In recent years, the proportion of genital ulcer disease (GUD) caused by bacterial pathogens has decreased dramatically in sub-Saharan Africa, whereas herpes simplex virus type 2 (HSV-2) has become the most common cause of GUD1 and is one of the most prevalent sexually transmitted infections (STIs) worldwide.2,3 Prior HSV-2 infection significantly increases the risk of HIV acquisition by 2- to 3-fold.4 HIV-1 and HSV-2 dually infected individuals, with or without symptomatic genital herpes, experience more frequent genital shedding of both viruses,5 thereby fa cilitating the risk of sexual transmission of each virus. These reciprocal and synergistic relationships suggest the importance of controlling HSV-2 for HIV prevention,6 although HSV-2 therapy trials have not had an effect on HIV acquisition or transmission. The shift in GUD etiologies has been accompanied by decreased cure rates, which may undermine the confidence of patients and providers in the effectiveness of the syndromic approach to GUD management.7
The prevalence of GUD etiologies such as syphilis and chancroid are important determinants of HIV transmission and STI regimens.8 Given that many countries cannot conduct laboratory tests to determine those specific etiologies in routine practice, the World Health Organization (WHO) recommends syndromic management for patients presenting with GUD in low-resource settings.9 Traditionally, GUD management has consisted of antibiotics targeting Haemophilus ducreyi and Treponema pallidum,10 with treatments for other bacteria, such as the L1–L3 strains of Chlamydia trachomatis (lymphogranuloma venereum, or LGV) and Klebsiella granulomatis (donovanosis), being added depending on local epidemiology. More recently, the WHO recommended the addition of acyclovir for the treatment of herpetic ulcers in countries with an HSV-2 prevalence greater than 30%.9 Many countries including Malawi have added acyclovir to their GUD algorithms,11 although implementation has been incomplete because of lack of drug availability, lack of clinical retraining, and conflicting local data.
The WHO also advocates regular monitoring of STI syndrome etiologies to adjust treatment algorithms. To assist the Malawian Ministry of Health in monitoring and reviewing their algorithm for GUD and to provide regional data, we conducted a randomized controlled trial comparing adding acyclovir for episodic treatment of genital herpes to standard syndromic management. Episodic HSV-2 treatment with acyclovir added to syndromic management did not produce a significant clinical benefit in this African population; however, among HIV-1/HSV-2 coinfected individuals, acyclovir reduced the frequency of lesional HIV-1 shedding and detection of seminal plasma HIV-1 RNA.12 Secondary objectives of this trial were to assess the prevalence of GUD etiologies among trial participants and to assess the association with HIV serostatus.
The study was an individually randomized double-blind placebo-controlled clinical trial of acyclovir 800 mg twice daily for 5 days added to syndromic management of GUD in Malawi. The protocol was approved by the National Health Sciences Research Committee in Malawi and the ethics committees of the London School of Hygiene and Tropical Medicine, the University of North Carolina, and the Centers for Disease Control and Prevention (CDC).
Detailed procedures have been described elsewhere.12 Briefly, consecutive adult patients 18 years and older, residing in Lilongwe, and presenting with genital ulcers or vesicles at the Kamuzu Central Hospital STI clinic between September 2004 and June 2006 were eligible for enrollment. Pregnant, lactating, and menstruating women were excluded, as were previously enrolled participants.
Patients who provided written consent were interviewed using a standardized questionnaire and examined to obtain a detailed description of their ulcer(s). History of self-treatment was obtained. The size of the largest ulcer on the external genitalia was measured using paper tapes, and the ulcer surface area (in millimeters squared) was calculated. Cotton-tipped sterile swabs were used to collect lesional fluids from the base of the largest ulcer to determine ulcer etiology and detect HIV-1 RNA. We did not collect swabs from blisters only. Ulcer swabs were stored at −20°C and kept frozen before laboratory testing. Additional genital samples included a cervical swab and a cervicovaginal lavage from women and a urine sample from men. Blood samples were also collected.
