K. Aoun

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.

Cutaneous Leishmaniasis in North Africa: a review

In North African countries, cutaneous leishmaniasis transmission has been increasing since the 1980s, with a significant increase in the incidence of cases and a spread of the geographical distribution. The disease currently represents a major public health problem with a productivity gap and an impediment for development, which results in dramatic socioeconomic and psycho-sanitary impacts. The incidence is more than thousands of cases every year in Algeria, Libya, Morocco, and Tunisia. In Egypt, only a few dozen cases per year are reported, mainly in the Sinai Peninsula. Three Leishmania species, associated with distinct eco-epidemiological and clinical patterns, are involved, namely Leishmania infantum, L. major, and L. tropica. However, L. major is by far the most frequent in Algeria, Libya, and Tunisia, with more than 90% of the registered cases. It is mainly encountered in rural areas under semi-arid, arid and Saharan climates. Leishmania tropica is more prevalent in Morocco, reaching 30–40% of isolates in some districts. Much data is still missing concerning the risk factors of the infection and the lesion development, as well as vector and reservoir ecology and behavior. The knowledge of such parameters, following multidisciplinary and integrated approaches, is crucial for better management and control of the disease, that also faces a lack of resources and efficient control measures.

Population structure of Tunisian Leishmania infantum and evidence for the existence of hybrids and gene flow between genetically different populations

Twenty-seven strains of Leishmania infantum from north and central Tunisia belonging to the three main MON zymodemes (the MON-typing system is based on multilocus enzyme electrophoresis (MLEE) of 15 enzymes) found in this country (MON-1, MON-24 and MON-80) and representing different pathologies (visceral, cutaneous and canine leishmaniasis) have been studied to understand the genetic polymorphism within this species. Intraspecific variation could be detected in L. infantum by the use of 14 hypervariable microsatellite markers. In addition to microsatellite repeat length variation, a high degree of allelic heterozygosity has been observed among the strains investigated, suggestive of sexual recombination within L. infantum groups. The two major clusters found by using Bayesian statistics as well as distance analysis are consistent with the classification based on isoenzymes, dividing Tunisian L. infantum into MON-1 and MON-24/MON-80. Moreover, the existence of hybrid strains between the MON-1 and the non-MON-1 populations has been shown and verified by analysis of clones of one of these strains. Substructure analysis discriminated four groups of L. infantum. The major MON-1 cluster split into two groups, one comprising only Tunisian strains and the second both Tunisian and European strains. The major MON-24 cluster was subdivided into two groups with geographical and clinical feature correlations: a dermotropic group of strains mainly from the north, and a viscerotropic group of strains from the centre of Tunisia. The four viscerotropic hybrid strains all originated from central Tunisia and were typed by MLEE as MON-24 or MON-80. To our knowledge, this is the first report describing relationships between clinical picture and population substructure of L. infantum MON-24 based on genotype data, as well as the existence of hybrids between zymodemes MON-1 and MON-24/MON-80, and proving one of these hybrid strains by molecular analysis of the parent strain and its clones.

Seroprevalence of Toxoplasma gondii infection among horses in Tunisia

BackgroundThe present study was conducted to investigate the serological survey of Toxoplasma antibodies in local.horses from three major regions: a neighbourhood of a city in the North (Sidi Thabet), a neighbourhood of a city on the coast (Monastir) and a neighbourhood of a city in the middle (Battan) of Tunisia (North of Africa).MethodsA total of 158 serum samples were obtained from clinically healthy horses which consisted of 111 (32 female, 79 male) 2-10 years old and 47 (11 female, 36 male) older than 10 years. All of the horses were tested for antibodies to T. gondii using the Modified Agglutination Test (MAT).ResultsAccording to MAT results, antibodies to T. gondii were found in 28 (17.7%) of 158 sera with the titers of 1:20 in 20 horses, 1:40 in 1 horse, 1:80 in 2 horses, 1:160 in 2 horses, 1:320 in 1 horse and ≥1:640 in 2 horses. Anti-T. gondii antibodies were found in 18 (16.2%) of 111 horses (2-10 years old) and 10 (21.2%) of 47 horses (older than 10 years old). Six (13.9%) out of 43 female had anti-toxoplasma antibodies and 22 (19.1%) from 115 males remained positive.ConclusionStatistically significant differences in age groups and genders were observed between the seropositive and seronegative horses using the Chi square X(2) test. Other statistical correlation was also reported concerning horse breed.

Diagnosis of Mediterranean Visceral Leishmaniasis by Detection of Leishmania Antibodies and Leishmania DNA in Oral Fluid Samples Collected Using an Oracol Device

ABSTRACT Current methods for diagnosis of visceral leishmaniasis (VL) require invasive sampling procedures such as visceral aspiration and/or blood drawing. The use of diagnostic tests using oral fluid, which is easier to collect, would be more simple and practical for VL diagnosis, especially under field conditions. Oral fluids from 37 VL patients and 40 healthy controls were collected using Oracol devices. Blood samples and oral fluid specimens from both groups were analyzed by recombinant protein K39 (rK39) enzyme-linked immunosorbent assay and quantitative real-time PCR. Detection of antibodies in the oral fluid had a sensitivity of 100% and a specificity of 97.5%. Antibody levels measured in serum and oral fluid showed a significant positive correlation (ρ = 0.655 and P = 0.01). Detection of Leishmania DNA in oral fluid had a sensitivity of 94.6% and a specificity of 90%. The median parasite load estimated in blood was 133 parasites/ml (interquartile range [IR], 10 to 1,048), whereas that in oral fluid specimens was 3 parasites/ml (IR, 0.41 to 92). However, there was no significant linear relationship between parasite loads assessed in the two biological samples (ρ = 0.31 and P = 0.06). VL diagnosis based on specific antibody detection and Leishmania DNA identification using oral fluid samples was equivalent in accuracy to that using blood and therefore is promising for clinical use.

