The role of herpes simplex virus type 2 (HSV-2) in enhancing sexual transmission of HIV-1 (HIV) has been increasingly recognized in recent years. Meta-analyses have shown prevalent HSV-2 infection to be associated with a two-fold to four-fold increased risk of HIV acquisition [1,2]. HSV-2 infection may also increase HIV infectiousness among HIV-infected persons with genital herpes because of the presence of HIV in ulcers . In addition, increased HIV replication and shedding in genital secretions may also occur during subclinical HSV-2 activation [4,5]. However, epidemiologic studies have not shown an increased risk of HIV transmission from HSV-2 coinfected persons.
Increased risk of perinatal HIV transmission has been demonstrated among women with genital ulcers [6,7] and those with reactive syphilis serology . Recently, a case–control study  showed increased perinatal HIV transmission risk among HSV-2 seropositive women. However, to date, no specific effect of HSV-2 shedding on perinatal HIV transmission has been demonstrated. Because HSV-2 seroprevalence is around 80–90% among HIV-infected women [1,5,7,9], an association between HSV-2 infection and perinatal HIV transmission could have important public health implications.
In this study, stored samples from HIV-infected women who participated in a perinatal HIV transmission prevention trial in Thailand  were tested for HSV-2 to assess the role of HSV-2 serology and genital shedding in perinatal HIV transmission.
The study population consisted of women participating in a clinical trial to assess the efficacy of short-course zidovudine for the prevention of perinatal HIV transmission. This study has been described in detail elsewhere . In short, from May 1996 to December 1997, 1140 HIV-infected pregnant women were screened for enrollment at two Bangkok hospitals serving a population of relatively low socio-economic status. At enrollment, sociodemographic characteristics and HIV-related symptoms were documented according to the World Health Organization clinical-staging system . A total of 397 eligible women were randomized to placebo or zidovudine, 300 mg orally twice daily from 36 weeks of gestation until onset of labor, and 300 mg every 3 h orally from onset of labor until delivery. Women were not systematically examined for the presence of genital ulcers. Mothers were given infant formula and asked not to breastfeed in accordance with national guidelines. Short-course zidovudine reduced perinatal HIV transmission by half and transmission was associated with high plasma viral load and cervicovaginal HIV shedding . Informed consent was obtained from participants in this study.
Blood was collected at 36 weeks of gestation for lymphocyte phenotyping using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, California, USA) as previously described . Plasma was collected at 38 weeks of gestation and at delivery. Cervicovaginal lavage (CVL) samples were collected at 38 weeks of gestation. Three milliliter of saline was used to flush the cervix and vaginal wall and recollected with a transfer pipette. Samples were transported on ice to the local laboratory and were centrifuged at 750–1000 g at 4°C for 15 min. During the first 6 months of the study, whole CVL samples were stored; thereafter CVL samples were separated into supernatant and pellets. All CVL samples were stored at −70°C for up to 9 years. Plasma and CVL HIV viral loads were determined using the Roche Amplicor HIV-1 Monitor Test, Version 1.5 (Roche Diagnostic System, Branchburg, New Jersey, USA) [10,12], which was developed to improve binding ability to HIV-1 non-B subtypes. HIV viral load values in CVL samples of more than 400 copies/ml were considered as quantifiable .
Infant whole blood was collected at birth and at 2 months of age to determine HIV infection status using a modification of the DNA Roche Amplicor HIV-1 PCR v1.0 assay (Roche Diagnostic System) [10,14], designed to improve the detection of HIV-1 non-B subtypes. In-utero transmission was defined as infant PCR-positivity for HIV at birth (within 72 h after delivery), and intrapartum transmission was defined as infant PCR-negativity at birth and PCR-positivity at the 2-month visit. None of the women breastfed and it was, therefore, presumed that there was no postpartum transmission. Elective caesarean section was defined as caesarean section before the onset of labor or rupture of membranes. Women who underwent elective caesarean section, those who transmitted HIV in utero and those lacking an infant birth PCR sample, were excluded from the intrapartum transmission analysis.
Herpes simplex virus type 2 evaluation
Stored 38 week-gestation plasma samples were tested for HSV-2 serostatus using the Focus ELISA (HerpeSelect 2 ELISA; Focus Technologies, Cypress, California, USA) which detects HSV-2 IgG. For women who were HSV-2 seronegative at 38 weeks, plasma samples from 6 months after partum (1 month after partum if 6-month sample not available) were tested to identify recent HSV-2 seroconverters.
