Herpes simplex virus type 2 (HSV-2) is a major cause of genital ulcer disease in sub-Saharan Africa, where HSV-2 seroprevalence among sexually active adults ranges from 50% to 90%.1–3 Herpes simplex virus type 2 seropositivity is particularly high among human immunodeficiency virus type 1 (HIV-1)-seropositive women in this region, exceeding 90% in some studies.1,4 Genital ulcers have been associated with an approximately three-fold increased risk of sexual HIV-1 transmission.5 Increased HIV-1 infectivity of the HIV-1/HSV-2 co-infected person may result from breaches in genital mucosa and mucosal ulceration or from an increase in genital tract HIV-1 secondary to recruitment of CD4 T cells.6 Herpes simplex virus type 2 reactivation has also been associated with higher plasma HIV-1 viral load.7
Maternal plasma and genital tract HIV-1 levels are major risk factors for HIV-1 transmission intrapartum, which accounts for more than 50% of infant HIV-1 infections.8 Although HSV-2 infection is recognized as an important risk factor for sexual HIV-1 transmission, it has not been well studied in the context of mother-to-child HIV-1 transmission. Genital ulcers during pregnancy have been associated with an approximately two-fold increased risk of HIV-1 transmission in a study performed before use of antiretrovirals to prevent mother-to-child transmission,9 and clinical history of genital herpes has been associated with a similar increased risk of infant HIV-1 acquisition in a retrospective study.10 However, data from African perinatal cohorts receiving antiretroviral prophylaxis are lacking, and there are limited data on HSV shedding during delivery.11
In this study, our primary goal was to determine whether HSV-2 infection, as assessed by HSV-2 seropositivity at 32 weeks of gestation, cervical HSV DNA shedding at delivery, and genital ulcers at 32 weeks of gestation were associated with risk of intrapartum HIV-1 transmission in a prospective cohort of HIV-1-infected pregnant women. We also aimed to define correlates of HSV-2 seropositivity, cervical HSV DNA shedding, and genital ulcers in this cohort.
MATERIALS AND METHODS
Between July 1999 and October 2002, 463 HIV-1-seropositive pregnant women were enrolled in a prospective cohort study in Nairobi and followed through delivery, as previously described.12,13 Institutional review boards of the University of Washington and the University of Nairobi approved the prospective study, and all study participants gave written informed consent. We conducted a nested case control study within this prospective cohort and selected mother-infant pairs with specimens available for testing as cases if infants acquired HIV-1 between birth and month 1, consistent with HIV-1-infection intrapartum. Control mother-infant pairs were randomly selected among infants not HIV-1 infected by 1 month postpartum at a ratio of two controls per case, with oversampling of controls in the event that specimens of some controls were unavailable for testing. Twenty-three infants originally thought to be HIV-1-infected intrapartum were identified as being infected in utero. Mother-infant pairs with infants subsequently determined to be infected in utero were excluded from the analysis of risk factors for intrapartum HIV-1 transmission but were included for the analysis of correlates of HSV-2 infection.
Women were enrolled at 32 weeks of gestation, and mother-infant pairs were followed for 12 months postpartum. Women were offered short-course zidovudine from 34 weeks of gestation per the Thai–Centers for Disease Control and Prevention (CDC) regimen.14 A pelvic examination was performed at enrollment, at which time the presence of either vulvar, vaginal, or cervical ulcers was recorded, and blood was obtained for HSV-2 serology, CD4 T cell count, HIV-1 RNA levels, and syphilis testing. At delivery, blood was collected for HIV-1 RNA assays, and cervical swabs were obtained for HIV-1 RNA and HSV DNA assays. To determine infant HIV-1 infection status, filter paper specimens of whole blood were collected at birth, 1, 3, 6, 9, and 12 months. Infant plasma was collected at these same time points to confirm timing of infant HIV-1 acquisition.
