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Original Study

Etiology of Genital Ulcer Disease. A Prospective Study of 278 Cases Seen in an STD Clinic in Paris

Hope-Rapp, Emilie MD*; Anyfantakis, Vassili MD*; Fouéré, Sebastien MD*; Bonhomme, Philippe MD*; Louison, Jean B. MD*; de Marsac, Thibault Tandeau MD*; Chaine, Benedicte MD*; Vallee, Pascale MD*; Casin, Isabelle MD, PHD; Scieux, Catherine MD, PHD; Lassau, François MD*; Janier, Michel MD, PHD*

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Sexually Transmitted Diseases: March 2010 - Volume 37 - Issue 3 - p 153-158
doi: 10.1097/OLQ.0b013e3181bf5a98
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Sexually transmitted diseases (STD) and in particular genital ulcer diseases (GUD) have a major impact on morbidity and mortality in many countries, partially because of their ability to enhance the rate of sexual transmission of HIV.1–6 The most common causes of GUD are herpes simplex virus (HSV)-2, HSV-1, Treponema pallidum and Haemophilus ducreyi.7–12 In developing countries, leading causes of GUD are H. ducreyi and T. pallidum but HSV-2 is increasing rapidly. There is a significant variability in morphologic presentation of GUD, making the clinical interpretation unreliable when used without confirmatory laboratory tests. In the past decade, amplification techniques such as PCR have been developed to detect a number of infectious agents, including HSV-1, HSV-2, T. pallidum, and H. ducreyi.13–16 PCR can be performed for each agent separately or, more efficiently, by a multiplex assay.8,16,17

The aim of this study was to determine the prevalence of Treponema pallidum, HSV and Haemophilus ducreyi among patients attending a Paris STD clinic for a genital ulceration and factors associated with these 3 major pathogens during a decade.

PATIENTS AND METHODS

Study Population

A total of 283 patients attended the STD clinic of Hôpital Saint Louis in Paris, from January 1995 to December 2005 for a genital (penis, scrotum, vulva, vagina, pubis, and perineum) or anal ulceration lasting for less than 6 weeks. Balanitis and identified dermatological lesions; obvious chemical, caustic, or mechanical lesions; and patients with genital vesicles (at least 1); or in situ clinical recurrent erosions (i.e., typical genital herpes) were excluded. Patients having received antibiotics were not excluded. All patients underwent interview and examination, with use of standardized questionnaires and examination forms. Location, diameter in mm, induration, pain, depth (≥5 mm or <5 mm), and aspect was recorded for each ulceration. Participants gave informed consent and provided demographic information and medical and sexual histories.

Bacteriological Study

Exudates from genital ulcers were examined in each case under dark field microscope (Dark Field Examination DFE) for detection of spirochetes. A smear was obtained for Gram stain. Inoculation onto culture mediums was made in that order: Thayer- Martin agar, Columbia blood agar (with sheep blood enrichment) and chocolate agar with 1% isovitalex and 20% fetal calf serum, the latter medium being inoculated with swab obtained from the border of the ulceration for detection of Haemophilus ducreyi.

Presence of Chlamydia trachomatis was detected by PCR using C. trachomatis Amplicor C. trachomatis test from 1995 to 2000 and Cobas Amplicor C. trachomatis test from 2001 to 2005 (Roche diagnostics) from genital ulceration samples collected in 2 SP medium and first void urine samples.

All genital ulceration specimens found to be C. trachomatis positive were sent to the national reference centre (University of Bordeaux) where molecular genotyping was performed.

Virological Study

HSV isolation in cell culture was performed from genital ulceration specimens in viral transport medium. These samples were inoculated onto human fibroblast cells (MRC5). Virus typing was determined on infected-cells culture by use of monoclonal antibodies against HSV-1 and HSV-2 (Microtrak, Dade Behring, Paris, France).

For culture-negative samples between 1995 and 1999, HSV DNA was amplified by PCR-EIA (an home-grown HSV PCR as described by Guffond and revelation by DNA enzyme immunoassay GEN-ETI-K-DEIA, Sorin Biomedica, Saluggia, Italy).18 Between 1999 and 2005, HSV-1 and HSV-2 DNA analyses were performed using commercial PCR Kit (Herpes consensus generic and Hybridowell herpes identification kits, Argene-Biosoft, Varilhes, France).

