HIV-1 and herpes simplex virus type-2 genital shedding among co-infected women using self-collected swabs in Chiang Rai,Thailand

Abstract

We analysed 528 genital self-collected swabs (SCS) from 67 HIV-1 and herpes simplex virus type-2 (HSV-2) co-infected women collected during the placebo month of a randomized crossover clinical trial of suppressive acyclovir in Chiang Rai, Thailand. In this first longitudinal study of HIV-1 and HSV-2 co-infected women using genital SCS specimens, we found frequent mucosal HIV-1 shedding. Overall, 372 (70%) swabs had detectable HIV-1 RNA with median HIV-1 viral load of 2.61 log₁₀ copies/swab. We found no statistically significant association between detectable HIV-1 RNA and HSV-2 DNA in the same SCS specimen (adjusted odds ratio [aOR] 1.40; 95% confidence intervals [CI], 0.78–2.60, P = 0.25). Only baseline HIV-1 plasma viral load was independently associated with genital HIV-1 RNA shedding (aOR, 7.6; 95% CI, 3.3–17.2, P < 0.0001). SCS may be useful for future HIV-1 and HSV-2 studies because this method allows for frequent genital sampling, and inclusion of genital sites other than the cervix.

Keywords

HIV herpes simplex virus type-2 HSV women co-infection shedding genital viral load genital shedding Thailand self-collected swabs

INTRODUCTION

Herpes simplex virus type 2 (HSV-2) infection is associated with increased HIV-1 infectiousness and transmission.¹ Transmission risk is likely increased in co-infected persons due to increased genital HIV-1 shedding with HSV-2 co-infection, increased HIV-1 plasma viral load or both.^2,3 Little is known, however, about the inter-relationship of HIV-1 and HSV-2 at the mucosal level, especially in the setting of asymptomatic HSV-2 genital shedding.

The interaction of HSV-2 and HIV-1 genital shedding has been evaluated predominantly in studies using cervical specimens. Some of these studies have shown that both asymptomatic and symptomatic HSV-2 shedding is associated with HIV-1 genital shedding;^4–8 few longitudinal studies have assessed this question, and none have used genital self-collected swab (SCS) specimens. Although trials demonstrated reductions in HIV-1 and HSV-2 genital shedding with suppressive antiviral therapy for herpes in co-infected individuals,^9–13 a recent large randomized controlled trial of HSV antiviral therapy did not show a statistically significant reduction in HIV transmission in the treatment group.¹⁴ Findings from this study suggest the need to better understand the natural history of genital HSV-2 and HIV-1 shedding. To further describe the natural history of genital viral shedding, we analysed genital SCS specimens collected from HIV-1 and HSV-2 co-infected women during the placebo month of a randomized clinical trial of suppressive acyclovir.

MATERIALS AND METHODS

Sixty-seven HIV-1- and HSV-2-infected women were enrolled in a randomized, placebo-controlled, crossover clinical trial of suppressive acyclovir in Chiang Rai, Thailand.¹² Women aged 18–49 years with regular or no menses and serum antibodies to HIV-1 and HSV-2, and not eligible for antiretroviral therapy (ART) by Thai national guidelines (i.e. CD4 count >200 cells/μL and without opportunistic infection) were eligible for the study. At baseline, women were tested for plasma HIV-1 viral load and reproductive tract infections. During this three-month trial, participants were randomized to receive either acyclovir or placebo the first month, no product the second month and then placebo or acyclovir the third month. Each study month started with a woman's menstrual cycle (day 1 being the start of menses) and ended with her next menses. Once each month, women were tested for reproductive tract infections and pregnancy and had a plasma specimen collected for HIV-1 viral load. Study details are published elsewhere.¹²

At the first screening visit, written informed consent was obtained in Thai. The study protocol was reviewed and approved by the Ethics Review Committee of the Thailand Ministry of Public Health and the Institutional Review Board of the Centers for Disease Control and Prevention. The trial was registered in the National Institutes of Health clinical trials database (http://www.clinicaltrials.gov, NCT00362596).

