Sarah Hawkes, Paz Bailey G, Sternberg M, Lewis DA and Puren A Acute HIV infections among men with genital ulcer disease in South Africa Sarah Hawkes, Paz Bailey G, Sternberg M, Lewis DA and Puren A 1. London School of Hygiene and Tropical Medicine; 2. NCHHSTP, CDC US; 3. STI Reference Centre, NICD 4. Specialized Molecular Diagnostic Unit, NICD, South Africa;

Rationale for study Genital ulcer disease (GUD) increases the risk of HIV acquisition and transmission Cohort studies and mathematical modeling show that the rate of HIV transmission is higher after acute HIV infection Most common method to diagnose acute HIV infection is documenting seroconversion (diagnosis in retrospect) or diagnosis in real time among HIV seronegative individuals using HIV nucleic acid amplification Numerous studies have identified higher relative risks of HIV infection with genital ulcer disease (GUD) than with other sexually transmitted infections (STI) [2-4]. In terms of HIV acquisition, GUD has been associated with a seven-fold to eleven-fold excess risk of HIV acquisition in cohort studies [3, 5, 6]. In terms of HIV transmission, HIV virus has been detected in genital ulcers. Analysis from Rakai highlighted the importance of recently acquired HIV for transmission among discordant couples. They found that the rate of HIV transmission within 2.5 months after seroconversion was 12 times higher compared to discordant couples with HIV-prevalent index partners. Mathematical modeling also suggests that, the very high viral load in semen that has been demonstrated in patients with acute HIV infection would result in an 8-10 fold increase in the risk of male-to-female transmission. The most common method to diagnose acute HIV infection is documenting antibody seroconversion (making the diagnosis in retrospect). Two types of results are needed to diagnose acute HIV infection in real time. The first is a positive screening test result, indicating HIV infection. This can be HIV nucleic acid amplification, HIV p24 antigen, HIV antibody, or combined p24 antigen-antibody screening test.

Objectives of the trial To determine the prevalence of acute HIV among men with genital ulcer disease To evaluate risk factors for acute HIV infection We investigated the prevalence of acute HIV among a series of patients with genital ulcer disease enrolled on a randomized controlled trial on the impact of acyclovir therapy in South Africa. The objectives of the study were to determine the prevalence of acute HIV among men with GUD and evaluate risk factors for acute HIV.

Methods At enrolment tested for HSV2, HIV, syphilis, GUD aetiology Baseline and follow-up data from a double blind randomized placebo controlled trial of acyclovir 400mgs TID for 5 days versus placebo Men with GUD presenting at three clinics in South Africa were enrolled after consent All patients received syndromic management for syphilis and chancroid Behavioral questionnaire was administered At enrolment tested for HSV2, HIV, syphilis, GUD aetiology Data for this study, comes form an individually randomized double blind placebo-control trial of the addition of Acyclovir. Patients presenting with genital ulcers were recruited at two primary health care clinics in Johannesburg and one in Pretoria. All patients received single dose therapy for GUD with benzathine penicillin 2.4 MU IM and ciprofloxacin 500 mg PO. At baseline, a questionnaire was administered to collect information on demographics, sexual behavior, history of a sexually transmitted infection (STI), and clinical findings. Blood samples were collected to test for HIV, HSV2 and syphilis by serology. Ulcer etiology was determined with a real time multiplex PCR (M-PCR) for Treponema pallidum, Haemophilus ducreyi and HSV and in-house PCR for Chlamydia trachomatis L1-L3.

