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No association between antepartum serologic and genital tract evidence of herpes simplex virus-2 coinfection and perinatal HIV-1 transmission
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OBSTETRICS
No association between antepartum serologic and genital tract evidence of herpes simplex virus-2 coinfection and perinatal HIV-1 transmission Katherine T. Chen, MD, MPH; Ruth E. Tuomala, MD; Clara Chu, PhD; Meei-Li Huang, PhD; D. Heather Watts, MD; Carmen D. Zorrilla, MD; Mary Paul, MD; Ron Hershow, MD; Philip Larussa, MD OBJECTIVE: The purpose of this study was to assess the risk of perinatal HIV-1 transmission in women who are coinfected with herpes simplex virus-2 (HSV-2).
transmission. There was also no association between antepartum genital tract evidence of HSV-2 coinfection and risk of perinatal HIV-1 transmission.
STUDY DESIGN: We performed a nested case-control study of 26
CONCLUSION: Women who were infected with HIV-1 with antepartum serologic and genital tract evidence of HSV-2 coinfection did not appear to have an increased risk of perinatal HIV-1 transmission. However, further investigations are needed to assess HSV-2 reactivation and the risk of perinatal HIV-1 transmission at the time of delivery.
women whose HIV-1 was transmitted to their infants and 52 control subjects whose HIV-1 was not transmitted. We assessed antepartum serologic evidence of HSV-2 by HSV-2 serostatus and genital tract evidence of HSV-2 by presence of HSV-2 DNA. RESULTS: There was no significant association between antepartum
serologic evidence of HSV-2 coinfection and the risk of perinatal HIV-1
Key words: herpes simplex virus 2 infection, perinatal HIV-1 transmission, pregnancy, transmitted infection
Cite this article as: Chen KT, Tuomala RE, Chu C, et al. No association between antepartum serologic and genital tract evidence of herpes simplex virus-2 coinfection and perinatal HIV-1 transmission. Am J Obstet Gynecol 2008;198:399.e1-399.e5.
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ncreasing evidence demonstrates a substantial link between the epidemics of HIV-1 and herpes simplex virus-2 (HSV-2) infection.1 Genital tract infection with HSV-2 in HIV-1–infected individuals leads to an increased risk for
sexual transmission of HIV-1.2 Given the potential role for HSV-2 in the sexual transmission of HIV-1, we hypothesized that maternal coinfection with HSV-2 may be a risk factor for perinatal HIV-1 transmission.
From the Departments of Obstetrics and Gynecology and Epidemiology (Dr Chen) and Pediatrics (Dr Larussa), Columbia University, New York, NY; the Department of Obstetrics, Gynecology, and Reproductive Biology (Dr Tuomala), Brigham and Women’s Hospital and Harvard Medical School, Boston, MA; the Clinical Trials & Surveys Corporation (Dr Chu), Baltimore, MD; the Department of Laboratory Medicine (Dr Huang), University of Washington, Seattle, WA; the Pediatric, Adolescent and Maternal Acquired Immunodeficiency Syndrome Branch, National Institute of Child Health and Human Development (Dr Watts), Bethesda, MD; the Department of Obstetrics and Gynecology, University of Puerto Rico School of Medicine (Dr Zorrilla), San Juan, Puerto Rico; the Departments of Family and Community Medicine and Pediatrics (Dr Paul), Baylor College of Medicine, Houston, TX; and the Department of Internal Medicine (Dr Hershow), University of Illinois, Chicago, IL. Received May 14, 2007; accepted Oct. 1, 2007. Reprints: Katherine T. Chen, MD, MPH, Department of Obstetrics and Gynecology and Epidemiology, Columbia University, 722 West 168th St, New York, NY 10032; [email protected]. This study was supported in part by Women’s Reproductive Health Research Career Development Center grant SK12 HD01275 from the National Institute of Child Health and Human Development (K.T.C.) and by grants U01 AI 034858, 9U01 DA 015054, U01 DA 015053, U01 HD 036117, U01 AI 034841, U01 HD 041983, N01 AI 085339, 1 U01 AI 05027401, and NIH GCRC RR000188 and GCRC RR000645. 0002-9378/$34.00 • © 2008 Mosby, Inc. All rights reserved. • doi: 10.1016/j.ajog.2007.10.784
The potential mechanism by which coinfection with HSV-2 could increase perinatal HIV-1 transmission is an increase in plasma and genital tract HIV-1 viral load during HSV-2 reactivation. Plasma HIV-1 viral load is one of the most important risk factors for perinatal HIV-1 transmission.3 In addition, previous studies have demonstrated that the presence of genital HIV-1 RNA4 and DNA5 in HIV-1–infected women who receive no or minimal antiretroviral therapy increases the risk of perinatal HIV-1 transmission. A previous study from the Women and Infants Transmission Study (WITS) showed that genital tract HIV-1 DNA in HIV-1–infected women who receive antiretroviral therapy may also be an independent risk factor for perinatal HIV-1 transmission.6 Because reactivation with HSV-2 is thought to increase both plasma7 and genital8-10 HIV-1 viral loads, HSV-2 infection may also be important in perinatal HIV-1 transmission. The studies on perinatal HIV-1 transmission in women who are coinfected with HSV-2 are few. One US study found
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no association of self-reported genital HSV infection in HIV-1–infected women with perinatal HIV-1 transmission.11 Another US study showed that a clinical diagnosis of genital HSV infection during pregnancy in HIV-1–infected women (with a majority of the diagnoses based on culture confirmation of HSV) is a significant risk factor for perinatal HIV-1 transmission.12 Both studies are limited in that genital HSV coinfection was defined by self-report or clinical diagnosis instead of serologic or genital tract evidence of HSV-2 infection. A recent study in Kenya showed that HSV-2 seropositive HIV-infected women who had genital ulcers had an increased risk of perinatal HIV-1 transmission after adjustment for plasma HIV-1 viral loads.13 We report, from the WITS cohort, our assessment of the risk of perinatal HIV-1 transmission in HIV-1–infected women with known genital tract HIV viral load status that was associated with serologic and genital tract evidence of HSV-2 coinfection in the antepartum period.
