Aetiology of genital ulcer disease and associated factors among Mthatha public clinic attendees

Background Genital ulcer disease (GUD) is a sexually transmitted disease characterised by ulcerating lesions. Despite the introduction of sexually transmitted infections (STIs) syndromic management approach into primary healthcare in South Africa (SA) in 1995, the prevalence of STIs in South Africa remains high. Objectives The study investigated the aetiology of GUD and factors influencing it among public community health centre (CHC) attendees in the Eastern Cape, South Africa. Method A total of 105 participants were recruited among individuals presenting with GUD from three CHCs located in the Eastern Cape Province, South Africa. Blood and genital ulcer samples were collected from consented participants. Blood samples with suitable sera were tested for human immunodeficiency virus (HIV) and syphilis. Herpes simplex virus types 1/2 (HSV–1/2), Chlamydia trachomatis, Treponema pallidum, Haemophilus ducreyi and Klebsiella granulomatis were detected in nucleic acid extracted from genital ulcer specimens. Results Out of the 98 samples with suitable sera, 55.1% and 8.2% were HIV and syphilis seropositive, respectively. Ulcerating STI pathogens were detected in 31.4% of the study participants. Herpes simplex virus type 2 was the most detected pathogen (16.2%) followed by Chlamydia trachomatis (10.5%), HSV-1 (8.6%), Haemophilus ducreyi (8.6%) and Treponema pallidum (6.7%). Multiple pathogens were detected in 13.3% of participants. Detected multiple ulcerating pathogens were common among HIV-positives (p = 0.016). Conclusion Molecular methods for diagnosing pathogens have the potential to improve the management of GUD. Data generated from this study would contribute to the limited data on GUD in the Eastern Cape Province. Further research with a larger sample size is recommended. Contribution Data generated would contribute to the limited data on GUD in the Eastern Cape province, South Africa.

http://www.sajid.co.za Open Access It was found that syphilis cases among antenatal care attendees were not declining as the cases rose from 1.6% in 2011 to 2.0% in 2015. 12 There is a reported noticeable decline in the prevalence of chancroid worldwide although the infection might still occur in some African and Caribbean regions. 13,14 Genital herpes was the relatively prevalent aetiology of genital ulcer syndrome. 15 Herpes simplex virus type 2 is the major cause of GUD and a highly prevalent STI, worldwide. 16 In one South African study, 65.2% of GUD cases had ulcer-derived STI pathogens, with HSV accounting for 60.7% of these cases, followed by Treponema pallidum (3.9%), Chlamydia trachomatis L1-3 (0.9%) and Haemophilus ducreyi (0.5%). 17 The proportion of GUD due to bacterial pathogens had dramatically become less in sub-Saharan Africa. 18,19 Genital ulceration may be a predisposing or concomitant factor in transmitting HIV. 3,20,21 With STIs, the risk of male-female HIV transmission increases; moreover, the female-male transmission becomes even higher. 22 The HIV infection enhancement can be because of various processes, such as the disturbance of normal epithelial barriers with ulcerations, causing the recruitment of HIV-susceptible T-lymphocytes or macrophages to the infected area as part of the host's immune response. 22,23 Human immunodeficiency virus-infected persons are more likely to be coinfected with chronic herpesviruses, which replicate periodically producing viable herpes virions. 24 A South African study revealed that contracting either HIV or HSV-2 will encourage infection by the other. 25 The GUD epidemiology is also influenced by sexual partners' gender, socioeconomic factors, multiple/increased sexual companions, status on HIV and local prevalence, drug use, limited prevention, inadequate knowledge of STIs, commercial sex and circumcision. 26 Young people are at increased risk of acquiring STIs because of their risky behaviour, which is an important health and social concern. 27, 28 High rate of unprotected sex among the heterosexual and homosexual population is driven by several factors, among which there is fear of being considered unfaithful in the relationship and money and gifts in exchange for sex. 29 The syndromic STI management approach is beneficial, but it limits the opportunities for diagnosing asymptomatic STIs. 9,30 Syndromic STI management is the diagnosis of STIs based on symptoms and signs, which subsequently leads to their treatment, with point-of-care therapies to treat the majority of microbes that produce specific syndromes without confirmation with the laboratory tests. 31 It was introduced into South African primary healthcare in 1995; despite this introduction, the burden of STIs remains high. The WHO recommended that syndromic management algorithms be regularly re-evaluated through performing aetiological and antimicrobial resistance periodically. 15 Acyclovir was added for genital herpes patients with first episodes of the disease 13 ; however, treatment failures were reported. 5,6 There is limited data on GUD in the King Sabata Dalindyebo local municipality (KSDLM), Eastern Cape province of SA, despite the strong evidence that GUD presents a major health challenge. The lack or shortage of evidence-based data in this locality triggered an interest in investigating the aetiology of GUD and its associated factors, and hence this study among public community health centre (CHC) attendees.