Methods (continued) HIV-negative men tested again for HIV at day 28 to detect HIV seroconversions HIV+ tested for CD4, HIV RNA in plasma and ulcers, and HSV2 in ulcers HIV-1 antibody negative serum were tested by HIV RNA PCR using the COBAS AmpliScreen HIV-1 Test v.1.5 (lower limit of detection 100 copies/ml) to detect acute HIV Assay was used on initial pools of 6 samples, if positive individual samples tested HIV-negative participants were asked to return on day 28 for a second HIV test. HIV-positive participants diagnosed at baseline or day 28, we also tested for CD4 counts and HIV-1 plasma viral loads. Stored serum was tested to detect acute HIV infections. All antibody negative were tested by HIV RNA PCR. The COBAS AmpliScreen HIV-1 Test v.1.5 was used for detection of HIV-1 RNA in pooled samples. The assay was used on initial primary pools of 6 samples. Positive pools were deconstructed and individual samples tested. The lower limit of detection for a positive sample in a pool of 6 was 100 copies HIV-1 RNA/ml.

Definitions and analysis HIV-1 seroconverters: HIV antibody negative at baseline and HIV antibody positive by two rapid tests at follow-up on day 28 HIV prevalent: 1) two concordant positive rapid antibody tests or 2) discordant rapid antibody tests with positive ELISA Acute HIV: 1) Antibody rapid test was negative and HIV RNA results were positive or 2) two rapid tests discordant, ELISA negative and HIV RNA positive and 3) HIV seroconverters as described above HIV prevalent cases and HIV acute cases were compared to HIV negative participants to detect risk factors For this analysis, HIV prevalent cases and HIV acute cases were compared to HIV negative participants to detect variables associated to prevalent and acute infection. HIV-1 seroconverters were defined as HIV antibody negative at baseline and HIV antibody positive by two rapid tests at follow-up on day 28. If discordant on rapid tests at baseline, an ELISA screen test was conducted and two ELISA confirmatory tests if the screen test is positive. If positive the patient classified as HIV prevalent infection. If the ELISA test was negative the patient was advice to be retested in 4 weeks. Prevalent HIV infection was defined as either 1) two concordant positive rapid antibody tests or 2) discordant rapid antibody tests with a positive ELISA test. On the stored serum an HIV RNA PCR test was ran. If positive the case was classified as acute HIV. Acute HIV was considered to be present if: 1) antibody rapid test was negative and HIV RNA results were positive or 2) two rapid tests discordant, ELISA negative and HIV RNA positive and 3) HIV seroconverters as described above. Individuals with negative or discordant antibody results and negative RNA were considered to be uninfected.

Results 615 men enrolled in the trial 63% HIV positive 36.5% had been tested for HIV before and only 20.7% HIV-positive knew their status No signs or symptoms of HIV seroconversion Of 228 HIV-negative men at baseline, there were 8 seroconversions at day 28 and 20 HIV RNA positive specimens Prevalence of acute HIV overall 4.6% (95% CI: 2.9 – 6.2, 28/615) Prevalence of acute HIV among HIV-negative 12.2% (95% CI: 8.0 – 16.5, 28/228) Among the 615 men enrolled in the trial, 63.0% tested positive for HIV. Overall, 36.5% reported having been tested for HIV before and only 80 (20.7%) of the HIV-positive men knew their status. There were a total of 228 HIV-negative men at baseline. Of these 8 tested positive for HIV-1 by rapid tests at day 28 and were classified as HIV-1 seroconverters. Baseline and day 28 serum for the remaining 220 HIV-seronegative men was tested by HIV-1 PCR to detect acute infections. There were a total of 20 HIV RNA positive specimens. All of these were positive at baseline. These preliminary results suggest that the prevalence of acute HIV in the overall study population was 4.6% (95% CI: 2.9 – 6.2) based on 28/615 (20 by HIV PCR and 8 seroconverters). The prevalence of acute HIV among HIV seronegative men at baseline was 12.2% (95% CI: 8.0 – 16.5) or 28/228.