M ATERIALS AND M ETHODS Study design For the present study, we analyzed serologic samples and genital tract specimens for evidence of HSV-2 coinfection (defined as presence of HSV-2 antibodies or presence of HSV-2 DNA) from the same cases and control subjects in the previously described nested case-control WITS study that assessed the effect of genital tract HIV viral load status on perinatal HIV transmission.6 WITS is an institutional review board–approved, multicenter, prospective cohort study of HIV-1–infected pregnant women in the United States that began in December 1989. Enrollment occurred at any time during pregnancy. Analyses for the present study included data that were obtained from study visits in the antepartum period (⬍ 19, 19-31, and ⱖ 32 weeks of gestation), at the time of delivery, and from the first or second postpartum visit. Study data were obtained by standardized questionnaires, medical record abstraction, physical examination, and phlebotomy at each study visit. 399.e2
www.AJOG.org Of particular interest to this study, information about anti-HSV treatment at any time during pregnancy was queried specifically. Cervicovaginal lavage (CVL) of the genital tract was performed at the time of enrollment and at the antepartum visit that occurred at approximately 32 weeks of gestation. CVL was done with 10 mL of sterile nonbacteriostatic saline solution in a 10-mL syringe to which a soft plastic cannula (plastic transfer pipette; Fisher Scientific, Pittsburg, PA) was attached. A continuous stream of saline solution was directed into the cervical os. The pooled lavage was aspirated from the posterior fornix, emptied through the catheter into a 15-mL conical polypropylene tube, and transported on ice to the laboratory within the same day in all cases (if possible, transport to the laboratory occurred within 4 hours of collection).
Selection of cases and control subjects In the previously described nested casecontrol WITS study that assessed the effect of genital tract HIV viral load status on perinatal HIV transmission,6 the cases were 26 HIV-1–infected women who delivered between June 1995 and July 1999, who transmitted HIV-1 to their singleton infants, and who had at least 1 antepartum CVL specimen obtained. The control subjects were 52 HIV-1–infected pregnant women from the same time period who did not transmit HIV-1 to their infants. Control subjects were frequency matched by clinical center to the case subjects in a 2:1 ratio.
Testing for HSV-2 by serologic evaluation Plasma that was obtained at enrollment was tested by indirect enzyme-linked immunosorbent assay (ELISA) for the qualitative detection of HSV-2 type-specific immunoglobulin G antibodies (HerpeSelect HSV-2 ELISA IgG; Focus Diagnostics, Cypress, CA), because typespecific ELISA immunoglobulin G has a higher sensitivity and specificity than the type-specific glycoprotein G immunoassay.14 In addition, the US Food and Drug Administration has approved the Focus
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Diagnostics ELISA for use in pregnant women. The sensitivity for HSV-2 detection in pregnant women is 100%, and the specificity is 96.1%. Two ELISA runs were performed for each plasma sample. Plasma with either equivocal results in both runs or discordant results underwent additional testing for HSV-2 by a rapid type specific glycoprotein G immunoassay kit (Sure-Vue HSV-2; Biokit USA, Lexington, MA). Confirmatory Biokit testing of ELISA results is effective in reducing false-positive HSV-2 results.15 All serologic assays were performed by one of the authors who was blinded to case or control status. If the study subject was HSV-2 seronegative at enrollment, an additional plasma sample that had been obtained at delivery was tested to detect seroconversion to HSV-2 during pregnancy. If the subject was HSV-2 seronegative at enrollment and at delivery, another plasma sample, which was obtained from the earliest postpartum visit, was tested to detect seroconversion to HSV-2 at the time of delivery or early postpartum period.
Testing for presence of HSV-2 DNA Among the study subjects who were found to be HSV-2 seropositive, the presence of HSV-2 DNA in CVL specimens that were obtained closest to delivery was assessed. Quantification of HSV-2 DNA CVL specimens was determined by one of the authors who was blinded to the case or control status. The procedure uses a high-throughput, semiautomated, quantitative fluorescent-based polymerase chain reaction technique as previously described.16-18 If the CVL specimen of a study subject contained HSV-2 DNA, the study subject was considered to have the presence of HSV-2 DNA in the genital tract.
Statistical methods Given the sample size of 26 cases and 52 control subjects, we calculated a priori that we would have ⬎ 80% power to detect differences in the percentage of those with serologic evidence of HSV-2 in either scenario: 95% cases and 70% control subjects were HSV-2 seropositive or
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www.AJOG.org 75% cases and 43% control subjects were HSV-2 seropositive. We also calculated that we would have ⬎ 80% power to detect differences in the percentage of those with genital tract evidence of HSV-2 in a range of scenarios from 50% of cases positive for HSV-2 DNA as compared with 15% of control subjects to 25% of cases positive for HSV-2 DNA as compared with 2% of control subjects. Chi-square statistics were used in the analysis of categoric variables (eg, HSV-2 serostatus or presence of HSV-2 DNA) and Wilcoxon rank sum tests were used in the analysis of continuous variables (eg, HIV viral loads). Results were considered significant at P ⬍ .05; Fisher’s exact test probability values were reported when cells had expected counts ⬍ 5. Analyses were performed with SAS software for Unix (v 9.0; SAS Institute Inc, Cary, NC).