Infant filter paper specimens were assayed for HIV-1 DNA using a polymerase chain reaction (PCR) assay, as described elsewhere.15 If a filter paper specimen was positive for HIV-1 DNA, infant plasma specimens were assayed for HIV-1 RNA to confirm timing of HIV-1 acquisition. Plasma HIV-1 RNA levels were determined using the Gen-Probe assay (Gen-Probe Inc, San Diego, CA), a transcription-mediated amplification method sensitive for HIV-1 subtypes common in Kenya.16 Infants were considered to have tested positive if more than 50 copies/assay and more than 100 copies/mL of HIV-1 RNA were detected with the Gen-Probe assay. If infants tested positive by the HIV-1 DNA or RNA assay within 48 hours of birth, they were categorized as having acquired HIV-1 infection in utero. Infants with negative HIV-1 DNA and RNA assays within 48 hours of birth but positive at 1 month were considered to have acquired HIV-1 intrapartum.
Maternal plasma and cervical HIV-1 RNA levels in secretions were measured using the same Gen-Probe assay.17 Human immunodeficiency virus type 1 plasma and cervical viral loads below the limit of detection (7 copies/mL plasma and 18 copies/cervical swab) were recoded as half the limit of detection and considered undetectable. Maternal HSV-2 seropositivity was determined by Focus enzyme-linked immunosorbent assay (HerpeSelect HSV-2 enzyme-linked immunosorbent assay [ELISA]; Focus Diagnostics, Cypress, CA), and all positive specimens were confirmed using Western blot.18 Cervical swabs were analyzed for the presence of HSV using a sensitive PCR assay at the University of Washington.19,20 Briefly, primers were directed at specific glycoprotein B genes of HSV and samples with 10 or more copies of DNA per reaction were considered positive. Maternal plasma was tested for syphilis using the rapid plasma reagin (Beckton Dickinson, Cockeysville, MD) and confirmed using the Treponema pallidum hemagglutination assay (Randox Laboratories Ltd, Ardmore, Crumlin, UK).
Human immunodeficiency virus type 1 and HSV copy numbers/mL were log10 transformed for all analyses. Linear tests for trend were conducted using Pearson product-moment correlations. The Mann-Whitney U test was used to compare medians for continuous variables, and dichotomous variables were analyzed using Cochran Mantel-Haenszel or Fisher exact tests. Multivariable logistic regression models were constructed to determine whether associations persisted after adjusting for maternal plasma HIV-1 RNA or adjusting for CD4 T cell counts less than 200 cells/mm3. Statistical significance was based on P<.05, and all tests were two-sided. Statistical analyses were conducted using SAS 9 (SAS Institute, Cary, NC).
A total of 175 mother-infant pairs were included in the study: 121 (69%) mother-infant pairs did not transmit HIV-1, 31 (18%) infants acquired HIV-1 intrapartum, and 23 (13%) acquired HIV-1 in utero. Correlates of HSV-2 seropositivity, genital ulcer disease, and cervical HSV DNA shedding were determined for all mother-infant pairs, and estimates for the risk of intrapartum HIV-1 transmission were determined for the subset of 152 mother-infant pairs, excluding those with HIV-1 acquisition in utero.
Demographic characteristics and sexual history for all 175 pregnant women are summarized in Table 1. Median maternal age was 24 years (interquartile range [IQR] 21–28), 155 (89%) women were married, and the median number of lifetime sexual partners was three (IQR 2–4) (Table 1). Median CD4 T cell count at 32 weeks of gestation was 419 cells/mm3 (IQR 299–620), and median plasma HIV-1 viral load at 32 weeks of gestation and delivery were 4.81 and 4.21 log10 copies/mL, respectively, (IQR 4.30–5.35 and IQR 3.64–4.85, respectively). Cervical shedding of HIV-1 RNA was detected in 160 (96%) of 167 women with delivery swabs, and the median cervical HIV-1 RNA level at delivery was 2.38 log10 copies/mL (IQR 1.79–3.37).
Overall, 152 (87%) women were HSV-2-seropositive. Among the 152 HSV-2-seropositive women, nine (6%) had genital ulcers at 32 weeks of gestation, and 13 (9%) had HSV DNA detected in cervical secretions at the time of delivery. Women with genital ulcers at 32 weeks of gestation were found to be mutually exclusive from the women who were shedding HSV DNA during delivery, and none of these women tested positive for syphilis. Genital ulcers were not detected in any of the HSV-2-seronegative women.