Study flowchart 615 men with GUD 387 positive by HIV rapid tests 228 negative by HIV rapid tests 8 sero-conversions at day 28 20 HIV RNA positive 200 HIV negative* 387 prevalent HIV infections 28 acute HIV infections

Results-Bivariate Analysis No. of men Acute HIV Prevalent HIV HIV - Chi square Characteristics N n (%) p-value Number of casual partners last three months 0-5 417 11 (2.6) 275 (66.0) 131 (31.4) 6+ 196 17 (8.7) 111 (56.6) 68 (34.2) 0.002 Circumcision No 249 8 (3.2) 175 (70.3) 66 (26.5) Yes 49 1 (2.0) 25 (51.0) 23 (46.9) 0.017 HSV-2 serology Negative 181 13 (7.2) 67 (37.0) 101 (55.8) Positive 434 15 (3.5) 320 (73.7) 99 (22.8) <0.001 First episodes of genital herpes 479 19 (4.0) 332 (69.3) 128 (26.7) 136 9 (6.6) 55 (40.4) 72 (52.9) Ulcer aetiology Unknown etiology 126 15 (11.9) 72 (57.1) 39 (30.9) TP, HD, CT and mixed etiology 48 4 (8.3) 24 (50.0) 20 (41.6) HSV only 439 9 (2.1) 291 (66.3) 139 (31.7) In unadjusted analysis, HIV acute infections were associated with a higher number of casual partners in the last three months. Both prevalent and acute infections were more common among uncircumcised men (p=0.02). Prevalent HIV was more common among HSV-2 seropositive men. By contrast acute infections were more frequent among first episodes of genital herpes (HSV-2 sero-negative but HSV-2 virus isolated from the ulcer). Acute infections were more common among men with ulcers of unknown etiology (p<0.001). Other factors associated with acute and prevalent HIV not presented in this table were age, education, and marital status. JUST FOR REFERENCE In the unadjusted analysis, the prevalence of acute HIV infections was higher among the youngest age group while the proportion of HIV prevalent infections increased with age (p<0.001) (Table 2). HIV acute infections were more common among men with high-school education, while HIV prevalent infections decreased with increasing education (p=0.002). HIV acute infections were more common among single men and prevalent HIV was higher among widowed or divorced men (p=0.001).

Acute and Prevalent HIV versus HIV negative-Multinomial regression Characteristics OR (95%CI) P-value (2 sided) Number of casual partners last three months None Reference One or more Acute 3.0 (1.3-6.7) 0.01 Prevalent 0.9 (0.6-1.3) 0.48 Ulcer etiology TP, HSV HD, CT and mixed etiology Unknown 5.0 (2.1-11.6) <0.001 0.8 (0.5-1.3) 0.46 HSV-2 serology Negative Positive 1.4 (0.6-3.2) 0.49 3.9 (2.6-5.8) Note: Adjusted by age, prevalent HIV associated with older age (p<0.001)

Conclusions High prevalence of acute HIV among men with GUD in South Africa The proportion of acute HIV was 4.6% among all study participants and 12.2% among persons without established HIV infection, higher to what has been reported for other groups HIV prevalent infections were associated with older age and HSV-2 seropositivity Acute HIV was associated with casual partners and ulcers of unknown etiology GUD patients represent an opportunity to identify acute infections and initiate interventions that may reduce the chances of HIV-1 transmission The 8 sero-conversions documented during the study plus the 20 cases of acute HIV diagnosed by RNA detection, are evidence of a very high prevalence of acute HIV among this group. The proportion of acute HIV was 4.6% among all study participants and 12.2% among persons without established HIV infection. This is higher to what has been reported among STI populations in the United States (0.03%) and from STI clinics in Malawi (1.5%-2.5%). We found that HIV prevalent infections were associated with older age and HSV-2 seropositivity. Acute HIV infections were more common among men with casual partners in the last three months and with ulcers of unknown etiology. GUD patients represent an opportunity to readily identify acute infections and initiate care and prevention interventions that may reduce the chances of HIV-1 transmission and provide clinical benefit to the patient.

Collaborators National Institute of Communicable Diseases in South Africa US Centers for Disease Control and Prevention London School of Hygiene and Tropical Medicine Reproductive Health and HIV Research Unit Special thanks to participating clinics and study participants