R ESULTS As previously reported, cases were significantly less likely than control subjects to have been treated with combination antiretroviral therapy, significantly more likely to have a detectable plasma HIV-1 RNA level at delivery, significantly more likely to have a higher cell CVL pellet HIV-1 DNA level, tended to have a lower mean CD4 count at delivery, and no difference in CD4 cell percentage at time of delivery (data not shown).6
HSV-2 serostatus Among the 78 study subjects, 65 women (83.3%) were found to be HSV-2 seropositive. Nineteen of the 26 cases (73.1%) were HSV-2 seropositive, and 18 of the 19 women were HSV-2 seropositive at enrollment. One of the 19 cases seroconverted around the time of delivery or the early postpartum period because this study subject was initially HSV-2 seronegative at enrollment and at delivery and then found to be HSV-2 seropositive at the 8-week postpartum visit. Forty-six of the 52 control subjects (88.5%) were HSV-2 seropositive at enrollment, and none of the control subjects seroconverted. There was no significant association between serologic evidence of HSV-2 coinfection in the an-
tepartum period and the risk of perinatal HIV-1 transmission (odds ratio, 0.4; 95% CI, 0.1-1.2; P ⫽ .1). HSV-2 seropositive subjects did not differ from HSV-2 seronegative subjects in plasma HIV-1 RNA level at delivery or CVL cell pellet HIV-1 DNA level (data not shown).
Presence of HSV-2 DNA Seventeen of the 19 HSV-2 seropositive cases (90.0%) and 40 of the 46 HSV-2 seropositive control subjects (86.9%) had CVL available from the repository for HSV-2 DNA analysis. The time at which CVLs were obtained in the antepartum period ranged from 10-32 weeks of gestation, with a mean of 16.4 weeks of gestation. None of the 17 HSV-2 seropositive cases and 2 of the 40 HSV-2 seropositive control subjects (5.0%) were positive for HSV-2 DNA. There was no association between genital tract evidence of HSV-2 coinfection in the antepartum period and risk of perinatal HIV-1 transmission (odds ratio, 0.4; 95% CI, 0.02-10.0; P ⫽ 1.0).
HSV-2 treatment Among the 65 study subjects who were HSV-2 seropositive, 6 women (9.2%) had anti-HSV treatment at any time during pregnancy (1 case and 5 control subjects). There was no difference between anti-HSV treatment and risk of HIV-1 transmission in the HSV-2 seropositive subjects (odds ratio, 0.5; 95% CI, 0.014.6; P ⫽ .7). Among the 6 HSV-2 seropositive subjects who had anti-HSV treatment at any time during pregnancy, all 6 women were negative for HSV-2 DNA in CVL.
HSV-2 neonatal infection In this WITS nested case-control study, there were no cases of HSV-2 neonatal infection.
C OMMENT Given the potential role of HSV-2 in enhancing sexual transmission of HIV-1, we hypothesized that coinfection with HSV-2 might increase perinatal HIV-1 transmission because infection with HSV-2 could increase plasma and genital tract HIV-1 viral loads. A previous study
Research
showed that a clinical diagnosis of genital HSV infection during pregnancy in a multicenter New York City cohort (most of the diagnoses were based on culture confirmation of HSV) significantly increased the risk of perinatal HIV-1 transmission.12 In our nested case control study from the WITS cohort, we found no association between serologic evidence of HSV-2 coinfection in the antepartum period and the risk of perinatal HIV-1 transmission. Among those HIV1–infected women who were HSV-2 seropositive, we found no association between the presence of genital tract HSV-2 in the antepartum period and risk of perinatal HIV-1 transmission. Our finding that HIV-1–infected pregnant women have a high prevalence of HSV-2 infection is consistent with another study of HIV-1–infected pregnant women in labor in the United States.19 This high HSV-2 seroprevalence in HIV1–infected pregnant women is in contrast to the prevalence of 30% reported in the general female population20 and 20% in pregnant women21 in the United States. Serologic evidence of HSV-2 infection is more accurate than self-report or clinical diagnosis because most individuals with HSV-2 infection are unaware of their status20 and because most HSV-2 infections do not cause the classic, visible ulcerative lesions that are recognized easily as genital herpes infection.22 Because we found such a high prevalence of HSV-2 coinfection in both cases and control subjects, we had only 40% power to detect a meaningful difference with our sample size. Because the potential mechanism of increased perinatal HIV-1 transmission with HSV-2 coinfection might be enhancement of plasma and genital tract HIV-1 viral load during an HSV-2 reactivation, we attempted to detect HSV-2 active replication by determining the presence of HSV-2 DNA in the genital tract from CVL specimens. However, we did not have CVLs available for analyses from every study subject. We did find 2 of the 57 CVL specimens (3.5%) to be positive for HSV-2 DNA, which is a rate that is lower than the 9.0% rate from the Kenyan study.13 The other limitation to the CVL specimens is that they were ob-
