Genital ulcer disease (GUD) has been reported to increase the risk for acquisition and transmission of human immunodeficiency virus (HIV).1,2 Herpes simplex virus (HSV) has been shown to be a common etiological agent found in patients with GUD and significantly associated with higher seroprevalence of HIV infection in India.3,4 Reynolds et al5 observed a high incidence of syphilis with an increased risk of HIV infection among a cohort of sexually transmitted infection (STI) clinic attendees in India. Currently in India, the diagnosis and management of GUD are based mainly on the clinical presentation of the ulcer itself since laboratory diagnosis is unavailable at most places. In 1991, World Health Organization (WHO) developed simple algorithms for prompt diagnosis and treatment of bacterial sexually transmitted infections, which included flow charts for the management of GUD.6 Following the rise in prevalence of HSV-2 in sub-Saharan Africa, GUD syndromic management flow charts were revised in 2003 to include antiviral treatment.7 The current national STI guidelines in India recommend classification and treatment of GUD syndrome according to the clinical presentation. In the presence of vesicles or multiple painful ulcers, a herpetic diagnosis is assigned and episodic therapy with antivirals initiated; other ulcers are diagnosed as nonherpetic and treated for syphilis and chancroid.8 The Indian national guidelines for GUD differs from the WHO syndromic guidelines, which advocate treatment of vesicles for HSV-2 (and syphilis if rapid plasma reagin [RPR] positive or not received syphilis treatment recently); and for sores/ulcers without vesicles, treatment for syphilis, chancroid (and HSV if HSV-2 seroprevalence is >30%).7
The objectives of this study were to determine the etiology of GUDs in men presenting to STI clinics and to assess the performance of the national algorithm for syndromic management of herpetic and nonherpetic GUDs in India.
A cross-sectional study was conducted during May 2008 to June 2009 among male patients attending 8 STI clinics located in 4 states of India—Andhra Pradesh, Maharashtra, Manipur, and Delhi. Three clinics were non-governmental organizations administered HIV prevention interventions—2 for long distance truckers’ (Nagpur, Delhi) and another for injecting drug users (Manipur). The other 5 selected sites were public sector facilities; 2 were STI clinics of district hospitals (Nellore, AP and Vakola, Maharashtra) and the rest were the STI outpatient department of medical college hospitals (Hyderabad, Thirupati, and Mumbai). The sites were selected based on the rate and spectrum of GUD cases reported in 2009. All males with complaints of genital ulcer/second were enrolled based on eligibility criteria, which included age at least 18 years and not under the influence of drugs and alcohol. Written or witnessed oral consent was obtained from the individuals found eligible. A detailed clinical history was elicited, and clinical examination of the anogenital area was carried out by the clinic physician who had been trained on study protocols, clinical examination, syndromic diagnosis, and treatment before initiation of the study at each site. Genital ulcers were classified based on clinical manifestations; presence of vesicles or multiple painful ulcers with or without a history of recurrence as “herpetic”; and other ulcers were diagnosed as or “nonherpetic” and treated as per the national GUD algorithm. In addition, participants received free medicines as per the national guidelines, counseling for partner treatment and risk-reduction along with free condoms.
During clinical examination, ulcers were cleaned with a sterile Dacron swab moistened with saline and ulcer swabs were collected in 2-sucrose-phosphate transport medium. The swabs were stored at –20°C at the local laboratory and were shipped periodically on dry ice to the National AIDS Research Institute, Pune, India, where they were stored at –70°C until testing. Multiplex polymerase chain reaction (mPCR) testing was performed to detect and diagnose single infection with Treponema pallidum (TP), HSV, and Haemophilus ducreyi (HD) and mixed infections because of two or more etiologies at National AIDS Research Institute, Pune, India, as described by Orle and Weiss.9 In each test batch, we used target DNA for TP, HD, and HSV as positive controls (provided by Dr. Ian Maclean, University of Manitoba, Canada) as well as a negative control, thus replacing the target DNA with lysis buffer provided in the kit. Samples negative for TP, HD, and HSV (all 3 targets negative) in which PCR inhibitors identified were processed by phenol chloroform extraction and ethanol precipitation method, and these samples were retested for GUD pathogens by mPCR.9,10 The assay was validated by blinding target DNA of TP, HD, and HSV, and inclusion of negative control and known positive control TP, HD, and HSV. All positive mPCR results were repeated and reconfirmed.
All sera from participants were tested for syphilis using the RPR method (Span Diagnostics, Surat, India); and the TP hemagglutination assay test (Syphagen, TPHA Biokit, SA). Syphilis seropositive was defined as being RPR nonreactive or reactive (any titer) and a positive TPHA test.
This study was approved by the Ethics Committee of the National AIDS Research Institute and the Protection of Human Subjects Committee of Family Health International, North Carolina.
The data were double entered in CSPro (ORC Macro, Calverton, MD) and analyzed using STATA 12.0 (StataCorp, College Station, TX). For comparison of proportions, Pearson χ2 test was used. Odds ratio (OR) and confidence intervals (CIs) from univariate logistic regression was used to assess identify risk factors associated with laboratory-confirmed herpetic GUD.