Research methods and design
Study setting and population The study was conducted in the KSDLM in the OR Tambo district municipality. Participants were from Gateway, Ngangelizwe and Stanford Terrace CHCs. These CHCs were selected from other KSD CHCs based on the highest number of patients seen between April 2016 and August 2016 presenting with STIs according to the statistics by the KSD health department.
Participants presenting with genital ulcers were recruited between May 2018 and July 2019 during their routine visits at the CHCs. Participants were recruited by educating all CHC attendees using GUD posters. Those who self-reported to have the signs and symptoms of GUD (visible, unhealed ulcers) were physically examined and included in the study. Recruitment and informed consent processes were conducted in English and isiXhosa, a locally spoken language. Trained HIV counsellors conducted the pre-and post-HIV counselling at the CHCs.
Participants were interviewed privately to collect sociodemographic and clinical information, followed by clinical examination and sample collection by a research nurse assisted by the investigator. Structured questionnaire forms were administered, explained and completed with the assistance of the research nurse and/or investigator. The nurse performed the clinical examination. Venous blood was collected into plain tubes. Genital ulcers were swabbed using sterile Dacron swabs (Clinical Sciences Diagnostics Ltd, Booysens, SA) and were put in transport medium, stored at −20 °C and transported to the University of KwaZulu-Natal, Microbiology laboratory, Durban, SA for analysis.  Klebsiella granulomatis was detected using conditions as described by Carter and colleagues. 32

Data analysis
All variables were captured and coded in Microsoft excel. Single infection was defined as infection with one pathogen type, while multiple infection was defined as two or more pathogen types in the same sample. GraphPad Prism Software v8.0.1.244 statistical software was used to perform chi-squared for trends, Fisher's exact to compare the proportion between variables and the relative risk (RR). A p value ≤ 0.05 was used to indicate statistical significance.

Ethical considerations
All study aspects were approved by the Human Research Ethics Committee of Walter Sisulu University (HREC: 015/2016), EC Department of Health (EC_2016RP26_934) and KSD Department of health sub-district, Mthatha. Written informed consent was obtained from participants.   Table 5). Among HIV-negatives, the proportion of the GUD population decreased with increasing age significantly (p < 0.001) but not among HIV-positives (p = 0.683, Figure 1). A high proportion of GUD was seen in the participants with partners aged < 18-39 years compared with > 40      (Table 5).