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tained in the antepartum period and not at the time of delivery. Genital HSV infection during the latter time period is likely to be a more important clinical predictor of perinatal HIV-1 transmission. The risk of seroconversion to HSV-2 among a general population of pregnant women is reported to be 1.7%.23 However, among HSV-2 seronegative women with HSV-2 seropositive partners, the risk is 20%.24 We had 1 HIV-1–infected study subject who seroconverted to HSV-2 around the time of delivery or the early postpartum period and who transmitted HIV to her infant. This study subject was initially HSV-2 seronegative at enrollment and at delivery and then found to be HSV-2 seropositive at the 8-week postpartum visit. The usual time from HSV-2 infection to the development of detectable antibodies is 2-12 weeks.25 We do not know whether the study subject had a primary HSV-2 infection or nonprimary first episode because we do not know her previous HSV-1 status. In addition, we do not have CVL specimens during pregnancy and at delivery for this study subject to test for the presence of HSV-2 DNA. The current study presents laboratory evidence of HSV-2 coinfection but is lacking in information about clinical episodes of HSV-2. Perhaps detectable clinical episodes of HSV-2 coinfections such as genital ulcers may lead to a mucosal milieu that favors perinatal HIV-1 transmission by recruitment of HIV-1– infected CD4⫹ cells into mucosal ulcerations.26 HIV-1 RNA can be detected consistently in genital ulcers that are caused by HSV-2.8 This study also did not evaluate HSV-1 coinfection because genital tract infections are more likely to occur with HSV-2 instead of HSV-1. Although we did not find an association between serologic and genital tract evidence of HSV-2 coinfection and the risk of perinatal HIV-1 transmission, we believe that further investigations that would assess (1) both clinical episodes and laboratory evidence of HSV-2 reactivation at the time of delivery, (2) their relationship to plasma and genital HIV-1 viral loads, and (3) the risk of perinatal 399.e4
www.AJOG.org HIV-1 transmission are needed. The use of potent combination antiretroviral therapy to reduce HIV-1 viral loads to undetectable levels is the most effective strategy to decrease perinatal HIV-1 transmission. However, in low resource settings in which highly active antiretroviral therapy is not available routinely for the prevention of perinatal HIV-1 transmission, the addition of a short-course treatment of HSV-2 coinfection around the time of delivery may provide another strategy to reduce perinatal HIV-1 transmission. Of note, a recent randomized double-blind, placebo-controlled trial of HSV suppressive therapy in coinfected women in Burkina Faso, West Africa, showed a significant reduction of genital and plasma HIV-1 viral loads.27 Our work suggests a need for a more complete evaluation of HSV-2 during pregnancy to gain insight into the interactions of HIV-1 and HSV-2, specifically regarding the risks of transmission and the course of disease of f both infections. ACKNOWLEDGMENTS Funding to the Women and Infants Transmission Study Principal investigators, study coordinators, and program officers is as follows: Clemente Diaz, Edna Pacheco-Acosta (University of Puerto Rico, San Juan, PR; U01 AI 034858); Ruth Tuomala, Ellen Cooper, Donna Mesthene (Boston/Worcester Site, Boston, MA; 9U01 DA 015054); Philip LaRussa, Alice Higgins (Columbia University, New York, NY; U01 DA 015053); Sheldon Landesman, Herman Mendez, Ava Dennie (State University of New York, Brooklyn, NY; U01 HD 036117); Kenneth Rich, Delmyra Turpin (University of Illinois at Chicago, Chicago, IL; U01 AI 034841); William Shearer, Norma Cooper (Baylor College of Medicine, Houston, TX; U01 HD 041983); Joana Rosario (National Institute of Allergy and Infectious Diseases, Bethesda, MD); Kevin Ryan, (National Institute of Child Health and Human Development, Bethesda, MD); Vincent Smeriglio, Katherine Davenny (National Institute on Drug Abuse, Bethesda, MD); and Bruce Thompson (Clinical Trials & Surveys Corp., Baltimore, MD, N01 AI 085339). The scientific leadership core includes Kenneth Rich (PI), Delmyra Turpin (Study Coordinator; 1 U01 AI 05027401). Additional support was provided by local Clinical Research Centers: Baylor College of Medicine, Houston, TX (NIH GCRC RR000188); Columbia University, New York, NY (NIH GCRC RR000645).
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REFERENCES 1. 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. 2. Gray RH, Wawer MJ, Brookmeyer R, 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. 3. Garcia PM, Kalish LA, Pitt J, 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. 4. Chuachoowong R, Shaffer N, Siriwasin W, et al. Short-course antenatal zidovudine reduces both cervicovaginal human immunodeficiency virus type 1 RNA levels and risk of perinatal transmission: Bangkok Collaborative Perinatal HIV Transmission study group. J Infect Dis 2000;181:99-106. 5. John GC, Nduati RW, Mbori-Ngacha DA, 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. 6. Tuomala RE, O’Driscoll PT, Bremer JW, et al. Cell-associated genital tract virus and vertical transmission of human immunodeficiency virus type 1 in antiretroviral-experienced women. J Infect Dis 2003;187:375-84. 7. 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. 8. 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. 9. McClelland RS, Wang CC, Overbaugh J, et al. Association between cervical shedding of herpes simplex virus and HIV-1. AIDS 2002;16:2425-30. 10. Mbopi-Keou FX, Gresenguet G, Mayaud P, 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. 11. Van Dyke RB, Korber BT, Popek E, et al. The Ariel project: a prospective cohort study of maternal-child transmission of human immunodeficiency virus type 1 in the era of maternal antiretroviral therapy. J Infect Dis 1999; 179:319-28. 12. Chen KT, Segu M, Lumey LH, et al. Genital herpes simplex virus infection and perinatal transmission of human immunodeficiency virus. Obstet Gynecol 2005;106:1341-8. 13. Drake AL, John-Stewart GC, Wald A, et al. Herpes simplex virus type 2 and risk of intrapartum human immunodeficiency virus transmission. Obstet Gynecol 2007;109:403-9.
