Leishmania major infection among Psammomys obesus and Meriones shawi: reservoirs of zoonotic cutaneous leishmaniasis in Sidi Bouzid(central Tunisia)

A study was undertaken between November 2008 and March 2010, in the focus of cutaneous leishmaniasis of Central Tunisia, to evaluate the role of Psammomys obesus (n=472) and Meriones shawi (n=167) as reservoir hosts for Leishmania major infection. Prevalence of L. major infection was 7% versus 5% for culture (p=not signifiant [NS]), 19% versus 16% for direct examination of smears (p=NS), and 20% versus 33% (p=NS) for Indirect Fluorescent Antibody Test among P. obesus and M. shawi, respectively. The peak of this infection was in winter and autumn and increased steadily with age for the both species of rodents. The clinical examination showed that depilation, hyper-pigmentation, ignition, and severe edema of the higher edge of the ears were the most frequent signs observed in the study sample (all signs combined: 47% for P. obesus versus 43% for M. shawi; p=NS). However, the lesions were bilateral and seem to be more destructive among M. shawi compared with P. obesus. Asymptomatic infection was ~40% for both rodents. This study demonstrated that M. shawi plays an important role in the transmission and the emergence of Leishmania major cutaneous leishmaniasis in Tunisia.     

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.     

Human cutaneous leishmaniasis due to Leishmania infantum in the Sidi Bourouis focus (Northern Tunisia): epidemiological study and isoenzymatic characterization of the parasites

The authors report an increase of the number of case of cutaneous leishmaniasis in the Sidi Bourouis region community of Siliana Governorate (Tunisia), with 38 cases diagnosed in 6 months (1st November 2000-30th April 2001), contrary to its usual sporadic character. The isoenzymatic identification of 15 isolated strains emphasizes the role of the Leishmania infantum zymodeme MON-1 as an important factor in the genesis of the sporadic cutaneous leishmaniasis of Northern Tunisia. In fact it was isolated in 6 cases while L. infantum MON-24, the usual agent was isolated in 9 cases.     

Cutaneous leishmaniasis in Tunisia: results of the iso-enzymatic characterization of 71 strains

Three clinico-epidemiological forms of cutaneous leishmaniasis (CL) exist in Tunisia: zoonotic cutaneous leishmaniasis (ZCL; epidemic in the centre and the south-west); sporadic cutaneous leishmaniasis (SCL; found in the north); and chronic cutaneous leishmaniasis (CCL; originally described from Tataouine, in the south-east). As few isolates of Leishmania from Tunisian cases of CL have been typed, isolates were collected, using NNN medium, from 71 such cases. Most (59) of the cases investigated came from the north of the country, including 16 from Sidi Bourouis, where there was an epidemic outbreak of SCL in early 2001; the other 12 cases were natives of the centre or south of the country. The 71 strains were then characterized, at the Centre National de Référence des Leishmania, in Montpellier, France, by iso-enzyme analysis. This revealed four zymodemes: two of L. infantum and one each of L. major and L. killicki. The MON-1 zymodeme of L. infantum, which is more usually associated with visceral leishmaniasis, was recovered from seven of the cases, including six natives of Sidi Bourouis. The MON-24 zymodeme of this species, which appears to be responsible for the SCL, was isolated from 48 cases, all of whom lived in the north of the country. Another 15 cases (nine from the centre, four from the north, and two from the south-east of the country) were found to be harbouring L. major MON-25, the zymodeme usually causing ZCL. Only a single isolate of L. killicki was made; this was of the MON-8 zymodeme responsible for the CCL, and came from a native of Gafsa, in the south-west. Six of the cases investigated (five infected with L. infantum MON-24 and one with L. major MON-25) showed involvement of their nasal and labial mucosae. These results increase the number of strains typed from Tunisian cases of CL more than four-fold, and should help to elucidate the geographical distribution and epidemiology of the various forms of the disease.     

Habitats of the sandfly vectors of Leishmania tropica and L. major in a mixed focus of cutaneous leishmaniasis in southeast Tunisia

From 2009 to 2010, 3129 sandflies were caught in CDC light traps placed in various habitats in Ghomrassen, Tataouine governorate, southeast Tunisia, a mixed focus of human cutaneous leishmaniasis caused by Leishmania tropica and Leishmania major. Species diversity was quantified in anthropogenic, semi-anthropogenic and semi-natural locations. Sandflies were identified according to morphological characters and also by the comparative sequence analysis of a fragment of the mitochondrial cytochrome b gene to distinguish between two putative local vectors of L. tropica, namely Phlebotomus chabaudi and Phlebotomus riouxi. The lowest sandfly diversities were found in L. major sites, where the incriminated vector P. papatasi predominated in the burrows of the rodent reservoir hosts (Meriones) as well as inside and outside houses of human cases. In L. tropica sites, the incriminated peri-domestic vector Phlebotomus sergenti was the most abundant species inside houses, whereas P. riouxi or P. chabaudi was the dominant species in the semi-natural rocky habitats favoured by the putative rodent reservoir, Ctenodactylus gundi. All specimens of P. chabaudi identified molecularly had the diagnostic cytochrome b characters of P. riouxi, indicating either that the latter represents only a geographical variant of P. chabaudi or that these two species may sometimes hybridize.     