The validity of the GUD flowchart was assessed by calculating the sensitivity, specificity, and positive predictive value (PPV) against the mPCR results (gold standard). The sensitivity is defined as the proportion of correct syndromic diagnosis and treatment of “herpetic” or “nonherpetic” infections as per the flowchart. The specificity is the proportion of patients without either “herpetic” or “nonherpetic” infections who were correctly identified by the flowchart as not having the condition. The PPV is defined as the proportion of syndromic diagnoses confirmed by etiological diagnosis.
During the study period, a total of 2862 STI cases attended the clinics. Two hundred thirty-five (n = 235) patients with GUD were recruited for this study. Of these, 41 patients were excluded from the analysis—12 patients refused study participation and ulcer swabs could not be collected in 29 patients. Thus, 194 patients for whom ulcer swabs were available and investigated for etiologies of genital ulcers were analyzed for the study. The demographic and behavioral characteristics are shown in Table 1. The profile of the participants (Table 1) shows that more than half (62.9%) had ever paid for sex with a commercial sex workers, three-quarters (76.3%) reported sex with nonpaid partner/second in the past and 29% in past 2 weeks. Reported condom use rates during commercial sex at the last sex act and consistent use was low.
Vesicles, a typical sign of genital herpes, were only observed in 18 (9%) patients. A large proportion (76.8%) of participants reported that the current genital ulcer was of >7 days duration; more than half (58.8%) had taken a prior treatment from a clinic, 5.7% had self-treated, whereas 35.6% had not had any prior medication. Patients with nonherpetic ulcers were more likely than those with herpetic ulcers to report recent sexual intercourse with a female sex worker (31% vs. 12%, P < 0.002). Patients with herpetic ulcers were more likely than those with nonherpetic ulcers to report a history of genital ulcer (55% vs. 29%, P < 0.01) and ulcer duration of >30 days (49 of 96, 51% vs. 28 of 98, 29%; P < 0.001). Laboratory-confirmed herpetic GUD showed significant associations with a higher age-group (>25 years; OR, 0.32; 95% CI, 0.16–0.63; P < 0.001), having history of genial ulcers (OR, 2.21; 95% CI, 1.21–3.98; P < 0.05), and those presented with painful ulcers (OR, 2.89; 95% CI, 1.58–5.30; P < 0.05).
The etiology was determined in 121 GUD cases (62%). Of 194 genital ulcer samples tested by mPCR, 93 (48%) had HSV followed by 44 (22.65%) TP and a case of HD. Seventeen (8.8%) ulcer specimens showed 2 etiological agents. Specimens from 73 (38%) cases were negative by mPCR test. The comparison between syndromic and etiological diagnoses is shown in Table 2. Among the cases clinically diagnosed as herpetic, 18.8% had TP while among those clinically diagnosed as nonherpetic, 34.7% had HSV in mPCR. The sensitivity (74% and 33%) and specificity (51% and 56%) of syndromic diagnosis of GUD were 74% and 33% for herpetic and 51% and 56% for nonherpetic ulcers, respectively. The overall sensitivity and specificity of the GUD flowchart were 68% and 52%, respectively, and the PPV was 50%. The sensitivity and specificity of vesicular lesions were 14% and 87%, respectively.
For all GUD patients, the overall syphilis serology reactivity (RPR nonreactive or reactive any titer and TPHA positive) rate was 17%. Syphilis seroreactivity rate was 30% (13 of 44) in mPCR positive versus 13% (20 of 150) among mPCR-negative TP cases.
In the study population, HSV (48%) was the most common etiology of GUD, consistent with findings from several nations indicating genital herpes as the leading cause of genital ulcers in Asia and Africa.3,4,11,12 However, a higher proportion of mPCR confirmed syphilis cases (23%), and a case of chancroid was identified in this series. In our series, a case of chancroid identified by mPCR in a long distance trucker, necessitates continuation of treatment of “nonherpetic” syndrome for syphilis and chancroid. In-depth study is also required to understand factors contributing to persistence of chancroid.