Serological Tests for Antibodies

All patients were screened for the presence of HIV, HSV-1, and HSV-2 antibodies and Hepatitis B surface (HBs) antigen.

Serologic tests for syphilis (STS) included TPHA (Bayer Diagnostics, Puteaux, France), VDRL (VDRL cardiolipin antigen, Behring, Marburg, Germany) and FTA—abs test.

HIV screening was performed by using 2 HIV-1/2 antibody immunoassays, including an antibody/antigen combination screening test from 2001 to 2005. Reactive samples were confirmed by western-blot tests (New LAV Blot-1, Biorad, France).

After 1999, type-specific IgG antibodies to HSV-1 and HSV-2 in sera were detected using recombinant enzyme immunoassays (Captia HSV1 IgG Type Specific EIA and Captia HSV2 IgG Type Specific EIA, Trinity Biotech). Total IgG antibodies to HSV in sera were detected using enzyme immunoassays (Captia Herpes group IgG, Trinity Biotech, Bray, Ireland).

Criterion of Diagnosis

Syphilis was defined by a positive dark field examination (definite diagnosis) or by a negative dark field examination with at least 1 positive test for syphilis (probable).

Genital herpes was defined by a positive culture or PCR on genital ulcer. Chancroid was defined by a positive culture for Haemophilus ducreyi (definite) or by a negative culture with a positive direct microscopic examination (probable).

LGV was defined by a positive PCR for Chlamydia trachomatis (L serotype) on ulcer specimen.

Statistical Analysis

Demographics, clinical data and results on laboratory tests were abstracted onto a standardized form and analyzed using the 8.0 Version of Stata for Windows (Stata Corporation, College Station, TX). Hypotheses about categorical data were tested using Fisher exact test and χ2 Test. Associations between continuous variables (i.e., age, TPHA and VDRL titers, number of lesions, mean lesion area, etc.) were assessed by nonparametric tests (Mann-Whitney U test). Two-tailed P values <0.05 were considered to be significant.

Variables significant at a level of 0.2 in univariate analysis were fitted into multivariate models. Multivariate logistic regression models were constructed through backwards elimination to select independent risk factors for syphilis and HIV separately, while controlling for potential confounding factors.

RESULTS

Study Population Characteristics

Five patients were excluded subsequently because of lack of major clinical data (3 cases) or protocol violation (presence of vesicles) in 2 cases. Thus the study focused on 278 patients (244 male and 34 female) with a median age of 34 years. Their characteristics are summarized in Table 1. Males were significantly older (P < 0.0001). Most of the patients were French (52%), 19% were African and 29% from other countries. Men had a higher number of sex partners in the previous 6 months and were more likely to have a history of previous STD.

T1-5
TABLE 1:
Demographic, Behavioral, Clinical and Microbiological Characteristics of Patients With Genital Ulcer Disease (GUD)

The median duration of ulcerative lesions was 10 days. Fifty-seven men (24%) were circumcised. A total of 162 patients (58%) had only 1 lesion, 44 (16%) had 2 lesions, 25 (9%) had 3 lesions, and 45 (16%) had 4 or more ulcerations (data not shown). Among men, ulcers were situated in the coronal sulcus (73, 33%), on the glans penis (51, 23%), shaft of the penis (44, 20%), more rarely on the foreskin (17, 8%) and anal region (24, 11%). Lymph node enlargement was present in 113 (44%) of patients (data not shown).

Sixty-eight patients (27%) were HIV infected, GUD revealing HIV infection in 15 (22%). Prevalence of HIV-infection was 33% in syphilitic GUD and 25.5% in herpetic GUD.

Only 17 (9%) reported always using a condom for vaginal or anal sex, none for oral sex (data not shown).

Microbiologic Diagnosis

The overall prevalence of primary syphilis, genital herpes, chancroid, and LGV were 35%, 27%, 3%, and 1%, respectively, and another microbiologic diagnosis (herpes zoster, secondary syphilis, ecthyma, impetigo) was established in 4% (Table 1). All the investigations were negative in 32% (of whom 2 had aphthae and 1 a fixed drug eruption). Five patients, all male, had dual infections: 3 had both primary syphilis and genital herpes on the same ulceration, and 2 had chancroid respectively associated with herpes and syphilis.

