HUMAN IMMUNODEFICIENCY VIRUS type 1 (HIV-1) is spread primarily by heterosexual transmission in sub-Saharan Africa, where other sexually transmitted diseases (STDs) are also common.1 One explanation for the difference between the rate of heterosexual transmission of HIV-1 in Africa and the rates in industrialized countries is that other STDs act as cofactors facilitating the sexual transmission of HIV.2–4 Untreated STDs, especially genital ulcer disease (GUD), could increase both the susceptibility of uninfected persons to HIV infection and the infectivity of persons already infected with HIV.5 In two studies, HIV RNA was frequently detected in ulcer specimens6 and in urethral exudates from men7; those studies also reported a decreased rate of HIV RNA detection after successful treatment of urethritis and herpes simplex virus (HSV) infections.6,7 The biological mechanisms by which GUD and nonulcerative STDs facilitate the transmission of HIV remain to be fully delineated; however, macrodisruptions or microdisruptions of the epithelium and the presence of CD4+ inflammatory cells in these lesions are factors that may be involved in HIV transmission.8 Several studies conducted among African, Asian, and North American populations have supported the role of STDs as risk factors in HIV infection2,4,9–13; however, other studies have failed to show that ulcerative STDs14 or nonulcerative STDs15 increase the risk of HIV infection.
The primary etiologic agents of GUD among STD clinic attendees include Treponema pallidum, Haemophilus ducreyi, and herpes simplex virus type 2 (HSV-2); however, the proportion of genital ulcers that are caused by each of these agents varies geographically and temporally.16–18 In the United States, HSV-2 is probably the most common cause of genital ulcers, with one in five persons 12 years of age or older HSV-2-seropositive19; however, results of several studies conducted in South Africa during the 1980s suggest that HSV is not a major cause of GUD in men.20 For example, Coovadia et al21 isolated HSV from only 9% of ulcers in men attending an STD clinic in Durban in 1984. Another cross-sectional study conducted in Durban between 1988 and 1989 revealed a similar prevalence of HSV among men with GUD.3 In a study by Dangor et al22 HSV was isolated from 3.3% of genital ulcers in mineworkers attending a clinic in Carletonville, South Africa in 1989. The low isolation rates in previous studies may have reflected problems with specimen transport and culture methods; a more recent study in southern Africa that used a multiplex polymerase chain reaction assay (M-PCR) showed a higher prevalence of HSV among patients with GUD.23
The development of nucleic acid amplification technologies has facilitated the establishment of a definitive diagnosis in cases of GUD. A M-PCR amplification assay that can simultaneously detect the presence of HSV, H ducreyi, and T pallidum in a single ulcer specimen has been developed, and is more sensitive than conventional laboratory tests used for the diagnosis of GUD.18 Additionally, type-specific serologic assays for herpes simplex virus type 1 (HSV-1) and HSV-2 have been developed using glycoprotein G-1 (gG-1) and G-2 (gG-2) as the target antigens.24 Population-based seroprevalence studies have indicated that HSV-2 infections are widespread, but that the great majority of people with serologic evidence of HSV-2 infection have no history of genital herpes.19,25 Recent studies have reported a positive association between serologic evidence of HSV-2 infection and HIV infection in African populations.11,23,26
The purpose of this study was to determine (1) the etiology of GUD in HIV-infected and HIV-uninfected men attending STD clinics in Durban (DBN), Johannesburg (JHB), and Cape Town (CT), South Africa; and (2) the association of previous and current sexually transmitted infections with HIV infection in men with ulcerative and nonulcerative STDs. Our results demonstrate that HSV-2 infection has emerged as a major cause of GUD in South African men, and is strongly associated with HIV seropositivity.
Materials and Methods
Patients and Definitions for Clinical Diagnoses
Genital ulcer and serum specimens were obtained from approximately 200 consecutive male patients with GUD presenting at STD clinics DBN, JHB, and CT between October 1993 and January 1994. Serum specimens were also collected from consecutive non-GUD patients with urethritis presenting at each of these clinics during this period. Written informed consent was obtained from all patients, and guidelines of the Department of Health, South Africa were followed in the conduct of this study.