Genital SCS were collected daily from participants during the intervention and placebo months of the study. Specifically, during the clinical trial each participant was asked to collect a SCS daily beginning the day after menstrual bleeding stopped through the day before the next menstrual period began. For women with no menses, collection of SCS began on a selected day and continued for 28 days. At the enrolment visit, each participant received instructions about the SCS specimen collection, and how to store the specimen in a cooler using twice-daily ice pack changes. The participant then demonstrated the collection technique to research staff. To collect a genital SCS specimen, the participant used a single Dacron swab which she sequentially swabbed over: (1) the vaginal area (approximately 2 inches into the vaginal vault), (2) the vulvar area and (3) the perianal region, for a total of 15 seconds. The participant then inserted the swab into a labelled tube containing a sponge impregnated with approximately 150 μL of Genelock^® (Sierra Molecular Corporation, Sonora, CA, USA) that included a proprietary DNA/RNA preservation solution, capped the tube, and placed it into a cooler which contained frozen ice packs and temperature monitoring strips (3M Monitor Mark, Cold Chain Technologies, Holliston, MA, USA).

To maintain the temperature within the cooler between approximately 4 and 17°C, each participant changed the cooler ice packs twice daily. A pilot evaluation demonstrated that swabs could be maintained at 4–17°C using these methods at ambient temperature in Thailand. A temperature monitoring strip placed in the cooler noted if temperatures exceeded 30°C. Participants transported coolers (containing swabs) weekly to their study visit. Research staff collected the swabs, recorded information from the temperature-monitoring strip within the cooler, and, within two hours of delivery, stored the swabs at −70°C. Swabs were then shipped frozen to the laboratory for evaluation.

We analysed viral shedding results from select SCS specimens collected by the 67 study participants during the trial's placebo month. In a previous analysis from this trial, no period effect was observed.¹² Five hundred and seventy-six SCS specimens, 42.8% of the total collected from the 67 participants, were analysed for HIV-1 RNA and HSV-2 DNA. These SCS specimens included those collected on the same days that cervicovaginal lavage (CVL) specimens were obtained (days 7, 14 and 21) and a random selection of 1–2 additional SCS per week (approximately 5–9 swabs/month/woman).

Laboratory methods

Genital swab specimens were prepared for HIV-1 and HSV-2 analyses as follows: (1) specimens were thawed; (2) the sponge and the swab were squeezed to obtain all fluid; (3) 600 μL of Amplicor Monitor kit lysis buffer was added to approximately 150 μL of the fluid from each tube; (4) this 750 μL of eluate was used for nucleic acid extraction. The same nucleic acid extract was used for both HIV-1 RNA and HSV-2 DNA quantitative assessment; HIV-1 and HSV-2 genital viral load values were reported as copies/swab.

HIV-1 extraction, amplification and quantification

HIV-1 RNA in SCS specimens was quantified using Amplicor HIV-1 Monitor version 1.5 (Roche Molecular Systems, Inc., Branchburg, NJ, USA). Nucleic acid extraction procedures were modified by adding freshly prepared dithriothreitol to 750 μL of sample eluate, to obtain a final concentration of 0.3 mmol/L. The sample was heated at 60°C for 10 minutes; the remaining extraction steps followed the manufacturer's instructions. Extracted lysate was re-suspended in 400 μL of elution buffer. Polymerase chain reactions (PCR) were performed in 100 μL by adding 50 μL of extracted lysate to 50 μL of PCR master mix. HIV-1 RNA lower limit of detection was 40 copies/swab, and lower limit of quantitation was 80 copies/swab. Swabs with invalid HIV-1 RNA quantification were a result of lack of PCR amplification of the internal control. Every specimen with detectable HIV-1 was evaluated for viral quantity.