HSV-2 DNA was detected using 200 μl stored whole or supernatant CVL from HSV-2 seropositive women using an in-house real-time duplex PCR assay (Manuscript in preparation). Briefly, DNA was extracted using the QIAamp DNA minikit (Qiagen, Valencia, California, USA), and HSV-2 DNA was detected using primers and probes for the HSV-2 glycoprotein G and RNase P genes (as the internal control). PCR was performed on a Rotor-Gene 3000 (Corbett Robotics, Mortlake, New South Wales, Australia). Commercial HSV-2 quantitated DNA (ABI Advanced Technologies, Inc., Columbia, Maryland, USA) was used to establish the standard curve for quantitation and as a positive control for each run. The analytical sensitivity of the real-time PCR assay was approximately 10 genomic copies per reaction mixture with a dynamic range of 10–100 000 copies per reaction. If the RNaseP gene was not amplified, the HSV-2 result was considered invalid. Genital shedding of HSV-2 was defined as the presence of a valid, detectable HSV-2 DNA result in CVL samples.
The association between HSV-2 seropositivity and genital shedding was studied in relationship to both overall and intrapartum perinatal HIV transmission. Exact 95% binomial confidence intervals were used for interval estimation of intrauterine and intrapartum transmission rates. For univariate analysis of potential risk factors for HIV transmission, two sample t-tests, Wilcoxon rank sum tests, χ2 tests and odds ratios (OR) with 95% confidence intervals (CI) were calculated and stratified for zidovudine treatment. Significant (P < 0.05) factors from univariate analyses were subsequently included in multivariate models using logistic regression; the Wald test was used to calculate P values. Two models were used to assess factors associated with overall HIV transmission; the first model evaluated the association between HSV-2 serology and transmission, the second model evaluated HSV-2 shedding and transmission. The Mantel–Haenszel χ2 test was used for trend analysis. In addition, associations between HSV-2 and HIV viral load at 38 weeks of gestation and between HSV-2 shedding and short-course zidovudine were studied. The Spearman's rank correlation coefficient was used to study associations between viral load levels.
The original trial protocol was reviewed and approved by the ethical review committee of the Thai Ministry of Public Health and by the institutional review board of the US Centers for Disease Control and Prevention (CDC). All women participating in the main study provided informed consent for storing samples for future research purposes. Personal identifiers were removed from all study materials in 2004. This evaluation of stored samples was reviewed and approved by both institutions.
Three hundred and eighty-three women completed the short-course zidovudine study with infant HIV test results. Stored plasma and CVL samples were available for HSV-2 testing from 307 (80.2%) women; 76 women who did not have stored samples available were not included in this evaluation.
The median number of weeks for gestation was 40 and 38 weeks for the women who were and who were not included in the HSV-2 study, respectively (P < 0.001). Sixty-two (81.6%) of the 76 women who did not have stored samples available delivered before 38 weeks of gestation, which was the scheduled time for CVL collection.
The median age was 24 years (range: 17–39 years) for the 307 women evaluated; overall HIV transmission was 15.3% (47/307; 95% CI, 11.6–19.7) and intrapartum HIV transmission was 10.5% (28/267; 95% CI, 7.2–14.6).
Herpes simplex virus-2 evaluation
Two hundred and twenty-eight (74.3%) women tested HSV-2 seropositive at 38 weeks of gestation. CVL samples of these 228 HSV-2 seropositive women (190 had supernatant and 38 had whole CVL samples available) were tested for the presence of cervicovaginal HSV-2; 23 (10.1%) of these women had detectable cervicovaginal HSV-2 DNA (HSV-2 shedding) at 38 weeks of gestation.
Of the 79 HSV-2 seronegative pregnant HIV-infected women, 76 women had plasma samples from 6 months post partum, three had only a 1-month postpartum sample available. Of these 79 samples, seven were HSV-2 seropositive (all from 6 months post partum); one of the seven recent HSV-2 seroconverters had a CVL sample with detectable HSV-2 viral load. Thus, a total of 24 (7.8%) women had detectable HSV-2 DNA.
No difference in HSV-2 detection between supernatant samples and whole CVL samples was observed (10.0 vs. 10.5%; P = 0.9); all CVL samples tested were valid.
Overall HIV transmission
A higher proportion of HSV-2 seropositive women transmitted HIV to their infants than HSV-2 seronegative women (18.0 vs. 7.6%; P = 0.03). HSV-2 seropositivity was an independent risk factor for overall perinatal HIV transmission in multivariate analysis [Adjusted OR (AOR), 2.6; 95% CI, 1.0–6.7] as shown in Table 1. A higher proportion of women shedding HSV-2 transmitted HIV to their infants compared with women not shedding HSV-2 (33.3 vs. 13.8%; P = 0.02). HSV-2 shedding did not remain associated with overall HIV transmission after controlling for HIV viral load and treatment group (P = 0.09, Table 1). The seronegative woman who was shedding HSV-2 at 38 weeks did not transmit HIV to her infant.