The criteria used for clinical diagnoses were defined prospectively and have been described previously.23 A diagnosis based on clinical findings was made before knowledge of the results of microscopic or laboratory tests, and patients were treated according to established guidelines.27
Specimen Collection and Laboratory Tests
Specimens were collected from the base of each ulcer with a cotton-tipped swab for use in the M-PCR assays, which were performed at the Centers for Disease Control and Prevention (CDC). When multiple ulcers were present, the specimen was collected from the largest ulcer. The swab was agitated either in 0.2 ml sterile distilled water or in 1 ml of AMPLICOR™ Specimen Transport Medium (Roche Diagnostic Systems, Branchburg, NJ) and discarded.23 Specimens were stored frozen at −70 °C until analyzed. Additional specimens were obtained for conventional laboratory testing, including darkfield microscopy and culture for H ducreyi and HSV.23
Serum specimens obtained by venipuncture were tested by the quantitative rapid plasma reagin test (RPR; Omega Diagnostics, Alloa, UK) and the fluorescent treponemal antibody absorption test (FTA-ABS; Murex, Dartford, UK). Human immunodeficiency virus serology was determined by enzyme-linked immunoabsorbent assay (ELISA; Abbott Laboratories, Abbott Park, IL), with low positive results confirmed by Western blot assays (HIV blot 2.2; Diagnostic Biotechnology, Singapore) and high positive results confirmed by a direct fluorescent antibody test (DFA; Serofluor; Virion, Ruschlikon, Switzerland).
Herpes simplex virus type-specific antibodies were determined at CDC as described previously using Western blot analysis of recombinant-derived, baculovirus-expressed HSV gG1 and gG2 from HSV-1-infected and HSV-2-infected Sf9 cells, respectively.24,25
Polymerase Chain Reaction Amplifications
Swab specimens were tested by M-PCR as described previously,18 except that 10 U of Taq polymerase (Ampli Taq; Perkin-Elmer, Norwalk, CT) was used, and PCR inhibition was analyzed using an internal control plasmid. Precautions were used to prevent contamination. Specimens containing PCR inhibitors were treated as previously described23; HSV amplified products were typed using immobilized type-specific capture probes.28 Classification of lesions with positive M-PCR for HSV as primary, initial nonprimary, or recurrent genital herpes was based on HSV serology and DNA typing results29; the lesion was classified as primary if antibodies to HSV-1 and HSV-2 were both absent, initial nonprimary if there were antibodies to HSV-1 but not to HSV-2, and recurrent if antibodies to HSV-2 were present.
Demographic and clinical data and results of laboratory tests were abstracted onto a standardized form and analyzed using EpiInfo Version 6 (CDC, Atlanta, GA).30 Prevalence rate ratios (RR) were calculated as estimates of relative risk, and 95% CI and P values were reported. Continuous variables were compared using the Kruskal-Wallis test.
Study Population Characteristics
A total of 558 GUD patients (198 from CT, 160 from JHB, and 200 from DBN) and 603 urethritis patients (196 from CT, 206 from JHB, and 200 from DBN) were enrolled in the study. Overall, the mean ages of men presenting with GUD and urethritis were 26.3 years (range 15-65 years) and 26.9 years (range 16-54 years), respectively. All enrolled patients were African.
The majority of patients tested (98.5%) had antibodies to HSV-1, as indicated by IgG reactivity with gG-1 on Western blot analysis; HSV-1 seroprevalence was similar in men with GUD and men with urethritis (98.2% versus 98.7%). Examination of the age-specific seroprevalence rates suggested that HSV-1 was acquired at a young age, because the seroprevalence rates approached 100% among men 15-19 years presenting with either syndrome (Figure 1).
The overall seroprevalence of antibodies to HSV-2, as indicated by IgG reactivity with gG-2 on Western blot analysis, was 45.4% (570 of 1052 patients). Men with GUD were more likely to be HSV-2 seropositive than men with urethritis (245 of 498 patients, 49.2% versus 237 of 554 patients, 42.1%; RR = 1.16, 95% CI 1.01, 1.37; P = 0.04). The age-specific seroprevalence rates for HSV-2 increased with age, and appeared to stabilize in GUD and urethritis patients older than 30 years (Figure 1).
