Circumcision Status and Incident HSV-2 Infection , Genital Ulcer Disease , and HIV Infection 1

Word Count = 243) Objective: We assessed the protective effect of medical male circumcision (MMC) against HIV, HSV-2, and GUD incidence. Design: 2784 men aged 18–24 years living in Kisumu, Kenya were randomly assigned to circumcision (n=1391) or delayed circumcision (n=1393), and assessed by HIV and HSV-2 testing and medical examinations during follow-ups at 1, 3, 6, 12, 18, and 24 months. Methods: Cox regression estimated the risk ratio (RR) of each outcome (incident HIV, GUD, HSV-2) for circumcision status and multivariable models estimated HIV risk associated with HSV-2, GUD and circumcision status as time-varying covariates. Results: HIV incidence was 1.42 per 100 person-years. Circumcision was 62% protective against HIV [RR=0.38; 95% CI: 0.22 0.67], and did not change when controlling for HSV-2 and GUD [RR=0.39; 95% CI: 0.23 0.69]. GUD incidence was halved among circumcised men [RR=0.52, 95% CI: 0.37 0.73]. HSV-2 incidence did not differ by circumcision status [RR=0.94; 95% CI: 0.70 1.25]. In the multivariable model, HIV seroconversions were tripled [RR=3.44; 95% CI: 1.52 7.80] among men with incident HSV-2 and 7 times greater [RR=6.98; 95% CI: 3.50 13.9] for men with GUD. Conclusion: Contrary to findings from the South African and Ugandan trials, the protective effect of MMC against HIV was independent of GUD and HSV-2 and MMC had no effect on HSV-2 incidence. Determining the causes of GUD is necessary to reduce associated HIV risk, and to understand how circumcision confers protection against GUD and HIV. Introduction Three randomized controlled trials in Africa demonstrated that adult medical male circumcision (MMC) is effective in reducing HIV acquisition by 50-60%. The mechanisms by which this is thought to occur include: reduction in HIV target cell populations through removal of mucosal foreskin, increased keratinization of penile skin other than the foreskin, and reduction of cofactors for infection, such as HSV-2 and genital ulcer disease (GUD). In the MMC trial in Rakai, Uganda, circumcision resulted in a 46% reduction in GUD and a 28% reduction in Herpes simplex virus type 2 (HSV-2) acquisition, similar to the 30% reduction in HSV-2 incidence observed among Orange Farm, South Africa, MMC trial participants. An estimated 11% of HIV acquisition among Rakai trial participants may have been due to symptomatic GUD, and among Orange Farm trial participants, authors estimated that 28% of HIV infections may have been due to HSV-2 infection. In the Orange Farm trial, the effect of MMC on HIV acquisition was not modified by HSV-2 status; whereas in the Rakai trial MMC was not protective against HIV for men who were HSV-2 infected at enrollment. We examined the effect of MMC on HSV-2 and GUD incidence, and whether HSV-2 or GUD modified the protective effect of MMC against HIV acquisition among participants of the randomized trial of MMC to reduce HIV incidence in Kisumu, Kenya. Methods The Kisumu trial enrolled 2,784 men aged 18-24 years. For inclusion men had to be: uncircumcised, HIV-negative, sexually active in the last 12 months, and aged 18-24 years; have a hemoglobin > 9·0 mmol/L; and reside in Kisumu District. Exclusion criteria included: foreskin covering less than half of the glans, a bleeding disorder, keloid formation, other conditions that might unduly increase the risks of elective surgery, or a medical indication for circumcision. Participants with sexually transmitted infections or other treatable medical conditions were deferred until treated. Trial recruitment, enrollment, reasons for refusing enrollment, and followup have been previously described. Following written informed consent, participants were randomized 1:1 to either immediate circumcision or delayed circumcision after a two-year follow-up period (the control group). Both groups underwent STI and HIV risk reduction counseling and were provided unlimited supplies of free condoms. Personal interview, medical examination, and laboratory testing for HIV were conducted at baseline, 1, 3, 6, 12, 18 and 24 months from randomization for both the circumcision and the control groups. The study was approved by the Institutional Review Boards of the University of Illinois at Chicago, the Kenyatta National Hospital, RTI International, and University of Manitoba, and was overseen by a Data and Safety Monitoring Board (DSMB). Detection of HSV-2 and Syphilis Specimens were collected for HSV-2 and syphilis testing at baseline, 6, 12, 18, and 24 months from randomization for all participants. Serum specimens were tested for HSV-2 antibody (Kalon HSV-2 IgG ELISA, Kalon Biological Limited, Aldershot, United Kingdom), using the manufacturer’s recommended cut-off. In men who were initially HSV-2 seronegative who seroconverted to HSV-2, the last available sample was tested, and then previous samples were tested to determine the visit at which HSV-2 seroconversion occurred. Syphilis infection was assessed