Primary syphilis was the most common etiology of GUD among men (40%) and especially among men who have sex with men (MSMs) (71%). Only 1 woman had primary syphilis. Among patients with primary syphilis: 41 (44%) were DFE+, VDRL was reactive in 69 (73%), and TPHA in 83 (87%).

Genital herpes was more prevalent in women (50%) than in men (23%) (P < 0.0001). Culture was positive in 61 (HSV-1 in 23 and HSV-2 in 38). PCR was positive in 9 (HSV-1 in 3 patients and HSV-2 in 6). In 4 cases both culture and PCR were negative but the patients experienced subsequent recurrences typical of genital herpes (data not shown).

Eight patients had chancroid. Of them, 5 were African, 2 from Maghreb, and 1 from France. They all had a positive direct microscopic examination for Haemophilus ducreyi, and 7 of 8 were confirmed by culture. All cases of chancroid were observed before 2002.

LGV accounted for only 1 case of GUD in 2004, proctitis being in the recent epidemics far more common than genital ulcers. No case was identified before 2004 although Chlamydia trachomatis was searched by PCR in the ulcerations of all patients. However, 11 patients (4%) had a positive PCR for C. trachomatis in the FCU.

Clinical Predictive Factors for Syphilis, Herpes and Chancroid

There was no major difference in the clinical characteristics of GUD according to the microbiologic diagnosis.

Univariable Analysis.

For the 3 main etiologies (syphilis, herpes and chancroid), the localization of chancre (glans penis, foreskin, shaft, or anus), the mean and median number of lesions (2.3 and 1 for syphilis and 3 and 2 for genital herpes, respectively), the median duration of the ulceration (10 days), the lesion diameter, induration and depth of the ulcer, the presence and aspect of lymph nodes, were not different. Pain was the only item slightly more frequent in herpes than in syphilis (66% vs. 51%, P = 0.06) although it was not significant in the multivariable model.

Multivariable Analysis.

The only clinical characteristic significantly associated with an etiology in the multivariate model was a diameter >10 mm in syphilitic GUD (OR: 9.2 [95% CI: 2.9-30.7], P < 0.001) (Table 2).

T2-5
TABLE 2:
Risk Factor for Syphilitic GUD (Multivariable Analysis)

Sexual Habitus

Comparison between MSMs and men who have sex with women (MSWs) are presented in Table 1.

Compared to MSWs, MSMs were more frequently French, HIV infected, and had both more sexual partners and history of STDs. Primary syphilis was more frequent in MSMs, chancroid and genital herpes being more frequent in MSWs but only syphilis remained significantly associated with a sexual habit (MSM) in the multivariable model (OR: 51.3 [95% CI: 14.7-178.7], P < 0.001) (Table 2).

HIV-Status

Due to the small number of women, the analysis focused on men (Table 3).

T3-5
TABLE 3:
Demographic, Behavioral, Clinical and Microbiological Characteristics of Male Patients With GUD According to HIV Status

HIV infected men were more likely of African origin, more likely homosexual and had more frequently prior STDs. All these factors remained significant in the multivariable model (Table 4).

T4-5
TABLE 4:
Risk Factor for HIV Infection (Multivariable Analysis)

Both men with and without HIV infection had similar duration of ulceration, depth, induration, pain, and other functional symptoms. Size of ulceration was significantly greater in HIV + patients (OR: 3.6 [95% CI: 1.3-9.7], P = 0.012) (Table 4). Enlarged lymph nodes were more frequent in HIV + males (59% vs. 38%, P = 0.004) but this was not confirmed in the multivariable analysis.

A higher prevalence of syphilis observed in HIV + males in the univariate analysis was not confirmed in the multivariable analysis (homosexuality being the confounding factor). No difference was observed in the clinical characteristics of syphilitic GUD, according to HIV status.

Prevalence of genital herpes was similar in HIV infected and non-infected males but was higher in the subgroup of HIV infected MSWs compared to non infected (OR: 24.4 [95% CI: 2.4-247.7], P = 0.007). No association was found between clinical characteristics of herpetic GUD and HIV status (number of lesions were lower in HIV infected males—1.4 vs. 3.6, P = 0.01—but this was not confirmed in the multivariable model). H. ducreyi was isolated similarly in HIV infected and non infected men. Finally a pathogen (whatever) was found more frequently in HIV infected patients (85% vs. 64%, P = 0.004).