The overall HIV seroprevalence rate was 30.1% (344 of 1143 patients). Men with GUD were significantly more likely to be HIV seropositive than men with urethritis (220 of 558 patients, 39.4% versus 129 of 603 patients, 21.4%; RR = 1.84, 95% CI 1.53, 2.22; P ≤ 0.001). The age-specific HIV seroprevalence rates differed significantly between patients with GUD and patients with urethritis (Figure 1) and between patients in different cities (Figure 2). The HIV seroprevalence rates were 1.3-fold to 6.5-fold greater among men with GUD than among men with urethritis in all age groups examined.
The overall FTA-ABS positivity rate was 37.5% (388 of 1035 patients); patients with GUD were more likely to have a positive FTA-ABS test result than patients with urethritis (257 of 534 patients, 48.1% versus 131 of 501 patients, 26.1%; RR = 1.84, 95% CI 1.55, 2.18; P ≤ 0.001). If GUD patients with lesion-positive syphilis, as defined by a positive M-PCR result for T pallidum, were excluded from the analysis, the association of a positive FTA-ABS test result with GUD patients remained significant (158 of 428 patients, 36.9%; RR = 1.41, 95% CI 1.16, 1.71; P ≤ 0.001). A similar percentage of HSV-2-seropositive and HSV-2-seronegative men had a positive FTA-ABS test result (38.5% versus 37.1%); however, among patients with a positive FTA-ABS test result, 47.6% were HSV-2 seropositive. This percentage did not change significantly when patients with current syphilis were excluded from the analysis.
Etiology of Genital Ulcer Disease
In the majority of GUD patients (538 of 558 patients, 96%), ulcer etiology was examined by M-PCR (Table 1). The most common agent identified in ulcer specimens was HSV (193 of 538 patients, 35.9%); HSV DNA was detected in 157 of 376 (41.8%) ulcers from which a single agent was identified, and in 36 of 45 (80%) ulcers containing multiple agents. The proportion of GUD patients with HSV varied significantly by city; 43 of 180 patients (23.9%) in CT, 72 of 199 patients (36.2%) in DBN, and 78 of 159 patients (49.1%) in JHB (P ≤ 0.001). The HSV was typed by PCR, and all HSV-positive lesions contained HSV-2 DNA.
Genital herpes was clinically diagnosed in only 29.5% of men with genital ulcers that were M-PCR-positive for HSV. Among men with GUD, genital herpes was more likely to be clinically diagnosed in those who were infected with HIV than in those who were not infected with HIV (36.3% versus 22%; RR = 1.65, 95% CI 1.04, 2.63; P ≤ 0.001). In GUD patients with HSV-positive lesions, chancroid was the most common clinical diagnosis (37.3%), occurring more often in patients infected with HIV than in patients not infected with HIV; however, this difference was not statistically significant (44% versus 29.7%; P = NS).
The presence of H ducreyi DNA was detected by M-PCR in 171 of 538 (31.2%) specimens, including 132 of 538 (24.5%) ulcers that contained a single agent and 39 of 45 (86.7%) ulcers that contained more than one agent. The prevalence of H ducreyi varied significantly among the city locations; 17.2% in CT, 21.4% in JHB, and 53.3% in DBN (P ≤ 0.001) (Table 1). The presence of T pallidum DNA was detected by M-PCR in 106 of 538 (19.7%) ulcer specimens, including 87 of 376 (23.1%) specimens that contained a single agent and 19 of 45 (42.2%) specimens containing more than one agent. The prevalence of T pallidum also varied significantly by city; 11.9% in JHB, 14.6% in DBN, and 32.2% in CT (P ≤ 0.001).
Forty-five of 538 (8.3%) patients with GUD had multiple agents; 33 of 199 patients (16.1%) in DBN, 6 of 180 patients (3.4%) in CT, and 6 of 159 patients (3.8%) in JHB. The proportion of patients with an indeterminate diagnosis by M-PCR was 21.7% (117 of 538 patients), which varied significantly by city; 30.0% in CT, 21.4% in JHB, and 14.6% in DBN (P ≤ 0.001) and was highest in the city with the lowest HIV prevalence rate (CT) (Table 1, Figure 2). Among patients with an indeterminate diagnosis, 52 of 104 (50%) had antibodies to HSV-2, and 50 of 117 (42.7%) had a reactive FTA-ABS test result.