At the end of the baseline visit (day 0), participants were randomized to acyclovir or placebo, then received directly observed syndromic treatment following the Malawi Syndromic Management Guidelines for chancroid (ciprofloxacin 500 mg orally single dose) and syphilis (intramuscular injection of benzathine penicillin 2.4 million units single dose), STI and HIV counseling, partner notification slips, and a supply of condoms. Participants were asked to return to the clinic on day 2 and/or day 4 (women only) and days 7, 14, and 28, although this article focuses on results of the baseline visit only. Patients whose ulcers had not healed or remained symptomatic after 7 days received second-line treatment with a single dose of azithromycin (1 g orally). HIV-positive participants were counseled about their status and referred to the adjoining HIV treatment center (Lighthouse Clinic) for further care. At the time of this study, the Malawi guidelines recommended patients with CD4 count below 200 cells/mm3 start antiretroviral treatment (ART), and all those patients were directly referred to the Lighthouse Clinic to determine eligibility.
Blood serum samples were tested for HIV, HSV-2, and syphilis serologies. HIV-1 serostatus was determined at the clinic using 2 parallel rapid tests (Determine [Abbott Laboratories] and Uni-Gold [Trinity Biotech]). Discordant results were resolved using an HIV ELISA (Genetics Systems), and these results were taken as final. Herpes simplex virus type 2 serology was performed using HerpeSelect IgG2 ELISA (Focus Technologies, Cypress Hill, CA) with high sensitivity in African settings.13 Syphilis serology was performed using a nontreponemal test (rapid plasma reagin [RPR], Macro-Vue; Becton-Dickinson, Sparks, MD) with reactive samples confirmed by T. pallidum hemagglutination assay (Serodia-TPHA; Fujirebio, Tokyo, Japan).
Among HIV-1–seropositive participants, plasma HIV-1 RNA was quantified using Roche Amplicor HIV-1 Monitor Test, version 1.5 (Roche Diagnostic Systems, Branchburg, NJ) with a lower limit of detection of 400 copies/mL. CD4 T-lymphocyte count was performed using standard fluorocytometry (FACScan; Becton-Dickinson). All tests were performed at the University of North Carolina reference HIV/STI laboratory in Lilongwe.
DNA from the ulcer specimens was extracted using QIAamp DNA Mini Kit (Qiagen Inc), and the ulcer etiologies were determined at the Reference Laboratory and Research Branch, Division of STD Prevention, CDC (Atlanta, GA). Real-time multiplex polymerase chain reaction (PCR) was used for the molecular diagnosis of HSV-2, H. ducreyi, and T. pallidum on TaqMan technology using a CDC in-house protocol. The assay was performed on a Rotor-Gene 3000 (Qiagen Inc). First-episode and first-exposure HSV-2 ulcers were defined as the presence of lesional HSV-2 DNA in patients with negative HSV-2 serology; recurrent HSV-2 ulcers were defined as the presence of lesional HSV-2 DNA in patients with positive HSV-2 serology (regardless of GUD history). Lymphogranuloma venereum diagnosis was based on a C. trachomatis L1–L3 strain-specific real-time PCR assay developed by the CDC.
Other Genital Infections
Urine samples (men) and cervical swabs (women) were tested for Neisseria gonorrhoeae and C. trachomatis using Amplicor NG/CT PCR (Roche Diagnostic Systems).
All analyses were carried out using SAS version 9.2 (The SAS Institute, Cary, NC) and Stata version 10.0 (Stata Corporation, College Station, TX). Categorical data were analyzed using χ 2 tests, with Fisher exact test where appropriate. Comparisons of continuous data between 2 groups used the t test if normally distributed and the Wilcoxon rank sum test otherwise. HIV-1 RNA quantities were log10 transformed for analyses.
Of 460 patients presenting with GUD at Kamuzu Central Hospital/STI clinic between September 2004 and June 2006, 422 (313 men [74%] and 109 women [26%]) were enrolled (Table 1). The median age in both men and women was 28 years. More men than women reported 2 or more sexual partners in the past year (60% vs. 29%, P < 0.001), and men were more likely than women to have a recent change of partner (50% vs. 19%, P < 0.001). Both sexes reported infrequent recent condom use (18% reported using a condom at last sex). Approximately a third of patients reported experiencing prior GUD within the previous year. The reported median duration of the present ulcer was 6 to 7 days, with no difference by sex. Most lesions (69%) were multiple. Prevalence of N. gonorrhoeae was lower in men than in women (6% vs. 15%, P = 0.02). C. trachomatis was very low in both men and women, consistent with historical values from Malawi.14 Among the HIV-1–infected patients, HIV-1 viral load was high overall (4.85 log10 copies/mL) with no difference in mean log viral loads between men and women (4.90 vs. 4.74 log10 copies/mL, P = 0.15). At the enrollment visit, fewer than 5% of HIV-infected participants were on ART and the median CD4 cell count was 232 cells/mm3.