HSV-2 amplification and quantification

HSV-2 DNA in SCS samples was quantified using an in-house TaqMan-based realtime duplex PCR assay. Primers and probes specific for amplification of HSV-2 glycoprotein G gene and human RNaseP gene (as the internal control) were used in the realtime PCR assay. The realtime PCR assay was performed using 10 μL of extracted lysate in a 25-μL PCR reaction. The final PCR reaction mix contained five units of AmpliTaq Gold DNA polymerase (Perkin-Elmer, Foster City, CA, USA), 1× PCR Gold buffer, 4 mM MgCl₂, 400 nmol/L of each HSV-2 forward primer (5′GGC CTC CCC TGC TCT AGA TA3′), reverse primer (5′CCA AAG TTG TGC TGC CAA G3′), and FAM-labelled probe (5′FAM-CGG CGT TCG TTT GTC TGG TC3′BHQ1), 80 nmol/L of each RNaseP forward primer (5′AGA TTT GGA CCT GCG AGC G3′), reverse primer (5′GAG CGG CTG TCT CCA CAA GT 3′) and CY5-labelled probe (5′CY5-TT CTG ACC TGA AGG CTC TGC GCG BHQ33′), 200 μmol/L dNTP, 600 μmol/L dUTP (Roche Applied Science, Mannheim, Germany) and 0.5 unit of AmpErase UNG (USB, Cleveland, OH, USA). A Rotor-Gene 3000 instrument (Corbett Robotics, Mortlake, NSW, Australia) was used and PCR amplification consisted of two hold cycles: 50°C for two minutes and 95°C for 10 minutes followed by 50 cycles of 95°C for 20 seconds; 60°C for 60 seconds. The sensitivity of PCR assay was approximately 10 copies per PCR reaction. HSV-2 DNA lower limit of detection was 40 copies/swab, and lower limit of quantitation was 400 copies/swab. No swabs had invalid HSV-2 quantitation results. Every specimen with detectable HSV-2 was evaluated for viral quantity.

Statistical analysis

We analysed participant demographic, clinical, and laboratory characteristics, and overall qualitative and quantitative summary results of HIV-1 and HSV-2 genital shedding from SCS specimens using SAS version 9.2. We used the Kaplan–Meier¹⁵ approach to estimate median HIV-1 RNA and HSV-2 DNA shedding to account for the left censoring, and calculated the 95% confidence intervals (CI) for medians using percentile bootstrap methods.

To estimate the unadjusted and adjusted associations between same-day cervicovaginal HIV-1 RNA and HSV-2 DNA shedding, we created binary variables and applied generalized estimating equations (GEE) with a binomial distribution, an exchangeable working correlation matrix and a logit link to estimate the odds ratio (OR) and perform statistical inferences. HIV-1 RNA and HSV-2 DNA were coded as ‘non-detectable virus’ if the reported value was below the limit of detection and ‘detectable virus’, otherwise. In the multivariate analysis, we adjusted for continuous covariates including baseline plasma log₁₀ HIV-1 viral load and log₁₀ CD4 count. The log transformation of these covariates was used to improve the assumption that the log odds was linearly related to the covariate under study. We also adjusted for dichotomous covariates, including genital HSV-2 shedding (yes/no), baseline history of HSV-2 symptoms (yes/no), prior knowledge of HSV-2 diagnosis (yes/no), and menstrual cycle phase for those with menses and who were not receiving hormonal contraception (proliferative or secretory, ≤day 14 or day >14, respectively). The empirical variance-covariance matrix was used for all inferences and CI.¹⁶ Statistical significance was evaluated when the P value from the Wald chi-square statistic for Type III GEE analysis was ≤0.05.

RESULTS

The median age of participants was 33 years (range, 22–46 years); at baseline, median CD4 count was 366 cells/μL (range, 209–930 cells/μL) and median HIV-1 plasma viral load 4.6 log₁₀ copies/mL (range, 2.9–5.7 log₁₀ copies/mL). Twenty-eight (42%) women reported prior history of symptoms consistent with genital herpes. The mean number of SCS specimens analysed per women was 8 (range, 1–9).

