There is strong evidence that herpes simplex virus type 2 (HSV-2) enhances HIV-1 sexual transmission.1-5 In countries where both HIV-1 and HSV-2 are frequently cocirculating, HSV-2 suppression by antiviral drugs has been proposed as a potential intervention to reduce HIV-1 transmission.6 Randomized controlled trials to demonstrate the efficacy of such interventions should also include measurements of HIV-1 and HSV-2 genital shedding as trial endpoints. However, there is little current consensus as to how this should best be done. The aim of the present research was to prospectively study HIV-1 and HSV-2 shedding in the genital tract of women in Bobo Dioulasso, Burkina Faso, to assess a new sample collection method, and to estimate the optimal frequency of HIV-1 and HSV-2 shedding measurement to be used in subsequent intervention trials.
POPULATION AND METHODS
Forty-two volunteers from a cohort of female sex workers were selected for this pilot observational study. The study was approved by the Institutional Review Board of Centre Muraz, Bobo-Dioulasso, the Ethical Committee of the Ministry of Health of Burkina Faso, and the Research Ethics Committee of the London School of Hygiene and Tropical Medicine. All participants provided informed signed consent. All women (median age, 34 years; range, 23-45 years) were HSV-2 seropositive and 22 were also seropositive for HIV-1. None of these women were taking any antiretroviral treatment, although 4 (18%) were in World Health Organization AIDS stages III/IV, nor HSV-specific treatment, as these were not available in Burkina Faso at the time of the survey. Few women (4/42, 9.5%) were taking oral or injectable contraceptives. Study participants were seen weekly for 3 weeks, and genital samples were collected for a total of 4 time points. A cervicovaginal lavage (CVL) was performed using 2-mL physiological saline for 60 seconds and collected into a cryotube. A polyester swab was applied to the cervical os and rotated 360°, avoiding any mucosal trauma, then stirred in the CVL cryotube and fluid expressed on the tube walls, thereby creating an “enriched” CVL (eCVL), which was immediately dispensed into 0.5-mL aliquots and frozen at −80°C.
HSV DNA was extracted from 200 μL of eCVL fluid using the QIAamp DNA minikit (Qiagen, Courtaboeuf, France), then eluted in 100 μL buffer. HSV subtyping was based on real-time polymerase chain reaction (PCR) using the LightCycler technology (Roche Diagnostics, Meylan, France) with differentiation of HSV-1 and HSV-2 by melting curve analysis as previously described.7 HSV-2 DNA was amplified from 5 μL of eluate by TaqMan (Applied Biosystems, Courtaboeuf, France) real-time PCR using the ABI Prism 7000 Sequence Detection Systems (Applied Biosystems, Courtaboeuf, France). Primers were HSV Pol F and HSV Pol A7 and the probe was (6-FAM)_5′-CCAAGAAAAAGTACATCGGCGTCATCGCGG-3′_(TAMRA). PCR consisted of 45 cycles and HSV-2 DNA was quantitated by reference to a standard curve.
HIV-1 RNA was extracted from 200 μL of eCVL fluid using the QIAamp viral RNA minikit (Qiagen) and a 20-μL aliquot from the 60-μL elution fluid was used for amplification using the same equipment as for HSV-2 DNA. Primers were NEC 152: 5′-GCCTCAATAAAGCTTGCCTTGA-3′ (forward) and NEC 131: 5′-GGCGCCACTGCTAGAGATTTT-3′ (reverse), and the probe was 6-FAM_5′-AAGTAGTGTGTGCCCGTCTGTTRTKTGACT-3′_TAMRA. The target sequence was located within the 5′ long terminal repeat of HIV-1. PCR consisted of 50 cycles and HIV-1 RNA was quantitated using an external standard provided by the Agence Nationale de Recherche sur le SIDA (ANRS). Detection of sperm traces was based on Y-chromosomal Short Tandem Repeat (STR) identification by PCR as described,8 and presence of blood in the eCVL was ascertained visually by a physician and a laboratory technician, independently. A sample was considered to be contaminated by blood on agreement of these 2 persons. Concurrent genital infections were tested: cultures were done on specific media to detect Neisseria gonorrhoeae and Candida albicans, while Trichomonas vaginalis and bacterial vaginosis were diagnosed using immediate wet mount microscopy and Nugent score of a Gram-stained smear,9 respectively. Active or recent syphilis was defined as dual seroreactivity to Rapid Plasma Reagin (Human Gmbh, Wiesbaden, Germany) and Treponema pallidum hemagglutination assay tests (Newmarket Laboratory, Ltd., Kentford, UK). Molecular diagnosis of Chlamydia trachomatis infection was not available at the time of the study but this infection had been shown to be rare (<2%) by PCR in our study population.10 CD4 lymphocyte counts were measured from plasma samples using a standard flow cytometric method.