Variation in transmission risk according to HSV-2 status and intervention with short-course zidovudine is shown in Table 2. Transmission rates according to HSV-2 status (HSV-2 shedders – HSV-2 seronegatives) ranged from 33.3 to 12.5% among women receiving placebo (trend P value = 0.09) and from 33.3 to 2.6% among women receiving short-course zidovudine (trend P value = 0.03, Table 2).
Intrapartum HIV transmission
A total of 267 mother–infant pairs were evaluated for intrapartum HIV transmission; women who transmitted HIV in utero (n = 19), who underwent elective caesarean section (n = 14), and who did not have an infant blood sample within 72 h of delivery (n = 7) were excluded from this analysis. Intrapartum transmission was 12.4% for HSV-2 seropositive women and 5.5% for seronegative women (P = 0.1; Table 3). A higher proportion of women shedding HSV-2 transmitted HIV during the intrapartum period than women who were not shedding HSV-2 (27.3 vs. 9.0%; P = 0.01). Women shedding HSV-2 had nearly a four-fold higher risk for intrapartum HIV transmission compared with both HSV-2 seronegative and HSV-2 seropositive nonshedders (OR, 3.7; 95% CI, 1.3–10.5, Table 3), and a six-fold higher risk compared with HSV-2 seronegative women alone (OR, 6.1; 95% CI, 1.5–24.6). In multivariate analysis, women shedding HSV-2 were nearly three times more likely to transmit HIV to their infants intrapartum compared with those either not shedding or seronegative for HSV-2 (OR, 2.9, 95% CI, 1.0–8.5).
In-utero HIV transmission
The proportion of HSV-2 seropositive women transmitting HIV in utero was 7.6% compared with 2.6% for HSV-2 seronegative women (OR, 3.0; 95% CI, 0.7–13.4). HSV-2 shedding was not associated with in-utero HIV transmission (OR, 1.3; 95% CI, 0.3–6.3).
Median plasma HIV viral load at 38 weeks of gestation was higher among women shedding HSV-2 compared with those not shedding (4.2 vs. 4.1 log10copies/ml; P = 0.05) and tended to be higher among HSV-2 seropositive women compared with seronegative women (4.1 vs. 4.0 log10copies/ml, P = 0.09). A higher proportion of women shedding compared to those not shedding HSV-2 had quantifiable cervicovaginal HIV viral load (62.5 vs. 34.3%; P = 0.006). Among the 112 women with quantifiable cervicovaginal HIV viral load, the median cervicovaginal HIV viral load was similar among women shedding compared with those not shedding HSV-2 (both 3.6 log10copies/ml; P = 0.7). Among the 24 women shedding HSV-2, the HSV-2 and HIV CVL viral loads were not associated (Spearman's rank correlation coefficient 0.3).
Nine (8.0%) of 113 women receiving zidovudine were shedding HSV-2 compared with 15 (12.9%) of 116 women receiving placebo from 36 weeks of gestation onward (OR, 0.6; 95% CI, 0.2–1.4). Among the 24 women shedding HSV-2, the median HSV-2 viral load was 3.2 log10copies/ml among women receiving zidovudine compared with 4.3 log10copies/ml among those receiving placebo (P = 0.1).
Our findings show that maternal HSV-2 coinfection is associated with an increased risk of perinatal HIV transmission. Women who were HSV-2 seropositive had a two-fold to three-fold overall greater risk for HIV transmission than seronegative women. Consistent with this observation, women with HSV-2 genital shedding had a three-fold greater risk for intrapartum HIV transmission compared with those not shedding, after adjusting for other risk factors. These data suggest that HSV-2 promotes mucosal HIV transmission.
Recently, a case–control study in Zimbabwe showed increased risk for intrapartum HIV transmission among HSV-2 seropositive women compared with seronegative women . The OR of HSV seropositivity for intrapartum HIV transmission was 1.5 in this population not receiving antiretroviral treatment during sample collection as part of the 1997–2000 randomized vitamin A trial. We found a slightly higher risk of HSV seropositivity for HIV transmission (OR = 2.4) in our study population, with no significant difference between those randomized to zidovudine versus placebo.
Drake and colleagues  assessed the relationship between HSV-2 infection and perinatal HIV transmission and found an increased risk for intrapartum HIV transmission among Kenyan women with genital ulcers, but not for women seropositive for or shedding HSV-2. They noted that the detection of HSV-2 shedding by cervical swabs might be an underestimation of genital HSV-2 shedding. In addition, the smaller sample size of 152 Kenyan women and the fact that all were receiving antiretroviral prophylaxis compared with only half in our study may have reduced the power to show an association between HSV-2 shedding and intrapartum HIV transmission.