Relationship Between HIV Infection and GUD Etiology
Herpes simplex virus type 2 DNA was detected in a significantly higher proportion of ulcer specimens from HIV-seropositive patients than from HIV-seronegative patients (102 of 215 patients, 47.4% versus 91 of 323 patients, 28.2%; RR 1.68, 95% CI 1.35, 2.11; P ≤ 0.001), whereas T pallidum DNA was detected significantly less frequently in ulcer specimens from HIV-seropositive patients than from HIV-seronegative patients (22 of 215 patients, 10.2% versus 84 of 323 patients, 26%; RR = 0.39, 95% CI 0.25, 0.6; P ≤ 0.001). No association was found between M-PCR-positive results for H ducreyi and HIV seropositivity (74 of 215 patients, 34.3% versus 97 of 323 patients, 30%; RR = 1.15; P = NS). The associations between GUD etiology and HIV seropositivity were similar when patients who had more than one agent detected in their ulcers (as determined by M-PCR) were excluded from the analysis.
Relationship Between HSV and HIV Infections
Serum specimens for type-specific antibody determinations were available from 172 of 193 (89%) GUD patients with a positive M-PCR assay for HSV. Of the 172 patients with HSV-positive M-PCR results, 89 (51.7%) had antibodies to HSV-1 only, 81 (47.1%) had antibodies to both HSV-1 and HSV-2, and only 2 patients (1%) lacked antibodies to either virus. There was no difference in the mean ages of the HIV-uninfected or HIV-infected men who were experiencing recurrent or initial nonprimary HSV-2 infections. Recurrent HSV-2 infections were significantly more prevalent among patients infected with HIV than among HIV-uninfected patients with GUD (55 of 191 patients, 28.8% versus 26 of 290 patients, 9.0%; RR = 3.21, 95% CI 2.09, 4.93; P ≤ 0.001) (Table 2). Among GUD patients with genital herpes, recurrent infections were significantly more prevalent in patients infected with HIV (55 of 91 patients, 60.4%) than in patients not infected with HIV (26 of 81 patients, 32.1%) (RR =1.88, 95% CI 1.32, 2.69; P ≤ 0.001). When the analysis was restricted to GUD patients without antibodies to HSV-2, HSV-positive M-PCR results were still strongly associated with HIV seropositivity; 55 of 119 (46.2%) patients infected with HIV had HSV by M-PCR, versus 26 of 116 (22.4%) patients not infected with HIV (RR = 2.06, 95% CI 1.40, 3.05; P ≤ 0.001).
Association Between HSV-2 and T pallidum Antibodies and HIV Infection
Human immunodeficiency virus infection was strongly associated with HSV-2 infection, as measured by the presence of antibody to gG-2. Among all patients, men infected with HIV were significantly more likely to have HSV-2 antibodies (198 of 314 patients, 63.1%) than men not infected with HIV (284 of 737 patients, 38.5%) (RR = 1.64, 95% CI 1.44, 1.85; P ≤ 0.001). The strength of the association between HIV infection and HSV-2 seropositivity did not vary by city or by presenting syndrome; however, the difference was not statistically significant in CT, where the number of patients infected with HIV was low (Table 3).
Patients infected with HIV did not differ significantly from patients not infected with HIV when comparing rates of FTA-ABS seroreactivity (28.2% versus 32.1%). When the analysis was restricted to patients with GUD, the proportion of patients with a reactive FTA-ABS test result was significantly lower among those with HIV infection (32.2% versus 67.7%; RR = 0.69, 95% CI 0.56, 0.86; P ≤ 0.001); however, when men with M-PCR-positive syphilis lesions were excluded, the proportion of HIV-infected and HIV-uninfected GUD patients who were FTA-ABS reactive did not differ significantly (33.2% versus 39.9%, P = NS) (Table 3).