Correlates of HSV-2 infection are shown in Table 2. Women with genital ulcers had higher median maternal plasma HIV-1 RNA levels at delivery than women without ulcers (4.61 versus 4.17 log10 copies/mL, respectively, P=.02,), and we observed a borderline association for higher plasma HIV-1 viral load at delivery among women shedding HSV DNA at delivery compared with women not shedding HSV (4.73 versus 4.20 log10 copies/mL, respectively, P=.07). Women with maternal CD4 T cell counts of less than 200 cells/mm3 were more likely to have genital ulcers (odds ratio [OR] 5.8, 95% confidence interval [CI] 1.3–26.7, P=.01) and shed HSV DNA at delivery (OR 4.1, 95% CI 1.1–15.2, P=.02). Cervical HIV-1 viral load was not associated with HSV-2 seropositivity (P=.4), genital ulcers at 32 weeks of gestation (P=.9), or cervical HSV DNA shedding (P=.4) (Table 2). Among women shedding HSV DNA, the median HSV copy number was 3.48 (IQR 3.04–5.33) log10 copies/mL. Higher levels of cervical HIV-1 RNA were weakly correlated with higher levels of cervical HSV DNA (Pearson r=0.26, P=.4) but strongly correlated with higher levels of plasma HIV-1 RNA levels (Pearson r=0.43, P<.001).
A slightly larger proportion of women who transmitted HIV-1 in utero were HSV-2-seropositive compared with women who did not transmit HIV-1 in utero (96% of transmitters versus 85% of nontransmitters were HSV-2-seropositive, P=.2). Genital ulcers at 32 weeks of gestation (OR 0.8, 95% CI 0.1–6.7, P = .8) and cervical HSV shedding at delivery were also not associated with in utero HIV-1 transmission (OR 1.9, 95% CI 0.5–7.5, P=.4).
The presence of antibody to HSV-2 did not correlate with intrapartum HIV-1 transmission (Table 3). However, genital ulcers at 32 weeks of gestation were associated with an increased risk of intrapartum transmission (16% of transmitters versus 3% of nontransmitters had ulcers, P=.003). Among the eight women with genital ulcers at 32 weeks of gestation, five (63%) transmitted HIV-1 intrapartum and only one of those women had a CD4 T cell count less than 200 cells/mm3 or plasma HIV-1 viral load greater than 50,000 copies/mL. A somewhat greater proportion of women who were shedding HSV at the time of delivery transmitted intrapartum compared with women who were not shedding HSV (11% versus 7%, respectively, P=.5).
Other correlates associated with intrapartum transmission included maternal plasma HIV-1 RNA at delivery (P<.001), maternal cervical HIV-1 RNA level at delivery (P=.02), and maternal CD4 T cell count less than 200 cells/mm3 (P<.001). The proportion of women who transmitted intrapartum was higher among women who delivered by cesarean, the majority being emergent and after rupture of membranes, than among women who delivered vaginally (30% versus 13%, respectively, P=.03). Indicators of duration of delivery, including gestation, time from rupture of membranes to delivery, and time of stage of labor were not associated with intrapartum transmission.
To determine risk factors associated with intrapartum transmission adjusting for maternal plasma HIV-1 viral load at delivery or adjusting for maternal CD4 T cell count less than 200 cells/mm3, multivariable logistic regression models were constructed (Table 4). After adjusting for maternal plasma HIV-1 viral load at delivery, increased risk of intrapartum transmission was observed among women with genital ulcers (OR 5.1, 95% CI 1.1–24.1, P=.04). A nonsignificant association between genital ulcers and intrapartum transmission was also found after adjusting for maternal CD4 T cell count less than 200 cells/mm3 (OR 4.6, 95% CI 0.9–24.3, P=.07).
In this study we determined the relationship between HSV-2 infection and mother-to-child HIV-1 transmission among Kenyan women receiving zidovudine prophylaxis. This study demonstrates that, in the context of antiretrovirals, women with genital ulcers are more likely to transmit HIV-1 intrapartum than women without genital ulcers. The association between transmission risk and the presence of genital ulcers remained significant after adjusting for plasma HIV-1 viral load, a factor strongly associated with infant HIV-1 acquisition in our cohort and others.21,22 Although swabs from genital ulcers were not collected to detect HSV, genital ulcers observed in this study were most likely secondary to HSV-2 infection. Herpes simplex virus type 2 is the most common etiology of genital ulcers in sub-Saharan Africa,23,24 and all women with genital ulcers in this study tested negative for syphilis.