at each study visit using the rapid plasma reagin test (RPR) (Macro-VueTM, Becton Dickinson, New Jersey, United States), confirmed by the Treponema pallidum hemagglutination (TPHA) assay (Randox Laboratories Ltd., Ardmore, United Kingdom). All genital ulcers were tested for Haemophilus ducreyi by culture. Testing was conducted at the study clinic and the University of Nairobi Department of Medical Microbiology research laboratory. A random sample of clinically identified genital ulcers were tested for H. ducreyi, T. pallidum and HSV (did not distinguish between type 1 and type 2) by multiplex polymerase chain reaction (PCR) at the University of Manitoba Department of Medical Microbiology research laboratory. Self-Reported and Physical Examination Findings of GUD All consenting participants underwent standardized medical examination and history at all planned study visits. Participants were asked about the presence of painless sores and painful sores occurring in the genital region in the past six months and at the current visit. At all planned study visits, all participants underwent genital examination by trained clinicians, who recorded the presence or absence of genital ulcers, and the location and number of ulcers. Due to the broad range of clinical presentation for ulcerative infections in the genital region, a restricted definition was not used and clinicians were instructed to record any epithelial defect in the skin or mucosa of the genitalia. HIV Testing Testing for HIV infection was conducted using a parallel double rapid test protocol, using Determine® HIV 1/2 (Abbott Diagnostic Division, Hoofddorp, The Netherlands), and the UniGold RecombigenTM HIV Test (Trinity Biotech, Wicklow, Ireland). Men who were concordant negative were eligible for the study. Concordant positive results were confirmed by double ELISA and men were informed of their HIV status and followed-up at the study clinic or at the New Nyanza Provincial Hospital. Men with discordant results were followed up with additional tests to determine their HIV status, but were not enrolled. For determination of HIV seroconversion for analysis, positive rapid test and ELISA test results were confirmed by Health Canada National HIV Reference Laboratory (Ottawa, Canada) by line immunoassay (INNO-LIA HIV 1/2, Immunogenetics NV, Ghent, Belgium). Specimens indeterminate by line immunoassay were tested by PCR at Health Canada or the Fred Hutchinson Cancer Research Center (Seattle, WA, USA), with the PCR result deemed to be definitive. At the time of the DSMB meeting that halted the trial and in our published primary results, we reported that 4 men had been found to be HIV positive at baseline after detailed testing. After the paper was published, it was determined that 2 of the men who were labeled HIV positive at baseline in the circumcision group were HIV negative (therefore needing to be included in the analysis as HIV negative). Also, it was determined that one of the men who had been labeled HIV positive in the circumcision group at 3 months was positive at baseline (therefore needing to be excluded from analyses of HIV seroconversion). Also, one subject in the control group labeled positive at 12 months was determined negative (therefore remaining in analysis, but with a different outcome). Our current analysis reflects these updated results, which have been previously presented. Statistical Analysis Incident HSV-2 was defined as detection of HSV-2 antibody subsequent to a negative HSV-2 antibody test result at enrollment. Incident syphilis was defined as having a positive RPR test with positive TPHA test, subsequent to a negative RPR and negative TPHA test at enrollment. Men with baseline syphilis infection (n=27) were excluded to eliminate any potential uncertainty that may have arisen from whether these were truly new infections or whether these were baseline infections that were treatment failures. Prior exposure to syphilis was defined as being RPR negative with a positive TPHA. Genital ulcer disease was defined as having physical examination findings, or a current or past 6 months complaint of genital sores. As exposures, incident syphilis, GUD, and HSV-2 were treated as time-varying states in Cox regression models. As outcomes, observation was censored at first incidence of syphilis or GUD and at HSV-2 seroconversion. HIV seroconversion was defined as concordant positive results from the parallel test protocol. Circumcision status was analyzed as a fixed covariate based on treatment group (intention to treat, ITT) and as a time-varying covariate by status (as treated, AT). Because this was a secondary analysis and the trial was not designed to examine HSV-2 and GUD as endpoints, we present the results of AT analysis. The interpretation of results of ITT and AT analyses did not differ and only AT results are presented [results of ITT analysis available from the authors]. Thus results refer to circumcision by status rather than assignment or individual. There were 2,784 men enroll