DISCUSSION

Genital ulcerations, after exclusion of typical cases of genital herpes, occur in less than 5% of patients attending the 9 Sexually Transmitted Diseases Clinics in inner Paris (Dr N. Boo, personal communication). In a previous study of 75 cases of GUD collected in males in 1986-1987 in our clinic, primary syphilis, genital herpes, and chancroid accounted each for one-fourth of etiologies, the other 25% being of unknown origin.19 Since that time chancroid has disappeared in our country, the last case was seen in our Hospital in 2002. The number of GUD has decreased along the years with less than 20 cases per year between 1995 and 1999 and raised subsequently to 40 to 50 cases per year from 2000 on, due to an epidemic of early syphilis occurring mainly in MSMs, many of them being HIV-seropositive.20 Genital herpes was responsible for the majority of GUD cases during the mid 90s, and although it was stable along the years in absolute numbers, its proportion of the total annual burden of GUD decreased along with the recent syphilis epidemics. Moreover, lymphogranuloma venereum (LGV), the last cases of which had been observed in our country in 198921 has reappeared in 2003, although presenting much more often as a proctocolitis than a genital ulceration.

We had previously shown that the clinical aspect of the ulceration (namely pain, depth, size, and induration) was poorly correlated to the etiology, with the exception of buboes strongly associated to chancroid.22 This may be due in part to long delays before presentation (a median of 10 days) and to secondary colonization with bacteria such as staphylococci.12 Similar findings have been published in different settings, the positive predictive value of clinical examination being low in most of the studies, the higher values hardly reaching 70%.9,11,15 Another difficulty we did not confirm in the current study is the finding of multiple pathogens in the same ulceration, which accounted for only 5 cases (mainly syphilis associated with herpes) in our cohort but reached 23% in another series.11 In the present work, a diameter >10 mm was significantly associated with syphilis but no other clinical character was predictive of any etiology.

In one-third of the cases, the ulceration was of unknown origin i.e., DFE, STS, search for HSV by culture and PCR, PCR for C. trachomatis, and, finally, culture for H. ducreyi, were all negative. This compares both with other series8,11,17 and with our findings in 1990 (25%),22 although at that time we only used less sensitive laboratory exams such as culture for C. trachomatis and HSV.

Syphilis has reappeared in our country in 1999 in the gay community and has plateaued ever since, with some 500 annual cases of early syphilis reported in the French Voluntary Surveillance System of Sexually Transmitted Infections (ResIST network).20 Among the 2892 patients notified to the network from 2000 to 2007, 83% were MSMs, 45% were HIV-infected, and 46% asserted to always use condoms for penetrations, oral sex being alleged as the probable source of infection. Underestimation of the number of primary syphilis must be considered when both DFE and STS are negative but overestimation is also possible in DFE negative patients with positive STS. In the future years, use of PCR for T. pallidum will probably help.23

In this study, genital herpes was the second etiology of genital ulceration on a whole but still the most frequent in women and in MSW as already established in most studies, including in developing countries.24–26 In this cohort only ulcerations which were not typical of herpes were included i.e., we excluded patients with vesicles on physical examination and overt recurrent herpetic lesions. Inclusion of all cases of genital herpes during the same period would have given an overwhelming prevalence of genital herpes. To give an idea of the proportion, 255 cases of herpes have been collected in our clinic between 1999 and 2002 of whom only 16 were included in the current study.27 Although PCR is a sensitive technique,15 allowing us to diagnose 9 more cases of genital herpes in which culture was negative, underestimation is still likely. Most of the cases (63%) were HSV-2 positive and the majority (73%) were consistent with serologically-defined HSV-2 recurrences, although the majority of them experienced a first clinical episode. In a previous work, we had shown that among 255 consecutive cases of genital herpetic lesions, culture was only 85% sensitive when compared to PCR and that 37% of serologically—defined herpetic recurrences presented as a first clinical episode. These findings underline the importance of performing a search for herpes, preferably by PCR in all cases of genital ulceration, whatever its clinical appearance.