Discussion
The high number of GUD infections observed among the female participants in the present study could be because of the increased risk of GUD that is associated with the more fragile surface of female reproductive organs. 33 The observed low proportion of GUD infections among male participants could be because of less willingness to be involved in a research study or few men with GUD attending the CHC. However, it has been reported that among men, clinically GUD diagnosis is a predictor of re-visiting primary prevention facilities. 34 A third of the GUD population had ulcerating STIs. Studies revealed that the development of genital ulcers might not only be because of STIs but also non-infectious agents. 2,3 The observed low prevalence of ulcerating pathogens might be because of non-infectious agents 2,3 or low viral load of the pathogen that resulted in false-negative results. 35,36 There was more than one type of ulcerating STIs in some specimens, and this was prevalent among HIV-population. Previous studies reported that there could be more than one aetiological agent in any genital/anal/perianal ulcer. 28 Literature has documented that HIV-population is at increased risk of multiple STIs as pathogens share transmission routes. 37 Herpes simplex virus type 2 was reported as the most causative organism of GUD. Similar observations were observed in this study. A South African study also indicated that HSV-2 remained the leading cause of ulcerating STI pathogen, supporting the use of acyclovir in the STI syndromic management. 17 Herpes simplex virus type 1 was also one of the causes of GUD in this study. Studies previously conducted have revealed that HSV-1 can also be isolated in genital lesions, 5,6,7,26 and this could be because of the increased practice of orogenital sex. 38 This study revealed that Chlamydia trachomatis L1-3 was still a burden in SA. Previous studies have reported Chlamydia to be among pathogens causing a high burden of STIs among youth in SA. 11 Klebsiella granulomatis was not detected in this study, a finding similar to a previous study that was conducted among the South African population that also reported negative Klebsiella granulomatis in genital ulcer specimens. 39 The negative results are probably because of public recognition of Donovanosis as a health problem with control measures or as a result of the improvement of health services or living standards. 14 Haemophilus ducreyi was the third-highest ulcerating pathogen in this study. Studies conducted worldwide have reported a decline of chancroid; however, infections might still occur in some African and Caribbean regions. 13,14 This calls for review or monitoring of the STI syndromic management approach because Haemophilus ducreyi still seems to be burdensome. The high prevalence of ulcerating STIs among participants living in a semi-urban and rural area compared with participants in informal settlement and urban area residents has been observed in a study conducted in three clinical research sites in Durban, SA, where HSV-2 prevalence was also high among participants in rural/semi-rural areas of Durban. The higher prevalence in rural areas could be attributed to women who have less access to jobs and may engage in more transactional sex. Furthermore, rural areas have less access to healthcare, including STI treatment and free condoms. 40 Lower social status, lower education levels and lower income have been associated with increased risk of STIs. 41 The informal settlement population was more affected by the GUD and with a higher HIV-positivity rate compared with other population groups in this study; however, the prevalence of GUD pathogen was low.
The GUD prevalence was less dominant among participants with primary education, which is contrary to findings from other studies that reported lower education level as a risk factor for STIs. 40 Similarly, others have reported drug use as one of the factors influencing the epidemiology of GUD, 26 which is contrary to the current study's findings, which found no difference in the prevalence of ulcerating STIs among alcohol/dagga/cigarettes and those not using them. Supposedly, this might be because of false reporting. However, the HIV-positivity rate was a bit higher among those taking alcohol/drugs/cigarettes compared to the HIVnegativity rate. Factors such as socioeconomic factors, lack of adequate knowledge of STIs and multiple or increased sexual partners are reported to influence the epidemiology of GUD. 26 This is supported by high GUD or ulcerating STIs among the unemployed and no-condom use population in this study.
The HIV seroprevalence rate among GUD participants was high (55.1%). The relationship between genital ulcers and HIV acquisition is well documented in the literature. 3,20,21 Disruption of normal epithelial barriers with ulcerations causes recruitment of HIV-susceptible T-lymphocytes or macrophages to the infected area as part of the host's immune response. 22 South African researchers also found the prevalence of HIV co-infection among GUD patients to be high. 17 Syphilis serology had different results from the nucleic acid detection method. There was not much difference in percentages, but two samples were positive in both serological and nucleic acid tests. The reason could be that syphilis is a multistage STD. The primary stage is characterised by a chancre, where the test's sensitivity is high in collected nucleic acid test samples. Serological tests might be negative at the primary stage because of the window period between transmission and seroconversion, and as the stages progress, the serology test would be positive. 42

Limitations of the study
The small sample size in this study could be a limitation. Therefore, a larger sample size is recommended. The distribution of participants from the three recruitment sites was not equal, and there were few male participants. Therefore, the results could not be generalised to represent the three CHCs. Despite these limitations, this study remains valuable for the EC population. Further research with larger sample size is recommended.

Conclusion
Herpes simplex virus type 2 was the leading cause of GUD in KSDLM followed by Chlamydia trachomatis L1-3, HSV-1 and Haemophilus ducreyi and Treponema pallidum. The use of molecular methods in diagnosing ulcerating STIs can potentially improve the management of GUD. Sexually transmitted infections/STD and sexual behaviour education interventions remain essential to improve sexual behaviour and reduce the STD burden.