Our data demonstrate an increased HIV transmission risk (either sexual or perinatal) associated with HSV-2 seropositivity and HSV-2 genital shedding. Previous sexual transmission studies have identified an increased HIV transmission risk associated with genital ulcers  but not with HSV-2 seropositivity in the index partner . Acquisition and transmission of HIV need to be disentangled in sexual transmission studies as the HIV-uninfected partner of an HSV-2 coinfected person may also be HSV-2 seropositive, thereby increasing HIV acquisition risk. An advantage of the perinatal transmission context is that it facilitates a differentiation between HSV-2's role in HIV acquisition and transmission. In addition, perinatal studies allow evaluation of HIV exposure during a clearly defined time period.
Women who were shedding HSV-2 had higher HIV plasma viral loads and were more likely to have quantifiable cervicovaginal HIV viral loads compared with those not shedding HSV-2. This association suggests a biologically plausible mechanism for HSV-2 increasing perinatal HIV transmission. The importance of genital compartment HIV shedding [13,17,18] can also be seen in the effectiveness of caesarean section in preventing perinatal transmission, even in women receiving antiretroviral treatment with undetectable plasma HIV viral load [19,20].
HSV-2 seropositive women were more likely to transmit HIV to their infants. This observation cannot be fully explained by our study. However, HSV-2 seropositive women had higher HIV plasma viral loads compared with seronegative women, although not significantly in our limited sample size, potentially increasing the risk for HIV transmission.
A limitation of our study could be misclassification of HSV-2 shedder status. The assessment of HSV-2 shedding was done at 38 weeks of gestation rather than at delivery when the infant would be directly exposed to genital secretions. In addition, evaluation of HSV-2 in CVL samples does not capture shedding in the perivulvar and perianal areas, which are sites of more frequent shedding . Nevertheless, we were able to show a clear association between HSV-2 shedding and intrapartum HIV transmission.
Our findings, if replicated in other populations, suggest that HSV-2 suppressive antiviral therapy has a potential role in further reducing perinatal HIV transmission. Perinatal HIV transmission rates have declined in industrialized countries after the introduction of highly active antiretroviral treatment and elective caesarean sections . Transmission rates among formula-fed infants in Thailand have been reduced to 2.8% in a trial setting  and to 10.2% as part of the national monitoring program . However, the transmission rate in many resource-limited settings remains high, especially in sub-Saharan Africa [8,24]. Three recent trials found that HSV-2 suppressive antiviral therapy reduced HIV plasma and genital viral load among HSV-2 seropositive HIV-infected women [25–27]. Moreover, HSV-2 suppressive treatment further reduced genital HIV viral loads among women receiving antiretroviral treatment with good systemic viral control . Results from an ongoing trial determining whether HSV-2 suppression can reduce sexual HIV transmission in HIV discordant couples  may provide an additional impetus for studying this approach to reduce perinatal transmission. Acyclovir is well tolerated during pregnancy and was found safe for mothers and infants in a recent study  showing HSV-2 suppressive antiviral therapy to reduce the risk of neonatal herpes. The fact that acyclovir is cheap and no longer under patent protection makes it an appealing drug to consider for the prevention of perinatal HIV transmission in resource-limited settings.
The present study identified maternal coinfection with HSV-2 as a risk factor for perinatal HIV transmission, and is the first direct evidence of HSV-2 subclinical reactivations increasing the risk of HIV mucosal transmission. If our findings are confirmed among women receiving currently recommended perinatal antiretroviral drug regimens, HSV-2 suppressive treatment should be evaluated to further reduce perinatal HIV transmission in populations with high HSV-2 seroprevalence.
We would like to thank the participants in this study. Also, we thank the Bangkok Collaborative Perinatal HIV Transmission Study Group .
L.J.M.B. was the principal investigator, wrote the HSV-2 protocol and manuscript.
S.J.W. contributed to protocol development and writing of the manuscript.
P.A.M. participated in the study as data manager and statistics analyst.
W.L. performed the laboratory analysis. S.A. assisted with AZT study activities at Rajavithi Hospital. A.C. assisted with AZT study activities at Siriraj Hospital.
N.V. assisted with AZT study activities at Siriraj Hospital. T.C. supervised AZT study activities at the Children's Hospital.
J.M. supervised the laboratory studies and contributed to the writing of the manuscript. J.W.T. provided project direction and contributed to the writing of the manuscript. N.S. was the principal investigator of the AZT study and contributed to the writing of the manuscript. R.C. was the study director of the AZT study.
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the US Centers for Disease Control and Prevention.
Presented in part at the 14th Conference on Retroviruses and Opportunistic Infections (CROI 2007), February 25–28, 2007; Los Angeles, United States. Abstract number 75.
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