valacyclovir and placebo arms, respectively. All women had a mean HAART adherence above 90% during the month preceding randomization.
Impact on genital HIV-1 RNA
After adjustment for shedding during the baseline phase, there was no impact of valacylovir on the proportion of women with genital HIV-1 RNA detected at least once during the treatment phase [relative risk (RR) 1.09, 95% confidence interval (CI) 0.57, 2.08], nor on the frequency of HIV-1 shedding [odds ratio (OR) 0.90, 95% CI 0.31, 2.62]. These results were confirmed by repeated measures analyses (Table 2).
The mean quantity of HIV-1 RNA measured at visits during which genital HIV-1 RNA was detected was reduced by 0.33 log10 copies/ml in the valacyclovir group, after adjusting for the baseline phase, but this difference was not statistically significant (P = 0.19; Table 2).
Impact on plasma HIV-1 RNA
During the baseline phase, six (20%) women in the valacyclovir arm and four (13%) in the placebo arm had at least one detectable plasma HIV-1-RNA measurement, but only three women had detectable viral loads at all visits (Table 2). During the treatment phase, nine out of 30 (30%) women in the placebo arm and four out of 30 (14%) in the valacyclovir arm had detectable HIV-1 RNA, (RR 0.55, 95% CI 0.20, 1.52), and there was some reduction in the frequency of detectable plasma HIV-1 RNA (OR 0.23, 95% CI 0.04, 1.21; P = 0.08).
Plasma HIV-1 RNA was reduced by 0.41 log10 copies/ml in the valacyclovir arm when compared with the placebo arm after adjustment for the baseline phase, but this did not reach statistical significance (P = 0.39).
During the trial, 28/42 (67%) women with persistently undetectable plasma HIV-1 RNA shed HIV-1 at least once at the genital level. This pattern was not related to HAART duration (P = 0.65).
Impact on genital herpes simplex virus 2 DNA and episodes of clinical ulcer
The proportion of women shedding HSV-2 DNA at least once decreased from 56.7 to 30.0% between the two phases in the valacyclovir arm, while remaining constant (40.0 and 43.3%) in the placebo arm (RR 0.66, 95% CI 0.33, 1.31; Table 2). The impact was more apparent on repeated measures analysis, with some reduction in the proportion of visits with detectable HSV-2 DNA in the valacyclovir arm between the two phases (15.2–6.6%), with no change in the placebo arm (OR 0.37, 95% CI 0.13, 1.05). Moreover, valacyclovir reduced the quantity of HSV-2 DNA (when this was detected) by 1.18 log10 copies/ml compared with the placebo arm (P = 0.12).
There were very few women who experienced clinical ulcer episodes during the treatment phase in either arm, thus there was low power to detect any impact of the intervention (Table 2).
Impact on plasma and genital HIV-1 RNA among baseline genital HIV-1 RNA shedders
Analysis of the predefined subgroup of women with detectable genital HIV-1 RNA at least once over the baseline phase showed similar results for the frequency of detection and quantity of plasma HIV-1 RNA (data not shown). However, there was a significant impact of valacyclovir both on the proportion of visits with detectable HIV-1 shedding (OR 0.27; P = 0.05), and the quantity of genital HIV-1 RNA during these visits (−0.71 log10 copies/ml; P = 0.01; Table 2). This treatment effect tended to increase over time, with an average reduction in mean genital HIV-1 RNA of 0.10 log10 copies/ml (95% CI −0.24, 0.03; P = 0.12) every 2 weeks during the treatment phase.
The purpose of this trial was to determine the existence of a causal relationship between HSV-2 and HIV-1 genital shedding among women taking HAART. Overall, valacyclovir had no significant impact on both the frequency and quantity of genital HIV-1 RNA. However, this impact was demonstrated when using a pre-defined secondary analysis restricted to women with detectable genital HIV-1 RNA at baseline. These are the women in whom the demonstration of an independent effect of HSV-2 on genital HIV-1 RNA was most plausible. The absence of an overall impact on genital HIV-1 RNA is likely to be attributable to the substantial proportion of women without any genital HIV-1 shedding at baseline. Therefore, our results still support the hypothesis of an interaction between HSV-2 and HIV-1 replication. Several biological explanations have been proposed for this interaction. First, HSV and HIV can co-infect the same cells in vivo . Herpetic lesions generate an influx of activated CD4 cells , which have been shown to increase the HIV-1 plasma load . Furthermore, some HSV proteins such as ICP-0, ICP-27  and ICP-4  can interact with the HIV long terminal repeat region leading to the upregulation HIV replication. Similarly, HSV protein 16 was shown to increase HIV transcription through interactions with the HIV Tat protein .
Valacyclovir is likely to have had an impact on both the frequency and quantity of genital HSV-2 DNA, although this effect did not reach statistical significance. To our knowledge, the efficacy of valacyclovir (or acyclovir) on these outcomes among HIV-1-infected individuals has never been reported. Only one study using HSV-2 culture  showed that famciclovir, another antiherpetic drug, could reduce the frequency of HSV-2 genital shedding from 11 to 1% of days. We could not directly assess the impact of valacyclovir on clinical ulcer episodes because of the small numbers, but a randomized study conducted in a US population of HIV-seropositive patients, 93% of whom were taking HAART, had conclusively shown that valacyclovir prophylaxis could significantly increase the time to clinical ulcer episodes .