HIV, HSV-2, and Syphilis Serologies
Overall, 299 (72%) of 417 patients were HSV-2 seropositive, with lower prevalence in men compared with women (69% vs. 81%, P = 0.01); 61% were HIV-1 seropositive (55% in men vs. 77% in women, P < 0.001); and 21/422 (5%) had a positive syphilis serology (5% in men vs. 6% in women 6%, P = 0.77).
Genital ulcer disease etiology was obtained from 398 (94%) patients. Herpes simplex virus type 2 ulcers were most prevalent (67%), either as a single agent (54%) or among ulcers with multiple etiologies (13%). Among the HSV-2 ulcers, the majority (75%) were recurrent or previously exposed. H. ducreyi remains a significant cause of GUD in Malawi (15%). The prevalences of T. pallidum and LGV were both 6% (Table 2). Of the 21 patients who were RPR and T. pallidum hemagglutination seropositive, 10 (45%) were multiplex PCR T. pallidum negative. Among these 10, 9 (90%) were also positive for as follows: HSV-2 alone (n = 6), H. ducreyi alone (n = 1), HSV-2 and H. ducreyi (n = 1), or HSV-2 and LGV (n = 1).
GUD Etiology by HIV Subgroup and by Sex
In HSV-2–infected patients, HIV prevalence was high (67%). HIV seropositivity was significantly higher among patients with recurrent or previously exposed HSV-2 compared with patients with first-episode and first-exposure HSV-2 (78% vs. 39%, P < 0.001; Table 3). Fewer HSV-2–infected men were HIV positive than HSV-2–infected women (61% vs. 81%, P = 0.008).
In this cross-sectional analysis of the etiology of GUD in Malawi, HSV-2 ulcers were the most prevalent presentation, including three quarters of recurrent HSV-2 ulcers. Ulcers caused by H. ducreyi (15%) remain prevalent in Malawi, but the prevalence of T. pallidum is low and has declined compared with past data.15,16 To date, LGV (6%) has largely been ignored (and untested) as a cause of GUD in this setting and, consequently, has been treated less than optimally.
The prevalence and etiological distribution of GUD in sub-Saharan Africa over the past 2 decades have been influenced by the initiation and routine use of syndromic management and the persistent high prevalence of HIV in this population. Similar to the timeline in many developing countries, STI syndromic management was initiated in Malawi in the mid-90s following etiologic studies that set the stage for the development of country-specific algorithms.11 The widespread use of this management approach that treats all genital ulcers for both syphilis and chancroid has contributed to a decline in the proportional prevalence of these treatable STIs in Malawi. Between 1992 and 2007, the proportion of GUD attributable to T. pallidum and H. ducreyi fell from 18% to 5% and 26% to 16%, respectively.11 Correspondingly, GUD caused by HSV-2 has increased from 23% in 199217 to 67% in 2007.12
Where HIV-1 prevalence is high, the etiological profile of GUD often includes higher rates of HSV-2, as seen in our setting. Although both initial and recurrent HSV-2 cases are associated with an increased risk of HIV-1 transmission and acquisition, patients with recurrent HSV-2 infections have a higher prevalence of HIV,4 which reflects both the cumulative increased risk of HIV acquisition facilitated by multiple episodes of GUD and the higher rate of recurrences associated with HIV-associated immunosuppression.18–21
The WHO recommends adding episodic treatment with acyclovir to syndromic management in areas where the prevalence of herpetic ulcers is greater than 30%,9 based on a reduction in ulcer healing time found among patients who present early,2,22 and because acyclovir reduces HIV viral shedding in HSV-2 ulcer lesions12,23 and in genital secretions12,22 among those dually infected. As a direct outcome of our study, the treatment of genital herpes has been added to the treatment of GUD in Malawi since 2007.
