Afif Ben Salah

Topical Paromomycin with or without Gentamicin for Cutaneous Leishmaniasis

BACKGROUND There is a need for a simple and efficacious treatment for cutaneous leishmaniasis with an acceptable side-effect profile. METHODS We conducted a randomized, vehicle-controlled phase 3 trial of topical treatments containing 15% paromomycin, with and without 0.5% gentamicin, for cutaneous leishmaniasis caused by Leishmania major in Tunisia. We randomly assigned 375 patients with one to five ulcerative lesions from cutaneous leishmaniasis to receive a cream containing 15% paromomycin-0.5% gentamicin (called WR 279,396), 15% paromomycin alone, or vehicle control (with the same base as the other two creams but containing neither paromomycin nor gentamicin). Each lesion was treated once daily for 20 days. The primary end point was the cure of the index lesion. Cure was defined as at least 50% reduction in the size of the index lesion by 42 days, complete reepithelialization by 98 days, and absence of relapse by the end of the trial (168 days). Any withdrawal from the trial was considered a treatment failure. RESULTS The rate of cure of the index lesion was 81% (95% confidence interval [CI], 73 to 87) for paromomycin-gentamicin, 82% (95% CI, 74 to 87) for paromomycin alone, and 58% (95% CI, 50 to 67) for vehicle control (P<0.001 for each treatment group vs. the vehicle-control group). Cure of the index lesion was accompanied by cure of all other lesions except in five patients, one in each of the paromomycin groups and three in the vehicle-control group. Mild-to-moderate application-site reactions were more frequent in the paromomycin groups than in the vehicle-control group. CONCLUSIONS This trial provides evidence of the efficacy of paromomycin-gentamicin and paromomycin alone for ulcerative L. major disease. (Funded by the Department of the Army; ClinicalTrials.gov number, NCT00606580.).

Immunologic Determinants of Disease Evolution in Localized Cutaneous Leishmaniasis due to Leishmania major

Localized cutaneous leishmaniasis caused by Leishmania major is polymorphic in its clinical presentation and evolution. Clinical and parasitologic features and disease evolution of 112 Tunisian patients was evaluated. The expression of interleukin (IL)-4, IL-6, IL-10, IL-12 (p40), interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha mRNA was analyzed by reverse transcription-polymerase chain reaction in 73 biopsies. Cytokine mRNA expression varied individually over a wide range; TNF-alpha, IL-6, and IFN-gamma were detectable in >90% of lesions, IL-12 and IL-10 in 40% and 70%, respectively, and IL-4 in only 9%. Statistical analysis demonstrated positive association between the level of IL-12 and IFN-gamma and the presence of parasites in the lesions. Unfavorable evolution of the lesions was positively associated with high IL-10, IL-12, and IFN-gamma mRNA expression. These results indicate that an unfavorable clinical outcome was not related to an inadequate Th1 cell response and suggest that the macrophage-activating effect of IFN-gamma may be inhibited by the concomitant expression of IL-10.

WR279,396, a Third Generation Aminoglycoside Ointment for the Treatment of Leishmania major Cutaneous Leishmaniasis: A Phase 2, Randomized, Double Blind, Placebo Controlled Study

Background Cutaneous leishmaniasis (CL) is a disfiguring disease that confronts clinicians with a quandary: leave patients untreated or engage in a complex or toxic treatment. Topical treatment of CL offers a practical and safe option. Accordingly, the treatment of CL with WR279,396, a formulation of paromomycin and gentamicin in a hydrophilic base, was investigated in a phase 2 clinical study in Tunisia and France. Methods A phase 2, randomized, double blind, vehicle-controlled study was conducted to assess the safety and efficacy of topical WR279,396 when applied twice a day for 20 days as treatment for parasitologically confirmed CL. The study protocol established the primary efficacy end point as complete clinical response (CCR) defined as 50% or greater reduction in the ulceration size of an index lesion by day 50 (D50) followed by complete re-epithelialization by D100, and no relapse through D180. Results Ninety-two subjects were randomized. Leishmania major was identified in 66 of 68 isolates typed (97%). In the intent-to-treat population, 47 of 50 WR279,396 treated participants (94%) met the definition of CCR, compared with 30 of 42 vehicle-placebo participants (71%) [p = 0.0045]. Erythema occurred in 30% and 24% of participants receiving WR279,396 and placebo, respectively [p = 0.64]. There was no clinical or laboratory evidence of systemic toxicity. Conclusion Application of WR279,396 for 20 days was found to be safe and effective in treating L. major CL, and offers great potential as a new, simple, easily applicable, and inexpensive topical therapy for this neglected disease. Trial Registration ClinicalTrials.gov NCT00703924