Natural Infection of North African Gundi (Ctenodactylus gundi) by Leishmania tropica in the Focus of Cutaneous Leishmaniasis, Southeast Tunisia

North African gundis (Ctenodactylus gundi) were trapped in the Leishmania (L.) tropica focus of cutaneous leishmaniasis, situated in southeast Tunisia and evaluated for Leishmania infection by real-time kinetoplast DNA polymerase chain reaction (PCR). Species identification was performed by internal transcribed spacer one (ITS1)-PCR-restriction fragment length polymorphism (RFLP) and high-resolution melting (HRM) analysis of the 7SL RNA gene. Real-time PCR on blood was positive in 6 of 13 (46.2%) tested gundis. Leishmania tropica was identified in five infected gundis and Leishmania major in one specimen. Alignments of the ITS-1 DNA sequences and 7S-HRM curves analysis indicated that similar genotypes were present in humans, a sandfly, and gundis from the same region suggesting a potential role of this rodent as reservoir host of L. tropica in southeast Tunisia.

First report on natural infection of Phlebotomus sergenti with Leishmania promastigotes in the cutaneous leishmaniasis focus in southeastern Tunisia.

During September 2010, 133 female sand flies were caught inside houses of patients with cutaneous leishmaniasis in the focus for this disease in southeastern Tunisia and subsequently dissected. One specimen was positive for Leishmania protozoa. This sand fly species was identified as Phlebotomus sergenti, and the parasite was identified as L. tropica. This is the first report of P. sergenti involvement in transmission of L. tropica in Tunisia.

Actualités épidémiologiques de la leishmaniose viscérale en Tunisie

OBJECTIVE Visceral leishmaniasis is an important health problem in Tunisia. The aim of this study was to update the epidemiological and clinical features of the disease. DESIGN We performed a retrospective systematic sampling of epidemiological and clinical data collected from the medical records of 1,096 cases of visceral leishmaniasis diagnosed between 1996 and 2006 all over the country. RESULTS The mean annual incidence of cases was 99.6 cases/year. The mean annual incidence rate was 1.04 cases/100,000 inhabitants, showing an important increase compared to former studies. As expected, children under 5 years (866 cases) were the most affected with a mean annual incidence rate of 9.6 cases/100,000 (p<0.001). The geographical distribution of cases revealed the spreading of the disease from the Northern parts of the country to the Central and even to Southern ones. Rural cases (65.3%) were significantly more numerous than urban ones (34.7%), p<0.001. The sex ratio was 1.03. The diagnostic delay (average of 54 days) was considerably shortened during the study period compared to previous reports, and explains the decrease of the lethality rate (2.9%). CONCLUSIONS Visceral leishmaniasis has been present in central Tunisia since the early 1990 s. Its incidence and the distribution area have increased. This evolution is probably linked to the development of irrigation and agriculture favorable to the multiplication of vector sandflies and dogs reservoirs of Leishmania infantum.

Données épidémiologiques sur la leishmaniose viscérale infantile en Tunisie en 1993

Summary In order to list all cases of human visceral leishmaiasisdiagnosed in Tunisia during 1993, a retrospective study, based on the epidemiological record of the ministry of health and investigations in the most important hospitals on the north, was performed. 153 cases were reported essentially in children of 1 to 3 years of age (64%). The cases were distributed mainly in the north and the center of Tunisia. This study confirmed the increase of the incidence of visceral leishmaniasis and its geographical extention to the center of the country these last years.

Advantages and limits of real-time PCR assay and PCR-restriction fragment length polymorphism for the identification of cutaneous Leishmania species in Tunisia

Cutaneous leishmaniasis (CL), a public health problem in Tunisia, is associated to three species: Leishmania (L.) infantum, L. major and L. killicki. Accurate and sensitive procedures for the diagnostic of Leishmania infection and for species identification are required to enable adequate treatment and appropriate control measures. Several PCR-methods are applied for the diagnosis and the identification of Leishmania parasites such as PCR-restriction fragment length polymorphism (PCR-RFLP), DNA sequencing, hybridization probes and real-time PCR (RT-PCR). In this study, PCR-RFLP and RT-PCR were performed on skin scrapings from 27 patients with confirmed CL by microscopic examination, in order to compare their usefulness and efficiency for identification of Leishmania species in routine diagnostic laboratories. Identification of Leishmania species was successfully achieved in 96.3% and 81.5% respectively. Agreement between using internal transcribed spacer 1 (ITS1)-PCR-RFLP and kDNA-RT-PCR assays was 70% (19/27). Characterization problems using RT-PCR were mainly due to the difficulties in analyzing the melting temperatures. ITS1-PCR-RFLP and kDNA-RT-PCR presented an interesting alternative to conventional methods for the identification of Leishmania parasites from clinical samples. Both PCR assays can be used in a routine diagnostic, however, further prospective studies including largest sampling, are required to determine their performances in a routine use.