The 15% prevalence of H. ducreyi among patients with GUD in Malawi is higher than in most other areas in sub-Saharan Africa. In Botswana, H. ducreyi prevalence decreased from 23% in 1993 to 1% in 20027 (treating with a single intramuscular injection of ceftriaxone 250 mg) and was similarly low (1%) between 2002 and 2006, in Rakai, Uganda (treating with ciprofloxacin 500 mg twice daily for 3 days + erythromycin 500 mg per os, 4 times daily for 14 days).24 The relatively higher prevalence of H. ducreyi in Malawi may be related to the historic choice of treatment. In 1995, erythromycin 500 mg 4 times daily for 7 days had a cure rate (>95%) comparable with ciprofloxacin 500 mg as a single dose.25 Erythromycin was chosen for cost considerations, despite the risk of noncompliance because of gastrointestinal side effects. This regimen was used until 2005 when the Malawi STI guidelines replaced the multidose eryrthromycin regimen with a single dose of ciprofloxacin. Another etiologic survey will be necessary to determine and monitor the effect of this regimen change on H. ducreyi prevalence.
This evaluation was the first to assess the presence of LGV in Malawi. The proportion of GUD attributable to LGV was higher than a recent assessment in Namibia (2%)26 and an assessment of the trends among a mining community in South Africa, which ranged from 0.9% to 4.5% between 1986 and 1998.27 The prevalence in our population was similar to STI clinics in Bangkok (10%), Madagascar (8%), and Lesotho (7%) in the mid- to late 1990s.16,23 The standard treatment of LGV is doxycycline 100 mg twice daily for 21 days.28 The current Malawi guidelines do not include LGV treatment in their GUD treatment algorithm, although patients returning with unhealed ulcers or other related symptoms at 7 days are treated with a single 1 g dose of azithromycin. The effectiveness of this regimen on LGV is unproven.
The presence of T. pallidum in Malawi is low and continues to decline. This decline may be caused by almost 2 decades of systematically treating all presenting GUD with benzathine penicillin. Not only has the prevalence of genital ulcers caused by T. pallidum declined from 18% (1992)25 to 5% (2007),11 but also RPR seropositivity among women attending antenatal services countrywide declined from 7.0% in 1996 to 1.2% in 2010.29 Although the rates of T. pallidum are at historic lows, the risk of resurgence, including an increase in congenital syphilis, would be too high to recommend removing syphilis treatment from the GUD syndromic treatment guidelines.
We were unable to identify a specific etiology for 20% of the patients with GUD, which is within the range of other similar evaluations.21,24 Unidentified ulcers could result from nonsexually transmitted etiologies or might represent older, healing sexually transmitted ulcers, perhaps coinfected with non-STI agents.
Study participants were patients who presented for care at a semiurban STI clinic in Malawi, so our work is generalizable to similar populations but might not represent the distribution of GUD in the population. Patients who successfully self-treated their GUD or whose lesions would have spontaneously healed would be less likely to present to the clinic. This could include patients with ulcers too small or painless to notice, or indeed patients with recurrent HSV2 ulcers who may already know that their ulcers will heal with time alone. Patients with blisters only were excluded in the etiology analysis because swabs were not taken. The diagnostic tests have changed: the HSV-2 antigen detection method used in 1992 (EIA HERPCHECK) was less sensitive that the multiplex PCR used in subsequent years and could have contributed to the difference in prevalence found over time.
Nearly 2 decades of syndromic management of GUD seem to have contributed to a real reduction in the burden of some of the bacterial etiologies of GUD such as T. pallidum and H. ducreyi in Malawi, although the reduction in H. ducreyi has not been as pronounced as elsewhere because of suboptimal treatment. Although the WHO has attempted to respond to the burden of HSV/HIV coinfections by adding episodic acyclovir to the GUD algorithm, the failure of randomized clinical trials to show a benefit of suppressive HSV-2 therapy on acquisition and transmission of HIV hampers the implementation of episodic HSV-2 treatment at the country level. The clinical benefit of acyclovir for patients presenting with HSV ulcers is small but not debatable; however, more aggressive interventions would be required to prevent HIV transmission in this population. Broadly applied HIV testing and counseling and HIV and HSV-2 risk reduction counseling among all patients presenting with GUD and their sexual partners, in addition to the early initiation of ART when appropriate, are, for now, the best prevention measures for persons coinfected with HSV-2 and HIV and those at risk for exposure.
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