The major findings of this study are that (1) HSV-2 is a more common etiology of GUD than has been suggested by previous studies conducted in South Africa; and (2) serologic evidence of HSV-2 infection and current initial or recurrent genital herpes are strongly associated with HIV infection among men who present to STD clinics with GUD or urethritis. No such associations were seen either with current or prior syphilis or with current chancroid. Among GUD patients, a negative association was observed between a positive FTA-ABS test result or presence of T pallidum DNA, as determined by M-PCR, and HIV infection. At least one study that reported an association between HIV and HSV-2 infections did not find an association between HIV infection and syphilis31; however, these protective effects disappeared after men with M-PCR-positive syphilis lesions were excluded from the analysis.
Previous studies18,23 have demonstrated that M-PCR provides a more reliable and sensitive diagnosis of GUD than either clinical examination or use of standard laboratory tests such as culture, microscopy, and serology. In this study, M-PCR was more reliable and sensitive than culture or clinical examination for the diagnosis of genital herpes; M-PCR detected HSV DNA in 36% of ulcer specimens, a 4-fold to 10-fold increase over isolation rates reported 5 to 10 years earlier.3,21,22 It is not known whether this difference represents an increase in the prevalence of HSV-2 infection, more sensitive laboratory tests, or both.
Genital herpes was clinically underdiagnosed in men with GUD, largely because few men present with vesicular lesions typical of HSV infection.22 In addition, the observation that chancroid was the most common clinical diagnosis in men with genital herpes suggests either that many cases were superinfected with bacteria resulting in purulent lesions,22 or that atypical lesions occurred, possibly as a result of coexisting HIV infection.23
The presence of HSV-2 antibodies in 49% of men with GUD and 42% of men with urethritis indicates that infection with HSV-2 is common among South African men attending STD clinics. The overall HSV-2 seroprevalence recorded in this study was similar to that reported previously in Lesotho,23 where 43% of men had antibodies to HSV-2 and 74% of women with GUD had HSV-2 antibodies. Population-based19,25 and cross-sectional studies11 have also reported that the seroprevalence of HSV-2 antibodies are greater in women than in men; therefore, whereas we did not determine the presence of antibodies to HSV-2 in women attending STD clinics in South Africa, it is likely that their seroprevalence is similar, if not greater, than that observed in men.
The World Health Organization (WHO) has recommended the use of syndromic STD management protocols for GUD, especially among patients in areas with limited resources.32 According to the WHO algorithm, patients are treated for genital herpes if they present with vesicular lesions or with a history of vesicular lesions. This treatment does not include antiviral suppressive therapy, because the antimicrobial therapy is directed at chancroid and syphilis. The use of these criteria in South Africa should be reevaluated in light of the results of this study.
The most common agent identified in ulcer specimens was HSV-2; HSV-2 DNA was detected in 42% of ulcers in which a single agent was identified, and in 80% of ulcers containing multiple agents. Furthermore, our data suggest the majority of patients with HSV-2 did not present with typical lesions, which confirms the recent findings of Bogaerts et al.33 Results of serologic testing revealed that 45% of the participants in this study were seropositive for HSV-2; however, previous studies have indicated that only a small percentage of these HSV-2-seropositive persons report a history of genital herpes infection.19,25,34 Our results show that HSV-1 infection was widespread, and occurred in patients at a young age. It has been well demonstrated that previous HSV-1 infection may modify subsequent HSV-2 infection,29,35,36 with the severity of the initial episode of HVS-2 infection being milder in persons who acquire HSV-2 after HSV-1 infection.36
Both previous and current cases of GUD have been shown to be associated with HIV infection.5 This association does not necessarily reflect a causal relationship, because both GUD and HIV infection may reflect common risk factors. It is thought that GUD increases the risk of HIV transmission per sexual exposure by the shedding of HIV through genital lesions,6 and by providing an easy portal of entry for the virus into the host. Recently, a community-based, randomized trial in Mwanza, Tanzania showed that effective management of STDs using syndromic treatment algorithms reduced the incidence of HIV in both men and women by approximately 40% during a 2 year period.37 Genital ulcer disease represents approximately 22% of the diagnoses made at dedicated STD clinics in southern Africa38; however, a high prevalence of genital herpes infection in patients with GUD would limit the effectiveness of mass treatment for bacterial STDs as a means of preventing HIV. Such mass treatments have been attempted recently in Uganda and have been associated with declines in bacterial STD prevalence, but have had no effect on HIV incidence.39
Whereas acyclovir suppresses recurrent herpetic infections, cost may preclude its widespread use, even in patients presenting with overt lesions. Nevertheless, a trial to determine the effectiveness of acyclovir therapy in reducing the incidence of HIV infection among both clinically typical cases of herpes and HSV-2-seropositive persons is worthy of consideration, because most gG-2-seropositive persons intermittently reactivate HSV-2 on mucosal surfaces. The presence of small, often difficult-to-recognize lesions and lesions of short duration suggest that the prevalence of genital lesions due to HSV-2 among persons suspected of transmitting HIV may be underestimated.6 Thus, to lower the number of HIV infections attributable to genital herpes, it may be cost effective to identify high risk, HSV-2-seropositive persons and to provide them with counseling to encourage condom usage and to reduce their number of sex partners.