The rate of mixed infections (HSV and TP) in the current study demonstrates the challenges in clinically differentiating herpetic and nonherpetic infections. Despite using mPCR, a sensitive and specific test for common GUD pathogens, the etiology was not identified in 38% of the ulcers in this series. This rate is comparable with earlier studies conducted in India.3,4 Recently, several newer assays for detection of TP; real time PCR, multiplex real time PCR, and quantitative PCR were developed to improve turn-around time, sensitivity, specificity, and to minimize false positives.13–15 The reported specificity and sensitivity of these newer TP-PCR assays were 98% and 73% to 80%, respectively, for syphilitic ulcers. The quantitative PCR is shown to be highly sensitive (100%) and quantification of TP in ulcers improves our understanding of risk of acquisition/transmission HIV infection. Persistence of PCR inhibiting components (26%), despite our efforts for elimination and presence of other uncommon organisms, may have contributed to lower identification rates of etiological agents in our study.16
The sensitivity and specificity of the current national GUD flowchart in India is low. The sensitivity of the WHO algorithm for GUD (1995) was very low for genital herpes in Rwanda.17 The presence of vesicular lesions for diagnosing and other genital ulcers as syphilis and chancroid also yielded lower rates of sensitivity and specificity, but the sensitivity of GUD algorithm improved considerably with addition of treatment for herpes in all cases of genital ulcers.18 In the present series, there were 52 cases misclassified as either herpetic (18 cases) or nonherpetic (34 cases) GUD resulting in incorrect treatment. Almost a fifth of clinically diagnosed herpetic GUDs has syphilis infection, and would not have received the appropriate treatment. O’Farrell et al indicated the similarities between clinical appearances of various causes of GUD and presence of mixed infections may also result in inaccurate diagnosis.19
In the current study, discrepant results were obtained for 31 mPCR TP-positive patients who had a negative syphilis serology (RPR and TPHA). These results indicate that there might be low levels of viable or nonviable organisms that may have failed to elicit an immunologic response or may represent false-positive results. In the absence of a “gold standard” assay for diagnosis of primary syphilis, we compared the syphilis seroreactivity with TP mPCR results in this study. Few published studies have compared TP mPCR assays with serology results. Behets et al20 found that reactive syphilis (Toluidine Red Unheated Serum Test and Micro hemagglutination assay TP) serology was 75% sensitive and 85% specific compared with mPCR assay. A correlation of 88% with RPR or Venereal Disease Research Laboratory serology without confirmation by treponemal antibody test was reported by Orle et al.21 PCR assay was compared with a combination of RPR, TPHA/Treponema pallidum particle agglutination assay, FTA-Abs, and IgG and IgM Enzyme immunoassay and found a good correlation in patients with primary syphilis (94%–96%).13,14,22 Risbud et al4 reported a lower sensitivity of RPR and Fluorescent treponemal antibody–absorption (22% sensitivity and 98% specificity) among patients who presented with painless syphilitic ulcers. The low syphilis reactivity rate (30%; 13 of 44) among mPCR TP-positive ulcer cases could be explained by absence of antibodies in the standard nontreponemal and treponemal serological tests for syphilis until 1 to 4 weeks after the chancre appears.23 In the current series, >60% of cases had a duration of ulcer <30 days. Sixty three percent of study participants had received treatment from a local doctor or pharmacist before the consultation and might have resulted in immunologic nonresponse in these patients. Modern TP-specific assays (TPPA, IgM EIA, recombinant total antibody enzyme immunoassay) have shown high sensitivity in early syphilis, thereby use of a combination of tests would reduce the widely quoted “window period” in primary syphilis.14,23 In our study, high rates of TP-positive genital ulcers and presence or absence of syphilis serology in GUD patients suggest that all individuals with GUD should be treated empirically for syphilis, regardless of the clinical and serological diagnosis. Studies have shown an increased incidence and risk of HIV transmission among individuals, with bacterial and viral GUD and bacterial causes are easily treatable.1–5,24–26 Hence, it is important that the GUD algorithm does not miss curable causes (syphilis and chancroid) to avoid the long-term sequelae of these diseases and prevent HIV acquisition or transmission.
The US Center for Disease Control and Prevention (CDC) Sexually Transmitted Diseases Treatment Guidelines (2010) recommend antiviral therapy for recurrent genital herpes— either as suppressive therapy to reduce the frequency of recurrences or episodically to ameliorate or shorten the duration of lesions.27 Randomized control trials to assess the effectiveness of adding acyclovir in syndromic management of GUD in Africa yielded inconclusive results. But the pooled analysis indicated stronger effect among HIV-infected individuals suggesting acyclovir may be beneficial in HIV/HSV coinfected individuals.28 Although the WHO GUD algorithm recommends inclusion of treatment for HSV based on the prevalence, the cost of antiherpetic viral agents is a deterrent factor for inclusion in the management of all GUDs in India. However, inclusion of HSV treatment in GUDs among HIV-positive individuals may be considered. The presence of either herpetic or nonherpetic GUD in patients with unknown status is a good indication of the need for HIV testing as per the national guidelines in India.
Our study had some limitations. Biases might have introduced, as the sampling size was not developed on the estimated prevalence of GUD, which may have resulted in either over or underrepresentation of the specific syndrome in the study. However, enrollment of patients with GUD in select sites of primary, district, and tertiary health care facilities represented true distribution of “nonherpetic” and “herpetic” GUD cases in this study. Unfortunately, samples were not obtained from all eligible participants who were excluded in the study. Our study did not ensure follow-up of GUD patients. Hence, we could not detect seroconversion after 3 to 4 weeks to correlate with mPCR results. HIV testing was not done, which would have enabled us to correlate etiology and clinical manifestations of genital ulcers with HIV status.
In summary, the study findings suggest a revision of existing National GUD treatment guidelines in India to include treatment of syphilis for all GUD patients, irrespective of the clinical classification and syphilis serology. High detection rates of HSV and TP highlight the need for implementation of provider initiated HIV testing for patients with genital ulcer syndrome. Additional efforts are still needed for identifying pockets of persisting chancroid and making progress toward elimination of chancroid.
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