Our study had several strengths, including high levels of adherence with study procedures and the treatment regime. Repeated measurements of shedding before and after randomization allowed for the adjustment for within-person variability of HSV-2 and HIV-1 shedding, and provided a robust measure of treatment effect sustained over a 3-month duration. The main limitation of the study is its low power. The expected frequency of detection and the quantity of genital HIV-1 RNA, based on the only data available (from cross-sectional studies carried out among US  and Italian  women taking HAART), was slightly overestimated.
This is the first study reporting HIV-1 genital shedding among women taking HAART in Africa. Antiretroviral therapy provided good virological efficacy in this trial, with over 80% of women having an undetectable plasma viral load during the baseline phase, similar to previous African studies [28,29]. Following good systemic control, HAART can rapidly reduce HIV-1 genital shedding . Furthermore, cross-sectional studies conducted in the United States have shown that less than 33% of women with plasma HIV-1 RNA below 500 copies/ml  or 25% of women with plasma HIV-1 RNA below 80 copies/ml  would still have detectable HIV-1 shedding after several months on HAART. Our data, however, showed that this proportion was at least doubled when using a longitudinal approach with repeated sampling, which takes into account the high variability of HIV-1 genital shedding. To our knowledge, only one study had included weekly measurements of both plasma and genital HIV-1 RNA over an 8-week period in the United States , but the shedding pattern among women with sustained suppressed plasma HIV-1 RNA was not reported, and thus cannot be compared with our data.
This high frequency of HIV-1 shedding despite good virological control independently of HAART duration is a cause for concern, and suggests that many of these women have the potential to transmit HIV-1. Some experts have speculated that antiretroviral programmes might be a possible tool to control HIV transmission , whereas modelling studies have suggested a more limited impact, depending on the sexual risk behaviours of HAART recipients . This would be compounded by a lack of virological control at the genital level. However, the residual quantities of genital HIV-1 RNA were small, and it remains unclear whether this level of genital load can lead to HIV-1 sexual transmission. Nonetheless, our results underscore the need actively to promote prevention messages regarding safe sex among all patients taking HAART to prevent onward transmission. Second, an independent replication of HIV-1 at the genital level, previously suggested in men  and supported by in-vitro studies , carries the theoretical risk of the selection of resistant HIV mutants that would be sexually transmissible. It has been shown that some antiretroviral drugs included in the HAART first line regimens have poor penetration in the female genital tract. For example, a recent pharmacodynamics study showed that efavirenz and stavudine had much reduced concentrations in the female genital tract, 0.6 and 4% of their plasma concentrations, respectively . The selection of resistance mutations is more likely to occur in an environment with suboptimal antiviral concentrations , with potentially devastating effects on the future control of the epidemic. Further studies are required to assess and compare the resistance patterns of HIV-1 at both the genital and systemic levels.
In conclusion, HSV-2 facilitates residual genital HIV-1 replication among dually infected women taking HAART, which may partly explain the high proportion of women having intermittent genital HIV-1 shedding despite suppression at the systemic level.
The authors would like to thank the women and the organizations of individuals living with HIV/AIDS who participated in this study, and staff from Service d'Hygiene of Bobo-Dioulasso. They are grateful to members of the Data Monitoring Committee (chairman: Prof. Simon Cousens, LSHTM; Prof. Adama Traore, CHU Ouagadougou, Burkina Faso; Prof. Jean-Marie Huraux Hopital Pitie-Salpetriere, Paris, France); to Profs. David Mabey and Richard Hayes (LSHTM) and Prof. Myron Cohen (University of North Carolina, Chapel Hill, USA) for reviewing the manuscript; and to Dr B. Bazin, Prof. M. Kazatchkine and Prof. J.F. Delfraissy (ANRS) for their encouragement.
Sponsorship: The study was sponsored by Agence Nationale de Recherches sur le SIDA (ANRS, contract 2003-149), France. Additional financial support was provided through the UK's Department for International Development-funded Knowledge Programme on HIV/AIDS and STI of the London School of Hygiene and Tropical Medicine.
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Composition of the ANRS 1285 study group
Eloi Bahembera, Abdramane Berthe, Minata Coulibaly, Marie-Christine Defer, Ramata Diallo, Didier Djagbare, Issouf Konate, Florent Ky-Dama, Gilles T. M'Boutiki, Nicolas Meda, Ines Millogo, Nicolas Nagot, Abdoulaye Ouedraogo, Djeneba Ouedraogo, François Rouet, Anselme Sanon, Haoua Sawadogo, Roselyne Vallo, Laurence Vergne, Centre Muraz, Bobo-Dioulasso, Burkina Faso; Philippe Mayaud, Nicolas Nagot, Helen A. Weiss, London School of Hygiene and Tropical Medicine, London, UK; Pierre Becquart, Vincent Foulongne, Michel Segondy, Philippe Van de Perre, Montpellier University Hospital, and UMR 145, Institute for Research and Development, University of Montpellier 1, Montpellier, France; Jean-Baptiste Andonaba, Adrien Sawadogo, University Hospital of Bobo-Dioulasso, Burkina Faso.
Role of authors
N. Nagot, P. Mayaud, P. Van de Perre and A. Ouedraogo designed and supervised the study. A. Ouedraogo and N. Nagot implemented the study with I. Konate, A. Sanon and J-B. Andonaba. Laboratory analyses were conducted by L. Vergne and M-C. Defer in Bobo-Dioulasso, and by V. Foulongne in Montpellier, under the direction of M. Segondy and P. Van de Perre. Statistical analyses were performed by H.A. Weiss, N. Nagot and P. Mayaud. The first draft of the manuscript was written by N. Nagot, H.A. Weiss, P. Van de Perre and P. Mayaud. All authors reviewed and approved the final version of the manuscript.
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