One strength of our study is that we used clinical (presence of genital ulcers), virologic (cervical HSV DNA shedding), and serologic (HSV-2 antibody) measures of HSV-2 infection. Although we found cervical HSV shedding to be relatively low, the seroprevalence of HSV-2 was remarkably high among HIV-1-seropositive pregnant women in Nairobi, consistent with recent studies among HIV-1-infected African women.3,4 We did not find HSV-2 seropositivity to be associated with risk of intrapartum HIV-1 transmission, although high HSV-2 seroprevalence made any potential effect of HSV-2 seropositivity on risk of intrapartum HIV-1 transmission difficult to detect.
Several possible mechanisms may explain the increased risk of intrapartum HIV-1 transmission among women with HSV-2 infection. Both clinical and subclinical HSV-2 reactivation can cause recruitment of HIV-1-infected CD4 T cells to the mucosal surface of HSV-2 lesions, resulting in increased HIV-1 viral load in the female genital tract1,4 and in plasma.25,26 We observed a borderline association between HSV shedding and increased plasma HIV-1 levels, as has been shown by others.7 However, we did not find an association between HSV DNA and HIV-1 RNA in genital secretions. This may be because more than 95% of women in our cohort were shedding HIV-1, and we lacked power to detect a difference between those shedding and those not shedding as a result of this high proportion. Other studies of HIV-1-infected nonpregnant women have also found a large percentage of women shedding HIV-1 RNA in cervical secretions,1,4,27 and our results displaying no association between cervical HIV-1 shedding and HSV shedding are consistent with findings reported by Cowan et al.27
Herpes simplex virus type 2 infection may also result in ulceration, which would provide a route for HIV-1 transmission to the infant. The presence of significant quantities of HIV-1 RNA in genital ulcers has been reported,28 and our observation that clinical diagnosis of genital ulcers was the strongest predictor supports this as a potential mechanism for increased intrapartum HIV-1 transmission among pregnant women with active HSV-2 infection. Finally, HSV-2 reactivation has been associated with increased plasma HIV-1 RNA, and we did observe in our cohort that genital ulcers were associated with higher levels of viremia at delivery.7
A limitation of our study was that the number of women who transmitted HIV-1 intrapartum was small, as was the number of women who were shedding HSV. In addition, we may have underestimated the amount of genital ulcers and HSV shedding. The presence of genital ulcers was recorded during a pelvic examination at 32 weeks of gestation; women who did not have ulcers at 32 weeks of gestation may have developed ulcers by the time of delivery, or the ulcers detected at 32 weeks of gestation may not have persisted until the time of delivery. Schacker et al29 reported the median duration of genital ulcers in HIV/HSV-2 co-infected persons not receiving treatment for HSV infection was 11 days, and genital ulcers were detected in 14% of days observed. Therefore, even if ulcers did not persist until delivery, new genital ulcers could have developed by the time of delivery. Furthermore, the swabs at delivery were cervical and not vulvar or perianal, making them less sensitive for detecting HSV shedding. As a result, our assessment may underestimate both genital ulcers and HSV shedding at delivery and is subject to misclassification bias.
Another important consideration is that this study was conducted in the setting of zidovudine prophylaxis, which could attenuate the impact of HSV-2 on transmission risk by reducing plasma HIV-1 RNA. Yet, in our cohort of women receiving a short-course antiretroviral regimen, we observed that genital ulcers were associated with a similar degree of risk of perinatal HIV-1 transmission as an antiretroviral-naïve cohort,9 irrespective of plasma HIV-1 viral load. Furthermore, in our cohort the population-attributable risk of maternal HSV-2 ulcers to intrapartum HIV-1 transmission is 14% of infant infections. Together, these data suggest that genital ulcers play an important role in perinatal HIV-1 transmission and that HSV-2 suppression might be beneficial even in an antiretroviral setting.