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www.AJOG.org 14. Ashley RL. Sorting out the new HSV type specific antibody tests. Sex Transm Infect 2001;77:232-7. 15. Morrow RA, Friedrich D, Meier A, Corey L. Use of “biokit HSV-2 rapid assay” to improve the positive predictive value of Focus HerpeSelect HSV-2 ELISA. BMC Infect Dis 2005;5:84. 16. Ryncarz AJ, Goddard J, Wald A, Huang ML, Roizman B, Corey L. Development of a high-throughput quantitative assay for detecting herpes simplex virus DNA in clinical samples. J Clin Microbiol 1999;37:1941-7. 17. 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. 18. 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. 19. Hitti J, Watts DH, Burchett SK, 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. 20. Fleming DT, McQuillan GM, Johnson RE, et al. Herpes simplex virus type 2 in the United States, 1976 to 1994. N Engl J Med 1997;337:1105-11. 21. Xu F, Markowitz LE, Gottlieb SL, Berman SM. Seroprevalence of herpes simplex virus types 1 and 2 in pregnant women in the United States. Am J Obstet Gynecol 2007;196:43 e1-6. 22. Wald A, Zeh J, Selke S, et al. Reactivation of genital herpes simplex virus type 2 infection in asymptomatic seropositive persons. N Engl J Med 2000;342:844-50.
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23. Brown ZA, Selke S, Zeh J, et al. The acquisition of herpes simplex virus during pregnancy. N Engl J Med 1997;337:509-15. 24. Gardella C, Brown Z, Wald A, et al. Risk factors for herpes simplex virus transmission to pregnant women: a couples study. Am J Obstet Gynecol 2005;193:1891-9. 25. Ashley R, Cent A, Maggs V, Nahmias A, Corey L. Inability of enzyme immunoassays to discriminate between infections with herpes simplex virus types 1 and 2. Ann Intern Med 1991;115:520-6. 26. Celum CL. The interaction between herpes simplex virus and human immunodeficiency virus. Herpes 2004;11(suppl):36A-45A. 27. Nagot N, Ouedraogo A, Foulongne V, et al. Reduction of HIV-1 RNA levels with therapy to suppress herpes simplex virus. N Engl J Med 2007;356:790-9.
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No association between antepartum serologic and genital tract evidence of herpes simplex virus-2 coinfection and perinatal HIV-1 transmission Katherine T. Chen, MD, MPH; Ruth E. Tuomala, MD; Clara Chu, PhD; Meei-Li Huang, PhD; D. Heather Watts, MD; Carmen D. Zorrilla, MD; Mary Paul, MD; Ron Hershow, MD; Philip Larussa, MD OBJECTIVE: The purpose of this study was to assess the risk of perinatal HIV-1 transmission in women who are coinfected with herpes simplex virus-2 (HSV-2).
transmission. There was also no association between antepartum genital tract evidence of HSV-2 coinfection and risk of perinatal HIV-1 transmission.
STUDY DESIGN: We performed a nested case-control study of 26
CONCLUSION: Women who were infected with HIV-1 with antepartum serologic and genital tract evidence of HSV-2 coinfection did not appear to have an increased risk of perinatal HIV-1 transmission. However, further investigations are needed to assess HSV-2 reactivation and the risk of perinatal HIV-1 transmission at the time of delivery.
women whose HIV-1 was transmitted to their infants and 52 control subjects whose HIV-1 was not transmitted. We assessed antepartum serologic evidence of HSV-2 by HSV-2 serostatus and genital tract evidence of HSV-2 by presence of HSV-2 DNA. RESULTS: There was no significant association between antepartum
serologic evidence of HSV-2 coinfection and the risk of perinatal HIV-1
Key words: herpes simplex virus 2 infection, perinatal HIV-1 transmission, pregnancy, transmitted infection
Cite this article as: Chen KT, Tuomala RE, Chu C, et al. No association between antepartum serologic and genital tract evidence of herpes simplex virus-2 coinfection and perinatal HIV-1 transmission. Am J Obstet Gynecol 2008;198:399.e1-399.e5.
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ncreasing evidence demonstrates a substantial link between the epidemics of HIV-1 and herpes simplex virus-2 (HSV-2) infection.1 Genital tract infection with HSV-2 in HIV-1–infected individuals leads to an increased risk for
sexual transmission of HIV-1.2 Given the potential role for HSV-2 in the sexual transmission of HIV-1, we hypothesized that maternal coinfection with HSV-2 may be a risk factor for perinatal HIV-1 transmission.