A total of 576 SCS specimens were analysed from the 67 participants. Of these, 528 (91.7%) were maintained within the coolers at <30°C and were used for this assessment.

Overall, 372 (70%) swabs had detectable HIV-1 RNA. The median HIV-1 viral load was 2.61 log₁₀ copies/swab (range, 1.90–4.46 log₁₀ copies/swab). Overall, 139 (26%) swabs had detectable HSV-2 DNA. The median HSV-2 viral load for these swabs was 4.66 log₁₀ copies/swab (range, 1.90–8.68 log₁₀ copies/swab). Both HIV-1 RNA and HSV-2 DNA were detected in 114 (22%) swabs; HIV-1 RNA only was detected in 258 (49%) swabs; HSV-2 DNA only was detected in 25 (5%) swabs; and neither virus was detected in 131 (25%) swabs.

We found no statistically significant association between detectable HSV-2 DNA and HIV-1 RNA in the same SCS specimen (unadjusted OR, 1.30; 95% CI, 0.94–1.94, P = 0.10; adjusted OR [aOR], 1.40; 95% CI, 0.78–2.60, P = 0.25). The only factor independently associated with genital HIV-1 RNA shedding was baseline HIV-1 plasma viral load (P < 0.0001, aOR, 7.6; 95% CI, 3.3–17.2). We found no relationship between HIV-1 RNA quantity and HSV-2 DNA quantity among same SCS specimens (Figure 1). There was no difference in the median HIV-1 RNA log₁₀ copies/swab (estimated median difference = 0.37; 95% CI: 0.01–0.68) (Table 1) among specimens with and without detectable HSV-2 DNA.

Table 1

HIV-1 RNA from genital self-collected swabs among participants by herpes simplex virus type-2 DNA shedding status, Chiang Rai, Thailand

			HIV-1 RNA (all swabs)(log₁₀ copies/swab)
HSV-2shedding	No. ofswabsanalysed	No. of swabswith detectableHIV-1 (%)	Median(95% CI)	IQR
Yes	139	114 (82)	2.47 (2.13–2.71)	1.90–2.95
No	389	258 (66)	2.10 (1.90–2.36)	1.60–2.87

95% CI = 95% confidence interval; IQR = interquartile range

Figure 1

Relationship between quantity of HIV-1 RNA and quantity of herpes simplex virus type-2 (HSV-2) DNA from the same self-collected genital swab (estimated trend lines generated by a kernel smoother using a box kernel and a bandwidth of 2.0²⁰), Chiang Rai, Thailand

Among the 67 study participants, 61 (91%) participants shed HIV-1 RNA at least once and 46 (69%) participants shed HSV-2 DNA at least once. Specifically, 19 (28%) participants shed HIV-1 RNA only, four (6%) shed HSV-2 DNA only, and 42 (63%) shed HIV-1 RNA at least once and HSV-2 DNA at least once; two (3%) participants shed neither virus.

DISCUSSION

This is the first longitudinal study of HIV-1 and HSV-2 genital shedding in co-infected women using SCS specimens. HIV-1 genital shedding occurred more frequently than did HSV-2 genital shedding among these 67 co-infected women. We found a statistically significant association between plasma HIV-1 RNA and genital HIV-1 shedding, similar to other studies,^3,18 but not between same-day genital shedding of HIV-1 and HSV-2.

Few studies have evaluated HIV-1 and HSV-2 genital shedding among co-infected women.^4,5,8,19 These studies are heterogeneous by study design, specimen type, thresholds for viral detection and statistical methods; the different methodological approaches used to evaluate the relationship between HIV-1 and HSV-2 shedding make comparisons between studies difficult. Our genital sampling method, using SCS specimens, was unique, and our analysis included all analysed specimens from all women, regardless of shedding results. We used methods to appropriately account for the left censoring inherent in this type of study.