Temporal Dynamics and Impact of Climate Factors on the Incidence of Zoonotic Cutaneous Leishmaniasis in Central Tunisia

Background Old world Zoonotic Cutaneous Leishmaniasis (ZCL) is a vector-borne human disease caused by Leishmania major, a unicellular eukaryotic parasite transmitted by pool blood-feeding sand flies mainly to wild rodents, such as Psammomys obesus. The human beings who share the rodent and sand fly habitats can be subverted as both sand fly blood resource. ZCL is endemic in the Middle East, Central Asia, Subsaharan and North Africa. Like other vector-borne diseases, the incidence of ZCL displayed by humans varies with environmental and climate factors. However, so far no study has addressed the temporal dynamics or the impact of climate factors on the ZCL risk. Principal Findings Seasonality during the same epidemiologic year and interval between ZCL epidemics ranging from 4 to 7 years were demonstrated. Models showed that ZCL incidence is raising i) by 1.8% (95% confidence intervals CI:0.0–3.6%) when there is 1 mm increase in the rainfall lagged by 12 to 14 months ii) by 5.0% (95% CI: 0.8–9.4%) when there is a 1% increase in humidity from July to September in the same epidemiologic year. Conclusion/Significance Higher rainfall is expected to result in increased density of chenopods, a halophytic plant that constitutes the exclusive food of Psammomys obesus. Consequently, following a high density of Psammomys obesus, the pool of Leishmania major transmissible from the rodents to blood-feeding female sand flies could lead to a higher probability of transmission to humans over the next season. These findings provide the evidence that ZCL is highly influenced by climate factors that could affect both Psammomys obesus and the sand fly population densities.

Characterization of the Antibody Response to the Saliva of Phlebotomus papatasi in People Living in Endemic Areas of Cutaneous Leishmaniasis

Important data obtained in mice raise the possibility that immunization against the saliva of sand flies could protect from leishmaniasis. Sand fly saliva stimulates the production of specific antibodies in individuals living in endemic areas of parasite transmission. To characterize the humoral immune response against the saliva of Phlebotomus papatasi in humans, we carried out a prospective study on 200 children living in areas of Leishmania major transmission. We showed that 83% of donors carried anti-saliva IgG antibodies, primarily of IgG4 isotype. Positive sera reacted differentially with seven salivary proteins. The protein PpSP30 was prominently recognized by all the sera. The salivary proteins triggered the production of various antibody isotypes. Interestingly, the immunodominant PpSP30 was recognized by all IgG subclasses, whereas PpSP12 was not by IgG4. Immunoproteomic analyses may help to identify the impact of each salivary protein on the L. major infection and to select potential vaccine candidates.

Analysis of Granzyme B Activity as a Surrogate Marker of Leishmania-Specific Cell-Mediated Cytotoxicity in Zoonotic Cutaneous Leishmaniasis

The purpose of this study was to analyze Leishmania-specific cell-mediated cytotoxicity in individuals with active or healed zoonotic cutaneous leishmaniasis (ZCL). The (51)Cr-release assay revealed a significant cytotoxicity against L. major-infected autologous macrophages in individuals with active or healed ZCL. As a surrogate marker of cytotoxic response, a test based on the measure of granzyme B activity in parasite-stimulated peripheral blood mononuclear cells was optimized and evaluated in 88 individuals. Increased granzyme B activity was found in 62.5% of patients with active ZCL and in only 30% of individuals with healed ZCL. In both groups, granzyme B activity was significantly higher than in healthy, leishmanin skin test-negative control subjects (P<.05). These data provide additional evidence that Leishmania-specific cell-mediated cytotoxicity is part of the acquired immune response developed against the parasite. Its role in resistance to reinfection should be evaluated.