From the Departments of Obstetrics and Gynecology and Epidemiology (Dr Chen) and Pediatrics (Dr Larussa), Columbia University, New York, NY; the Department of Obstetrics, Gynecology, and Reproductive Biology (Dr Tuomala), Brigham and Women’s Hospital and Harvard Medical School, Boston, MA; the Clinical Trials & Surveys Corporation (Dr Chu), Baltimore, MD; the Department of Laboratory Medicine (Dr Huang), University of Washington, Seattle, WA; the Pediatric, Adolescent and Maternal Acquired Immunodeficiency Syndrome Branch, National Institute of Child Health and Human Development (Dr Watts), Bethesda, MD; the Department of Obstetrics and Gynecology, University of Puerto Rico School of Medicine (Dr Zorrilla), San Juan, Puerto Rico; the Departments of Family and Community Medicine and Pediatrics (Dr Paul), Baylor College of Medicine, Houston, TX; and the Department of Internal Medicine (Dr Hershow), University of Illinois, Chicago, IL. Received May 14, 2007; accepted Oct. 1, 2007. Reprints: Katherine T. Chen, MD, MPH, Department of Obstetrics and Gynecology and Epidemiology, Columbia University, 722 West 168th St, New York, NY 10032; [email protected]. This study was supported in part by Women’s Reproductive Health Research Career Development Center grant SK12 HD01275 from the National Institute of Child Health and Human Development (K.T.C.) and by grants U01 AI 034858, 9U01 DA 015054, U01 DA 015053, U01 HD 036117, U01 AI 034841, U01 HD 041983, N01 AI 085339, 1 U01 AI 05027401, and NIH GCRC RR000188 and GCRC RR000645. 0002-9378/$34.00 • © 2008 Mosby, Inc. All rights reserved. • doi: 10.1016/j.ajog.2007.10.784
The potential mechanism by which coinfection with HSV-2 could increase perinatal HIV-1 transmission is an increase in plasma and genital tract HIV-1 viral load during HSV-2 reactivation. Plasma HIV-1 viral load is one of the most important risk factors for perinatal HIV-1 transmission.3 In addition, previous studies have demonstrated that the presence of genital HIV-1 RNA4 and DNA5 in HIV-1–infected women who receive no or minimal antiretroviral therapy increases the risk of perinatal HIV-1 transmission. A previous study from the Women and Infants Transmission Study (WITS) showed that genital tract HIV-1 DNA in HIV-1–infected women who receive antiretroviral therapy may also be an independent risk factor for perinatal HIV-1 transmission.6 Because reactivation with HSV-2 is thought to increase both plasma7 and genital8-10 HIV-1 viral loads, HSV-2 infection may also be important in perinatal HIV-1 transmission. The studies on perinatal HIV-1 transmission in women who are coinfected with HSV-2 are few. One US study found
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no association of self-reported genital HSV infection in HIV-1–infected women with perinatal HIV-1 transmission.11 Another US study showed that a clinical diagnosis of genital HSV infection during pregnancy in HIV-1–infected women (with a majority of the diagnoses based on culture confirmation of HSV) is a significant risk factor for perinatal HIV-1 transmission.12 Both studies are limited in that genital HSV coinfection was defined by self-report or clinical diagnosis instead of serologic or genital tract evidence of HSV-2 infection. A recent study in Kenya showed that HSV-2 seropositive HIV-infected women who had genital ulcers had an increased risk of perinatal HIV-1 transmission after adjustment for plasma HIV-1 viral loads.13 We report, from the WITS cohort, our assessment of the risk of perinatal HIV-1 transmission in HIV-1–infected women with known genital tract HIV viral load status that was associated with serologic and genital tract evidence of HSV-2 coinfection in the antepartum period.
M ATERIALS AND M ETHODS Study design For the present study, we analyzed serologic samples and genital tract specimens for evidence of HSV-2 coinfection (defined as presence of HSV-2 antibodies or presence of HSV-2 DNA) from the same cases and control subjects in the previously described nested case-control WITS study that assessed the effect of genital tract HIV viral load status on perinatal HIV transmission.6 WITS is an institutional review board–approved, multicenter, prospective cohort study of HIV-1–infected pregnant women in the United States that began in December 1989. Enrollment occurred at any time during pregnancy. Analyses for the present study included data that were obtained from study visits in the antepartum period (⬍ 19, 19-31, and ⱖ 32 weeks of gestation), at the time of delivery, and from the first or second postpartum visit. Study data were obtained by standardized questionnaires, medical record abstraction, physical examination, and phlebotomy at each study visit. 399.e2
www.AJOG.org Of particular interest to this study, information about anti-HSV treatment at any time during pregnancy was queried specifically. Cervicovaginal lavage (CVL) of the genital tract was performed at the time of enrollment and at the antepartum visit that occurred at approximately 32 weeks of gestation. CVL was done with 10 mL of sterile nonbacteriostatic saline solution in a 10-mL syringe to which a soft plastic cannula (plastic transfer pipette; Fisher Scientific, Pittsburg, PA) was attached. A continuous stream of saline solution was directed into the cervical os. The pooled lavage was aspirated from the posterior fornix, emptied through the catheter into a 15-mL conical polypropylene tube, and transported on ice to the laboratory within the same day in all cases (if possible, transport to the laboratory occurred within 4 hours of collection).
Selection of cases and control subjects In the previously described nested casecontrol WITS study that assessed the effect of genital tract HIV viral load status on perinatal HIV transmission,6 the cases were 26 HIV-1–infected women who delivered between June 1995 and July 1999, who transmitted HIV-1 to their singleton infants, and who had at least 1 antepartum CVL specimen obtained. The control subjects were 52 HIV-1–infected pregnant women from the same time period who did not transmit HIV-1 to their infants. Control subjects were frequency matched by clinical center to the case subjects in a 2:1 ratio.
Testing for HSV-2 by serologic evaluation Plasma that was obtained at enrollment was tested by indirect enzyme-linked immunosorbent assay (ELISA) for the qualitative detection of HSV-2 type-specific immunoglobulin G antibodies (HerpeSelect HSV-2 ELISA IgG; Focus Diagnostics, Cypress, CA), because typespecific ELISA immunoglobulin G has a higher sensitivity and specificity than the type-specific glycoprotein G immunoassay.14 In addition, the US Food and Drug Administration has approved the Focus
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Diagnostics ELISA for use in pregnant women. The sensitivity for HSV-2 detection in pregnant women is 100%, and the specificity is 96.1%. Two ELISA runs were performed for each plasma sample. Plasma with either equivocal results in both runs or discordant results underwent additional testing for HSV-2 by a rapid type specific glycoprotein G immunoassay kit (Sure-Vue HSV-2; Biokit USA, Lexington, MA). Confirmatory Biokit testing of ELISA results is effective in reducing false-positive HSV-2 results.15 All serologic assays were performed by one of the authors who was blinded to case or control status. If the study subject was HSV-2 seronegative at enrollment, an additional plasma sample that had been obtained at delivery was tested to detect seroconversion to HSV-2 during pregnancy. If the subject was HSV-2 seronegative at enrollment and at delivery, another plasma sample, which was obtained from the earliest postpartum visit, was tested to detect seroconversion to HSV-2 at the time of delivery or early postpartum period.