Two cross-sectional studies compared genital HIV-1 shedding among HSV-2 shedders and non-shedders.^4,5 McClelland et al.,⁵ using endocervical swab specimens, found no association between HIV-1 RNA and HSV-2 DNA detection but did report a positive correlation between genital HIV-1 RNA and HSV-2 DNA quantities. In another cross-sectional study of HIV-1-infected women, Mbopi-Kéou et al. ⁴ identified a correlation between genital HIV-1 and genital HSV-2 in CVL specimens among the subset of 23 co-infected participants who shed HSV-2. Two longitudinal studies assessed HIV-1 and HSV-2 genital shedding; in one, Tanton et al. ⁸ assessed the correlation of HIV-1 and HSV-2 shedding among 487 HIV-1 and HSV-2 co-infected women in Tanzania who were evaluated over 24 months using CVL specimens. In that study, there was weak evidence for an association between HIV-1 RNA and HSV-2 DNA quantity in an analysis of specimens with detectable HSV-2, assigning HIV-1 non-shedders an arbitrary viral quantity and adjusting for HIV plasma viral load.

In our study, 91% of participants shed HIV-1 RNA at least once during a three-week period and 70% of SCS had detectable HIV-1 RNA; 86% of participants shed HIV during a three-week period reported by Nagot et al.,¹⁹ using CVL specimens enriched with cervical swabs. We detected HSV-2 DNA shedding at least once among a larger proportion (69%) of participants than did Nagot et al. (32%). However, we analysed a greater median number of specimens per woman which may account for this difference. A perianal, vulval and cervico-vaginal specimen may be optimal for HSV-2 DNA detection at these sites. Asymptomatic perianal HSV-2 shedding is particularly common²⁰ and would be missed with a cervical or cervico-vaginal specimen.

Genital SCS specimens have been widely employed and evaluated in studies of HSV-2 genital shedding,²⁰ but they have not been used to measure HIV-1 genital shedding. In this study, we used SCS specimens collected at home, placed in proprietary DNA/RNA preservation material, and stored in coolers. To detect HIV-1 using SCS specimens, we modified the Roche Amplicor Monitor kit extraction method. Using these methods, we found the proportion of women who shed HIV-1 at least once during the study similar to that reported in a study of similar length which utilized CVL specimens.¹⁹ While not reported here, we found that both HIV-1 RNA and HSV-2 DNA detection rates were comparable using SCS and CVL specimens (McNicholl, 2011, in preparation). Further studies are warranted to evaluate HIV-1 RNA detection using SCS specimens and to compare this genital sampling method with other methods. Since this SCS sampling method is easy and allows for capture of genital shedding at several sites, it may be useful in future studies of genital HIV-1 and HSV-2 shedding.

Several limitations should be considered. Although all participants received training on maintaining cooler ice packs, we were unable to confirm SCS storage temperatures in the home except those that deviated above 30°C. Few participants experienced symptoms of genital herpes, which precluded analysis of the relationship of same-day HIV-1 and HSV-2 genital shedding during symptomatic episodes. Our genital SCS specimen collection method (combined vaginal, vulvar and perianal sampling) prohibited evaluation of HIV-1 and HSV-2 shedding by specific genital site. If shedding differed by genital site, association between same-day HSV-2 and HIV-1 shedding may have been obscured.

In conclusion, using SCS specimens to detect both HIV-1 RNA and HSV-2 DNA genital shedding, we found that genital HIV-1 shedding was independently associated with plasma HIV-1, but was not associated with same-day genital HSV-2 shedding. HIV-1 shedding events occurred frequently and more often than HSV-2 shedding events during a three-week period. Results from clinical trials of HSV-2 suppression on HIV transmission or acquisition have been disappointing, highlighting the need to further understand both the compartment and systemic HSV-2 influence on HIV-1 transmission. SCS may be useful for future HIV-1 and HSV-2 studies because this method allows for frequent genital sampling, and inclusion of genital sites other than the cervix. Studies that follow greater numbers of participants of both sexes over longer time periods could provide a more complete understanding of genital mucosal shedding of HIV-1 and HSV-2.

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