The Predictive Validity of Naturally Acquired Delayed-Type Hypersensitivity to Leishmanin in Resistance to Leishmania major–Associated Cutaneous Leishmaniasis

To accurately quantify the different outcomes of Leishmania major infection and to evaluate the fraction of zoonotic cutaneous leishmaniasis (ZCL) cases prevented by naturally acquired leishmanin skin test (LST) reactivity, a cohort of 470 children was followed up in 2 endemic foci, Remada and Dhiba, in southern Tunisia. During May 1997, before the ZCL emergence season, LST was performed, and results were reassessed 12 months later. Active case detection during the ZCL emergence season showed a high incidence of ZCL: 57.0% in Remada and 13.7% in Dhiba. The preventive fraction of ZCL conferred by LST reactivity increased proportionally with the reaction size before the emergence season, revealing a dose-response effect of approximately 70%. In addition, asymptomatic L. major infection appeared to be a significant form of natural immunization, particularly in the context of relatively low transmission. These findings may help in the design and evaluation of vaccines.

Human cellular immune response to the saliva of Phlebotomus papatasi is mediated by IL-10-producing CD8+ T cells and Th1-polarized CD4+ lymphocytes.

Background The saliva of sand flies strongly enhances the infectivity of Leishmania in mice. Additionally, pre-exposure to saliva can protect mice from disease progression probably through the induction of a cellular immune response. Methodology/Principal Findings We analysed the cellular immune response against the saliva of Phlebotomus papatasi in humans and defined the phenotypic characteristics and cytokine production pattern of specific lymphocytes by flow cytometry. Additionally, proliferation and IFN-γ production of activated cells were analysed in magnetically separated CD4+ and CD8+ T cells. A proliferative response of peripheral blood mononuclear cells against the saliva of Phlebotomus papatasi was demonstrated in nearly 30% of naturally exposed individuals. Salivary extracts did not induce any secretion of IFN-γ but triggered the production of IL-10 primarily by CD8+ lymphocytes. In magnetically separated lymphocytes, the saliva induced the proliferation of both CD4+ and CD8+ T cells which was further enhanced after IL-10 blockage. Interestingly, when activated CD4+ lymphocytes were separated from CD8+ cells, they produced high amounts of IFN-γ. Conclusion Herein, we demonstrated that the overall effect of Phlebotomus papatasi saliva was dominated by the activation of IL-10-producing CD8+ cells suggesting a possible detrimental effect of pre-exposure to saliva on human leishmaniasis outcome. However, the activation of Th1 lymphocytes by the saliva provides the rationale to better define the nature of the salivary antigens that could be used for vaccine development.

A time series study on the effects of heat on mortality and evaluation of heterogeneity into European and Eastern-Southern Mediterranean cities: results of EU CIRCE project

BackgroundThe Mediterranean region is particularly vulnerable to the effect of summer temperature.Within the CIRCE project this time-series study aims to quantify for the first time the effect of summer temperature in Eastern-Southern Mediterranean cities and compared it with European cities around the Mediterranean basin, evaluating city characteristics that explain between-city heterogeneity.MethodsThe city-specific effect of maximum apparent temperature (Tappmax) was assessed by Generalized Estimation Equations, assuming a linear threshold model. Then, city-specific estimates were included in a random effect meta-regression analysis to investigate the effect modification by several city characteristics.ResultsHeterogeneity in the temperature-mortality relationship was observed among cities. Thresholds recorded higher values in the warmest cities of Tunis (35.5°C) and Tel-Aviv (32.8°C) while the effect of Tappmax above threshold was greater in the European cities. In Eastern-Southern Mediterranean cities a higher effect was observed among younger age groups (0–14 in Tunis and 15–64 in Tel-Aviv and Istanbul) in contrast with the European cities where the elderly population was more vulnerable. Climate conditions explained most of the observed heterogeneity and among socio-demographic and economic characteristics only health expenditure and unemployment rate were identified as effect modifiers.ConclusionsThe high vulnerability observed in the young populations in Eastern-Southern Mediterranean cities represent a major public health problem. Considering the large political and economic changes occurring in this region as well future temperature increase due to climate change, it is important to strengthen research and public health efforts in these Mediterranean countries.

Leishmaniasis: Middle East and North Africa research and development priorities.

The US-MENA Leishmaniasis conference provided an unprecedented opportunity for scientists to interact and focus on leishmaniasis research issues specific to the MENA region and resulted in several successful grant proposals funded through the Civilian Research and Development Foundation. The collaborations that arose from this conference will certainly influence leishmaniasis control in the MENA region specifically; moreover, the discussions and recommendations also have implications for the leishmaniasis field as a whole.