Testing for presence of HSV-2 DNA Among the study subjects who were found to be HSV-2 seropositive, the presence of HSV-2 DNA in CVL specimens that were obtained closest to delivery was assessed. Quantification of HSV-2 DNA CVL specimens was determined by one of the authors who was blinded to the case or control status. The procedure uses a high-throughput, semiautomated, quantitative fluorescent-based polymerase chain reaction technique as previously described.16-18 If the CVL specimen of a study subject contained HSV-2 DNA, the study subject was considered to have the presence of HSV-2 DNA in the genital tract.
Statistical methods Given the sample size of 26 cases and 52 control subjects, we calculated a priori that we would have ⬎ 80% power to detect differences in the percentage of those with serologic evidence of HSV-2 in either scenario: 95% cases and 70% control subjects were HSV-2 seropositive or
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www.AJOG.org 75% cases and 43% control subjects were HSV-2 seropositive. We also calculated that we would have ⬎ 80% power to detect differences in the percentage of those with genital tract evidence of HSV-2 in a range of scenarios from 50% of cases positive for HSV-2 DNA as compared with 15% of control subjects to 25% of cases positive for HSV-2 DNA as compared with 2% of control subjects. Chi-square statistics were used in the analysis of categoric variables (eg, HSV-2 serostatus or presence of HSV-2 DNA) and Wilcoxon rank sum tests were used in the analysis of continuous variables (eg, HIV viral loads). Results were considered significant at P ⬍ .05; Fisher’s exact test probability values were reported when cells had expected counts ⬍ 5. Analyses were performed with SAS software for Unix (v 9.0; SAS Institute Inc, Cary, NC).
R ESULTS As previously reported, cases were significantly less likely than control subjects to have been treated with combination antiretroviral therapy, significantly more likely to have a detectable plasma HIV-1 RNA level at delivery, significantly more likely to have a higher cell CVL pellet HIV-1 DNA level, tended to have a lower mean CD4 count at delivery, and no difference in CD4 cell percentage at time of delivery (data not shown).6
HSV-2 serostatus Among the 78 study subjects, 65 women (83.3%) were found to be HSV-2 seropositive. Nineteen of the 26 cases (73.1%) were HSV-2 seropositive, and 18 of the 19 women were HSV-2 seropositive at enrollment. One of the 19 cases seroconverted around the time of delivery or the early postpartum period because this study subject was initially HSV-2 seronegative at enrollment and at delivery and then found to be HSV-2 seropositive at the 8-week postpartum visit. Forty-six of the 52 control subjects (88.5%) were HSV-2 seropositive at enrollment, and none of the control subjects seroconverted. There was no significant association between serologic evidence of HSV-2 coinfection in the an-
tepartum period and the risk of perinatal HIV-1 transmission (odds ratio, 0.4; 95% CI, 0.1-1.2; P ⫽ .1). HSV-2 seropositive subjects did not differ from HSV-2 seronegative subjects in plasma HIV-1 RNA level at delivery or CVL cell pellet HIV-1 DNA level (data not shown).
Presence of HSV-2 DNA Seventeen of the 19 HSV-2 seropositive cases (90.0%) and 40 of the 46 HSV-2 seropositive control subjects (86.9%) had CVL available from the repository for HSV-2 DNA analysis. The time at which CVLs were obtained in the antepartum period ranged from 10-32 weeks of gestation, with a mean of 16.4 weeks of gestation. None of the 17 HSV-2 seropositive cases and 2 of the 40 HSV-2 seropositive control subjects (5.0%) were positive for HSV-2 DNA. There was no association between genital tract evidence of HSV-2 coinfection in the antepartum period and risk of perinatal HIV-1 transmission (odds ratio, 0.4; 95% CI, 0.02-10.0; P ⫽ 1.0).
HSV-2 treatment Among the 65 study subjects who were HSV-2 seropositive, 6 women (9.2%) had anti-HSV treatment at any time during pregnancy (1 case and 5 control subjects). There was no difference between anti-HSV treatment and risk of HIV-1 transmission in the HSV-2 seropositive subjects (odds ratio, 0.5; 95% CI, 0.014.6; P ⫽ .7). Among the 6 HSV-2 seropositive subjects who had anti-HSV treatment at any time during pregnancy, all 6 women were negative for HSV-2 DNA in CVL.
HSV-2 neonatal infection In this WITS nested case-control study, there were no cases of HSV-2 neonatal infection.