Additional research is needed to assess further the relationship between HSV-2 and mother-to-child HIV-1 transmission, as well as the potential for using acyclovir to modify the risk of intrapartum HIV-1 transmission. Acyclovir is an inexpensive, safe, well-tolerated drug that can be used during pregnancy30 and is no longer under patent protection, making it an appealing drug for preventing mother-to-child HIV-1 transmission programs. Currently, trials are ongoing to assess the role of acyclovir in preventing sexual HIV-1 transmission, and our study suggests that such an approach could be considered for preventing mother-to-child HIV-1 transmission.
1. Mbopi-Keou FX, Gresenguet G, Mayaud P, Weiss HA, Gopal R, Matta M, et al. Interactions between herpes simplex virus type 2 and human immunodeficiency virus type 1 infection in African women: opportunities for intervention. J Infect Dis 2000;182:1090–6.
2. Nahmias AJ, Lee FK, Beckman-Nahmias S. Sero-epidemiological and -sociological patterns of herpes simplex virus infection in the world. Scand J Infect Dis 1990;69:19–36.
3. Ozouaki F, Ndjoyi-Mbiguino A, Legoff J, Onas IN, Kendjo E, Si-Mohamed A, et al. Genital shedding of herpes simplex virus type 2 in childbearing-aged and pregnant women living in Gabon. Int J STD AIDS 2006;17:124–7.
4. McClelland RS, Wang CC, Overbaugh J, Richardson BA, Corey L, Ashley RL, et al. Association between cervical shedding of herpes simplex virus and HIV-1. AIDS 2002;16:2425–30.
5. Gray RH, Wawer MJ, Brookmeyer R, Sewankambo NK, Serwadda D, Wabwire-Mangen F, et al. Probability of HIV-1 transmission per coital act in monogamous, heterosexual, HIV-1-discordant couples in Rakai, Uganda. Lancet 2001;357:1149–53.
6. Corey L, Wald A, Celum CL, Quinn TC. The effects of herpes simplex virus-2 on HIV-1 acquisition and transmission: a review of two overlapping epidemics. J Acquir Immune Defic Syndr 2004;35:435–45.
7. Schacker T, Zeh J, Hu H, Shaughnessy M, Corey L. Changes in plasma human immunodeficiency virus type 1 RNA associated with herpes simplex virus reactivation and suppression. J Infect Dis 2002;186:1718–25.
8. Newell ML. Mechanisms and timing of mother-to-child transmission of HIV-1. AIDS 1998;12:831–7.
9. John GC, Nduati RW, Mbori-Ngacha DA, Richardson BA, Panteleeff D, Mwatha A, et al. Correlates of mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission: association with maternal plasma HIV-1 RNA load, genital HIV-1 DNA shedding, and breast infections. J Infect Dis 2001;183:206–12.
10. Chen KT, Segu M, Lumey LH, Kuhn L, Carter RJ, Bulterys M, et al. Genital herpes simplex virus infection and perinatal transmission of human immunodeficiency virus. Obstet Gynecol 2005;106:1341–8.
11. Hitti J, Watts DH, Burchett SK, Schacker T, Selke S, Brown ZA, Corey L, et al. Herpes simplex virus seropositivity and reactivation at delivery among pregnant women infected with human immunodeficiency virus-1. Am J Obstet Gynecol 1997;177:450-4.
12. Farquhar C, VanCott TC, Mbori-Ngacha DA, Horani L, Bosire RK, Kreiss JK, et al. Salivary secretory leukocyte protease inhibitor is associated with reduced transmission of human immunodeficiency virus type 1 through breast milk. J Infect Dis 2002;186:1173–6.
13. Farquhar C, Rowland-Jones S, Mbori-Ngacha D, Redman M, Lohman B, Slyker J, et al. Human leukocyte antigen (HLA) B*18 and protection against mother-to-child HIV type 1 transmission. AIDS Res Hum Retroviruses 2004;20:692–7.
14. Shaffer N, Chuachoowong R, Mock PA, Bhadrakom C, Siriwasin W, Young NL, et al. Short-course zidovudine for perinatal HIV-1 transmission in Bangkok, Thailand: a randomised controlled trial. Bangkok Collaborative Perinatal HIV Transmission Study Group. Lancet 1999;353:773–80.