Very few persons infected with HIV know when they became infected40; thus, the use of serologic tests attempts to infer a role for STDs present at the time of HIV infection, usually months or years before the HIV is identified. For STDs, the delay in ascertaining factors present at the time of HIV infection leads to exposure misclassification and bias in relative risk estimates.40 The problems are compounded because behavioral risk factors for STDs are similar, and positive associations between HIV seropositivity and a history of other STDs are expected, because of confounding by sexual behavior. Despite these limitations, we noted a significant association between previous and current HSV-2 infection and HIV infection. Both cross-sectional and population-based studies from Africa, Asia, and North America have observed a similar association between previous HSV-2 infection and HIV infection.10–12,23,25,26,31
Our data suggest that GUDs of different etiology may vary in their association with HIV infection. Rates of previous syphilis cases were the same among HIV-infected and HIV-uninfected patients; however, the reasons for this are unclear. Associations may have been obscured by the disproportionate contribution of previous syphilis cases from CT, which had the lowest rate of HIV infection. It is also possible that patients with syphilis and genital herpes infection exhibit different risk behaviors, or that recurrent disruptions of mucous membranes caused by HSV-2 may increase the risk of HIV infection more than the nonrecurrent genital lesions of syphilis.31 Kingsley et al14 suggested that HSV-2-seropositive persons were more sexually active than those with other STDs. Alternatively, treponemal tests may not be a sensitive marker of previous syphilis infection in persons with HIV infection. Haas et al41 observed that 7% of HIV-seropositive asymptomatic men and 38% of men with symptomatic HIV infection had loss of treponemal test reactivity, whereas none of the HIV-seronegative persons lost reactivity. The immune status of the HIV-infected participants in this study was unknown; thus, we were unable to assess this possibility.
It has been shown that HIV infection increases both genital shedding of HSV-242 and the frequency of recurrence43 in HSV-2-seropositive persons. The latter is consistent with our observation that HIV-infected men with genital herpes were approximately twice as likely to be experiencing a recurrent episode than HIV-uninfected men with genital herpes. O'Farrell and Tovey44 observed that the incidence of genital herpes was markedly higher in a cohort of HIV-infected men and women who had acquired HIV infection via heterosexual transmission than in new STD clinic attendees with HSV-2 from the general population. This finding suggests that HSV-2 may be responsible for a greater population-attributable risk of HIV infection among South Africans than previously thought.
Additional limitations of this study are that sexual behavioral factors were not addressed, and STD clinic data reflect relative prevalence versus true-population prevalence. This was a cross-sectional study, and we cannot imply causality from the results. Furthermore, the applicability of these results to other patient populations, particularly those in developed countries and in settings other than STD clinics, may be limited. Finally, no attempt was made to identify patients with urethritis due to HSV-2. This may have resulted in an underestimation of the association between HIV and active HSV-2 infection.
In conclusion, future approaches for reducing HIV transmission by STD control should include efforts to address HSV-2 infection, because HSV-2 appears to be the cause of GUD with the greatest potential impact on HIV infection.
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