C OMMENT Given the potential role of HSV-2 in enhancing sexual transmission of HIV-1, we hypothesized that coinfection with HSV-2 might increase perinatal HIV-1 transmission because infection with HSV-2 could increase plasma and genital tract HIV-1 viral loads. A previous study
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showed that a clinical diagnosis of genital HSV infection during pregnancy in a multicenter New York City cohort (most of the diagnoses were based on culture confirmation of HSV) significantly increased the risk of perinatal HIV-1 transmission.12 In our nested case control study from the WITS cohort, we found no association between serologic evidence of HSV-2 coinfection in the antepartum period and the risk of perinatal HIV-1 transmission. Among those HIV1–infected women who were HSV-2 seropositive, we found no association between the presence of genital tract HSV-2 in the antepartum period and risk of perinatal HIV-1 transmission. Our finding that HIV-1–infected pregnant women have a high prevalence of HSV-2 infection is consistent with another study of HIV-1–infected pregnant women in labor in the United States.19 This high HSV-2 seroprevalence in HIV1–infected pregnant women is in contrast to the prevalence of 30% reported in the general female population20 and 20% in pregnant women21 in the United States. Serologic evidence of HSV-2 infection is more accurate than self-report or clinical diagnosis because most individuals with HSV-2 infection are unaware of their status20 and because most HSV-2 infections do not cause the classic, visible ulcerative lesions that are recognized easily as genital herpes infection.22 Because we found such a high prevalence of HSV-2 coinfection in both cases and control subjects, we had only 40% power to detect a meaningful difference with our sample size. Because the potential mechanism of increased perinatal HIV-1 transmission with HSV-2 coinfection might be enhancement of plasma and genital tract HIV-1 viral load during an HSV-2 reactivation, we attempted to detect HSV-2 active replication by determining the presence of HSV-2 DNA in the genital tract from CVL specimens. However, we did not have CVLs available for analyses from every study subject. We did find 2 of the 57 CVL specimens (3.5%) to be positive for HSV-2 DNA, which is a rate that is lower than the 9.0% rate from the Kenyan study.13 The other limitation to the CVL specimens is that they were ob-
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tained in the antepartum period and not at the time of delivery. Genital HSV infection during the latter time period is likely to be a more important clinical predictor of perinatal HIV-1 transmission. The risk of seroconversion to HSV-2 among a general population of pregnant women is reported to be 1.7%.23 However, among HSV-2 seronegative women with HSV-2 seropositive partners, the risk is 20%.24 We had 1 HIV-1–infected study subject who seroconverted to HSV-2 around the time of delivery or the early postpartum period and who transmitted HIV to her infant. This study subject was initially HSV-2 seronegative at enrollment and at delivery and then found to be HSV-2 seropositive at the 8-week postpartum visit. The usual time from HSV-2 infection to the development of detectable antibodies is 2-12 weeks.25 We do not know whether the study subject had a primary HSV-2 infection or nonprimary first episode because we do not know her previous HSV-1 status. In addition, we do not have CVL specimens during pregnancy and at delivery for this study subject to test for the presence of HSV-2 DNA. The current study presents laboratory evidence of HSV-2 coinfection but is lacking in information about clinical episodes of HSV-2. Perhaps detectable clinical episodes of HSV-2 coinfections such as genital ulcers may lead to a mucosal milieu that favors perinatal HIV-1 transmission by recruitment of HIV-1– infected CD4⫹ cells into mucosal ulcerations.26 HIV-1 RNA can be detected consistently in genital ulcers that are caused by HSV-2.8 This study also did not evaluate HSV-1 coinfection because genital tract infections are more likely to occur with HSV-2 instead of HSV-1. Although we did not find an association between serologic and genital tract evidence of HSV-2 coinfection and the risk of perinatal HIV-1 transmission, we believe that further investigations that would assess (1) both clinical episodes and laboratory evidence of HSV-2 reactivation at the time of delivery, (2) their relationship to plasma and genital HIV-1 viral loads, and (3) the risk of perinatal 399.e4
www.AJOG.org HIV-1 transmission are needed. The use of potent combination antiretroviral therapy to reduce HIV-1 viral loads to undetectable levels is the most effective strategy to decrease perinatal HIV-1 transmission. However, in low resource settings in which highly active antiretroviral therapy is not available routinely for the prevention of perinatal HIV-1 transmission, the addition of a short-course treatment of HSV-2 coinfection around the time of delivery may provide another strategy to reduce perinatal HIV-1 transmission. Of note, a recent randomized double-blind, placebo-controlled trial of HSV suppressive therapy in coinfected women in Burkina Faso, West Africa, showed a significant reduction of genital and plasma HIV-1 viral loads.27 Our work suggests a need for a more complete evaluation of HSV-2 during pregnancy to gain insight into the interactions of HIV-1 and HSV-2, specifically regarding the risks of transmission and the course of disease of f both infections. ACKNOWLEDGMENTS Funding to the Women and Infants Transmission Study Principal investigators, study coordinators, and program officers is as follows: Clemente Diaz, Edna Pacheco-Acosta (University of Puerto Rico, San Juan, PR; U01 AI 034858); Ruth Tuomala, Ellen Cooper, Donna Mesthene (Boston/Worcester Site, Boston, MA; 9U01 DA 015054); Philip LaRussa, Alice Higgins (Columbia University, New York, NY; U01 DA 015053); Sheldon Landesman, Herman Mendez, Ava Dennie (State University of New York, Brooklyn, NY; U01 HD 036117); Kenneth Rich, Delmyra Turpin (University of Illinois at Chicago, Chicago, IL; U01 AI 034841); William Shearer, Norma Cooper (Baylor College of Medicine, Houston, TX; U01 HD 041983); Joana Rosario (National Institute of Allergy and Infectious Diseases, Bethesda, MD); Kevin Ryan, (National Institute of Child Health and Human Development, Bethesda, MD); Vincent Smeriglio, Katherine Davenny (National Institute on Drug Abuse, Bethesda, MD); and Bruce Thompson (Clinical Trials & Surveys Corp., Baltimore, MD, N01 AI 085339). The scientific leadership core includes Kenneth Rich (PI), Delmyra Turpin (Study Coordinator; 1 U01 AI 05027401). Additional support was provided by local Clinical Research Centers: Baylor College of Medicine, Houston, TX (NIH GCRC RR000188); Columbia University, New York, NY (NIH GCRC RR000645).
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