15. Panteleeff DD, John G, Nduati R, Mbori-Ngacha D, Richardson B, Kreiss J, et al. Rapid method for screening dried blood samples on filter paper for human immunodeficiency virus type 1 DNA. J Clin Microbiol 1999;37:350–3.
16. Emery S, Bodrug S, Richardson BA, Giachetti C, Bott MA, Panteleef D, et al. Evaluation of performance of the Gen-Probe human immunodeficiency virus type 1 viral load assay using primary subtype A, C, and D isolates from Kenya. J Clin Microbiol 2000;38:2688–95.
17. DeVange Panteleeff D, Emery S, Richardson BA, Rousseau C, Benki S, Bodrug S, et al. Validation of performance of the Gen-Probe human immunodeficiency virus type 1 viral load assay with genital swabs and breast milk samples. J Clin Microbiol 2002;40:3929–37.
18. Ashley RL, Militoni J, Lee F, Nahmias A, Corey L. Comparison of Western blot (immunoblot) and glycoprotein G-specific immunodot enzyme assay for detecting antibodies to herpes simplex virus types 1 and 2 in human sera. J Clin Microbiol 1988;26:662–7.
19. Jerome KR, Huang ML, Wald A, Selke S, Corey L. Quantitative stability of DNA after extended storage of clinical specimens as determined by real-time PCR. J Clin Microbiol 2002;40:2609–11.
20. Wald A, Huang ML, Carrell D, Selke S, Corey L. Polymerase chain reaction for detection of herpes simplex virus (HSV) DNA on mucosal surfaces: comparison with HSV isolation in cell culture. J Infect Dis 2003;188:1345–51.
21. Mofenson LM, Lambert JS, Stiehm ER, Bethel J, Meyer WA Whitehouse J, et al. Risk factors for perinatal transmission of human immunodeficiency virus type 1 in women treated with zidovudine. Pediatric AIDS Clinical Trials Group Study 185 Team. N Engl J Med 1999;341:385-93.
22. Garcia PM, Kalish LA, Pitt J, Minkoff H, Quinn TC, Burchett SK, et al. Maternal levels of plasma human immunodeficiency virus type 1 RNA and the risk of perinatal transmission. Women and Infants Transmission Study Group. N Engl J Med 1999;341:394–402.
23. Chen CY, Ballard RC, Beck-Sague CM, Dangor Y, Radebe F, Schmid S, et al. Human immunodeficiency virus infection and genital ulcer disease in South Africa: the herpetic connection. Sex Transm Dis 2000;27:21–9.
24. Ahmed HJ, Mbwana J, Gunnarsson E, Ahlman K, Guerino C, Svensson LA, et al. Etiology of genital ulcer disease and association with human immunodeficiency virus infection in two Tanzanian cities. Sex Transm Dis 2003;30:114–9.
25. Duffus WA, Mermin J, Bunnell R, Byers RH, Odongo G, Ekwaru P, et al. Chronic herpes simplex virus type-2 infection and HIV viral load. Int J STD AIDS 2005;16:733–5.
26. Mole L, Ripich S, Margolis D, Holodniy M. The impact of active herpes simplex virus infection on human immunodeficiency virus load. J Infect Dis 1997;176:766–70.
27. Cowan FF, Pascoe SJ, Barlow KL, Langhaug LF, Jaffar S, Hargrove JW, et al. Association of genital shedding of herpes simplex virus type 2 and HIV-1 among sex workers in rural Zimbabwe. AIDS 2006;20:261–7.
28. Schacker T, Ryncarz AJ, Goddard J, Diem K, Shaughnessy M, Corey L. Frequent recovery of HIV-1 from genital herpes simplex virus lesions in HIV-1-infected men. JAMA 1998;280:61–6.
29. Schacker T, Hu HL, Koelle DM, Zeh J, Saltzman R, Boon R, et al. Famciclovir for the suppression of symptomatic and asymptomatic herpes simplex virus reactivation in HIV-infected persons. A double-blind, placebo-controlled trial. Ann Intern Med 1998;128:21–8.
30. Management of herpes in pregnancy. Clinical management guidelines for obstetrician-gynecologists. ACOG Practice Bulletin 8. Int J Gynaecol Obstet 2000;68:165-73.