Binary variables among women were compared using the χ2 test and Fisher exact test where appropriate. For analyses involving all specimens, adjustments for nonindependence of samples within each woman were allowed for, using robust standard errors.
HIV-1 RNA was detected at least once in eCVL from 19 of 22 (86.4%) of the HIV-1-seropositive women. The proportion of HIV-1 RNA shedders at each of the 4 time points was 17/22 (77%), 16/22 (73%), 14/22 (64%), and 12/20 (60%), respectively (P = 0.60 for difference between time points). The average proportion of women shedding at any given time was 68.4%. Cumulative prevalence of HIV-1 shedding was calculated as the average proportion of HIV-1-seropositive women who shed any time over several measurement points, and this was 80.6, 87.1, and 90.0% over any 2, 3, and 4 time points, respectively. Among HIV-1 shedders, 10 of 19 women (53%) shed HIV-1 at each of the 4 time points, but 6 of 19 women (32%) shed at only 1 or 2 time points. The median level of HIV-1 RNA in eCVL among shedders was 3.01 log copies/mL (interquartile range, 2.15-3.89 log copies/mL).
HSV-2 DNA was detected at least once in eCVL from 12 of 42 women (28.6%). The proportion of HSV-2 DNA shedders at each of the 4 time points was 7 of 42 (17%), 5 of 41 (12%), 6 of 42 (14%), and 6 of 37 (16%), respectively (P = 0.93 for difference between time points). The average proportion of women shedding at any given time was 14.8%. The cumulative prevalences of women who shed any time over 2, 3, and 4 time points were 23.2, 27.9, and 32.4%, respectively.
As shown in Table 1, the proportion of women who shed HSV-2 at any time during follow-up was not statistically different between HIV-1-seropositive and seronegative women (32 vs. 25%, P = 0.74). However, there was evidence that HSV-2 genital shedding was more persistent among HIV-1-seropositive women. Indeed, 5 of 22 (23%) of the HIV-1-seropositive women shed on 3 episodes, compared with none of the 20 HIV-1-seronegative women (P = 0.05). The highest HSV-2 DNA loads in eCVL were observed in the HIV-1-seropositive women. Among 21 HIV-1-seropositive women with available CD4 cell measurements (median, 420 cells/μL), 5 had counts <200 cells/μL. HSV-2 shedding at any time point was significantly more common among women with CD4 counts <200 cells/μL (4/5 or 80% vs. 3/16 or 19%; P = 0.03).
Ten women (16 samples) had detectable Y-PCR and 7 women (10 samples) had blood contamination at ≥1 visits (Table 1). HIV-1 or HSV-2 shedding results were similar when data from the 16 Y-PCR-positive or the 10 blood-contaminated samples were excluded (data not shown). No gonococcal infection or syphilis cases were found at baseline and during follow-up. The baseline prevalences of C. albicans, T. vaginalis, and bacterial vaginosis were 23.8, 11.9, and 4.7%, respectively.
This pilot study focused on a limited number of participants. However, to our knowledge, it represents the first study assessing longitudinal shedding of HIV-1 and HSV-2 in the female genital tract in Africa. The point prevalence of HIV-1 and HSV-2 shedding as well as the levels of HIV-1 RNA and HSV-2 DNA were in agreement with previous studies.11-14 The higher frequency and quantity of HSV shedding among HIV-1-seropositive women have been reported elsewhere in Africa,15,16 but, importantly, this study highlighted the need for repeated sampling to identify women with intermittent viral shedding as reported elsewhere.11 Three measurement points (at weekly intervals in this study) seem to adequately allow detection of most HIV-1 or HSV-2 genital shedders.
Sample collection for detecting HIV-1 or HSV-2 genital shedding in women has been based mainly on the use of CVL or cervical swabbing. Both sampling modalities have been directly compared in only a few studies. Good correlation was observed for HIV-1 genital shedding in Central African Republic,13 while discrepant results were observed in a Kenyan study.12 In general, studies have shown that cervical swabbing would be a more adequate sample than CVL for detecting HIV-1 genital shedding, whereas CVL would be more adequate than swabbing for detecting HSV-2 shedding.12,14 The 2 sample collection modalities have therefore been combined in the present study. Shedding rates and frequencies for both viruses were within expected values. This probably indicates that combining CVL and cervical swabbing (eCVL) is suitable for assessing genital shedding of HIV-1 and HSV-2 on a single sample. Our findings might be useful for the design of HSV-2 intervention trials with HIV-1 and HSV-2 genital shedding as trial outcomes.
The authors thank staff at the Service d'Hygiene clinic and at the laboratories of Centre Muraz in Bobo-Dioulasso and the volunteers who participated in this study.
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