In the current study, we found a significant higher prevalence of syphilis in MSMs. Indeed MSMs account for the great majority of cases of early syphilis in developed countries, figures ranging from 50% up to 90%, according to studies. In the French National survey, this percentage remains very high (83%) although cases in heterosexual men and women are slowly increasing.28 This is true for both secondary and primary syphilis. MSMs also account for 100% of cases of LGV in the same survey. For comparison, MSMs account for 65% of cases of gonorrhea and 20% of men attending the STD clinic of our hospital. Several studies show that MSMs are keeping on with high risk behavior.29 Our study confirms this difference, the mean number of sexual partners in the previous 6 months being 15 in MSMs versus 2 in MSWs and 1 in female patients. According to a standardized national self-reporting study of gay men's sexual behavior, the number of reported acts of unprotected anal intercourse in the previous year doubled between 1997 and 2004 among both seropositive and seronegative men.29

The association between syphilis and HIV being no more significant in the multivariable analysis, no specific cause of GUD was significantly associated with HIV, apart from genital herpes in MSWs, but still the HIV prevalence was very high in all the groups studied underlining the close relationship between genital ulcerations and HIV. Moreover, it is noteworthy that 22% of HIV-positive patients of this study (15/68), mostly with infectious syphilis, were unaware of their seropositivity and still engaged in high risk sexuality. In a meta-analysis made in 2001, GUD were shown to enhance both susceptibility to HIV and infectiousness; GUD, syphilis, herpes, and chancroid had a combined effect estimate on susceptibility of 2.7, 2.5, 2.7, and 2.1, respectively, with higher effect in heterosexual males.3 The effect on infectiousness seems lower although HIV viral load is increased both in plasma and genital specimens from patients with concurrent STD, especially genital herpes.30,31 Several trials have, until now unsuccessfully, aimed at lowering the transmission of HIV by giving suppressive antiherpetic therapy to patients coinfected with HIV and HSV-2.32 Overall, 27% of our patients were HIV-infected, and HIV seropositivity reached 33% in syphilitic GUD and 40% in MSMs with syphilis, a percentage far higher than the prevalence of 2% seropositivity in our clinic attendees.33 More than 2000 new cases of HIV infection occurring in MSMs have been reported in 2007 in the French mandatory HIV Notification System. Although the annual number has stabilized, MSMs are the only group in which there is no decline.28

In the current study, HIV status was not associated with any particularity of genital ulcerations globally, apart from a greater size of the ulcers (OR: 3.6). In syphilitic ulcerations, TPHA titers were anecdotically slightly higher in HIV-positive patients, but no difference was found between HIV-positive and negative cases clinically. The mean and median number of lesions, the size of ulcers were not different according to HIV status. In previous works, both Rompalo et al and Rolfs et al34,35 had shown that multiple lesions were slightly more frequent in HIV-positive patients with syphilitic GUD, which we did not confirm. In herpetic ulcerations, only slight clinical differences were found, with a lower number of lesions in HIV-positive cases, which was not confirmed in the multivariable study and a tendency for a larger surface area which was not significant. This may be because most were treated with HAART and although we did not check systematically CD4 + cell count, very few were severely immune-suppressed. Moreover, we did not collect prospective data on the time required for healing, which may be longer in HIV- infected patients.36

We recognize that this study has several limitations. Although it was a prospective case series of genital ulcers, we cannot be certain that all cases of GUD seen during this period were included. Exclusion criteria such as presence of at least 1 vesicle and/or typical recurrent herpes are rather subjective and may differ from one investigator to another; moreover coinfection by syphilis or chancroid is possible in typical vesicular herpes. Second, several laboratory tests have changed along the decade and we have not used T. pallidum PCR. Third, the number of patients in each group was relatively low for statistical evaluation, specially chancroid and GUD in women. Finally, the study does not mean to be a true representation of the epidemiology and trends of GUD in France, with our particular selection bias of being a referral university department.

However, there are very few studies on GUD in developed countries, in particular in Europe and the important lessons from our work remains: (1) the absence of strong clinical predictive factors for a definite diagnosis of syphilis or herpes in patients with GUD and the necessity of performing laboratory tests for these 2 etiologies; (2) the important number of cases still remaining unexplained, which may be partly solved by using PCRs; and (3) the minor impact of HIV on the presentation of most GUD. During the past decade, the disappearance of chancroid and the resurgence of infectious syphilis have brought substantial changes to the epidemiology of GUD. Although, genital herpes remains the leading cause in heterosexuals and females, primary syphilis has become the most frequent etiology of genital ulcerations in MSMs. Risk reduction messages are more than ever necessary for targeting this group.

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