Geographical distribution and epidemiological features of Old World cutaneous leishmaniasis foci, based on the isoenzyme analysis of 1048 strains

A series of 1048 Leishmania strains from Old World cutaneous leishmaniasis foci, isolated between 1981 and 2005, were studied by isoenzyme analysis. The strains were obtained from humans, rodents, dogs and sandflies from 33 countries. The four typically dermotropic species, Leishmania major , L. tropica , L. aethiopica and L. killicki , were found. The viscerotropic species L. donovani and L. infantum, which can occasionally be responsible for cutaneous leishmaniasis, are not considered in this paper. Leishmania major was the least polymorphic species (12 zymodemes, 638 strains). Leishmania tropica was characterized by a complex polymorphism varying according to focus (35 zymodemes, 329 strains). Leishmania aethiopica , a species restricted to East Africa, showed a high polymorphism, in spite of a limited number of strains (23 zymodemes, 40 strains). Leishmania killicki , mainly restricted to Tunisia had a single zymodeme for 39 strains. Recently a parasite close to L. killicki (one zymodeme, two strains) was isolated in Algeria, which lead us to revise the taxonomic status of this taxon.     

Cutaneous leishmaniasis treatment

The causative species of cutaneous leishmaniasis determines the clinical features and courses, and treatments. Intralesional or systemic antimonials are the gold standard for the treatment of these diseases. However, as for visceral leishmaniasis, other therapeutic options appear promising. Paromomycin ointments are effective in Leishmania major, L. tropica, L. mexicana, and L. panamensis lesions. In L. braziliensis localized leishmaniasis, both paromomycin and imiquimod may be topically applied. Oral fluconazole and zinc sulfate are useful in L. major. Oral azithromycin, effective in vitro and in mice, needs further investigation in human leishmaniasis. On the contrary, data with oral itraconazole are disappointing. Oral miltefosine, which is very effective in visceral leishmaniasis caused by L. donovani, appears ineffective in L. major and L. braziliensis infections. Intramuscular pentamidine is required for L. guyanensis cutaneous leishmaniasis, for which systemic antimony is not effective. Liposomal amphotericin B could be an alternative to antimony in south American cutaneous leishmaniasis with mucosal involvement (especially L. braziliensis and L. guyanensis infections).     

Twenty-four new human cases of cutaneous leishmaniasis due to Leishmania killicki in Metlaoui, southwestern Tunisia: probable role of Phlebotomus sergenti in the transmission

Metlaoui district in the South-west of Tunisia is a classical focus of cutaneous leishmaniasis (CL) due to Leishmania major. Since 2005, a single case of CL due to L. killicki has been reported. We report twenty four human cases due to this parasite, affecting men and women from 2 to 70 years old. Leishmania killicki have been typed using molecular techniques: polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and gene sequencing. Four strains from patients have been successfully cultured on NNN medium and isoenzymatically typed as L. killicki MON-8. Our results strongly suggests that Metlaoui is a new L. killicki focus with a stable transmission cycle. Sand flies fauna in the same focus was also studied. 1400 Phlebotomine sand flies (785 males/615 females) have been caught during an entomological survey. Leishmania major DNA has been found in one P. papatasi female, the most abundant species, whereas L. killicki DNA has been found in one Phlebotomus sergenti female emphasizing the probable role of this species as vector of this zoonotic parasite.     

Epidemiologic and clinical features of cutaneous leishmaniasis in southeastern Tunisia

Species-specific diagnosis was performed in 66 patients with cutaneous leishmaniasis (CL) living in Tataouine focus in southeastern Tunisia. Leishmania DNA was extracted directly from dermal scrapings (n = 66) and from parasites obtained in culture (n = 12). Species were identified by using polymerase chain reaction-restriction fragment length polymorphism analysis for internal transcribed spacer region 1 and isoenzyme analysis. Leishmania tropica and L. major were identified in 31 (47%) and 35 (53%) cases respectively. Leishmania tropica CL cases were geographically scattered, and L. major CL cases were clustered. Lesions caused by L. tropica were mostly single (83.8%) and face-localized (55.8%), and lesions caused by L. major were multiple (57.1%; P < 0.001) and situated on limbs (83.7%; P < 0.001). For both species, most lesion onsets were reported during June-January. However, lesions that emerged during February-May were mainly caused by L. tropica (83.3%; P < 0.01). Moreover, the delay before seeking medical advice was higher for L. tropica infections than for L. major infections (P < 0.05).     

Cutaneous leishmaniasis in Jordan: biochemical identification of human and Psammomys obesus isolates as Leishmania major

As part of a series of epidemiological and ecological studies of leishmaniasis in Jordan, we have made functional studies of four isolates from human lesions and from ear lesions of three field-collected Psammomys obesus. Primary isolates were subcultured, frozen stabilates prepared and BALB/c mouse infectivity experiments initiated. Each mouse was inoculated with 4-8 x 10(4) promastigotes into a hind footpad. Quantitative evaluation of the footpads showed enlargement three to four weeks postinoculation. Amastigotes were readily identified in smears from footpad lesions and promastigotes in culture. At 47 days, liver and spleen samples grew out promastigotes. Biochemical characterization of these seven isolates was made by isozyme analysis using cellulose acetate membrane electrophoresis of fructokinase, phosphoglucose isomerase, phosphoglucomutase, aspartate aminotransferase, malate dehydrogenase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Reference isolates used for comparison were Leishmania major, L. tropica minor, L. donovani, L. aethiopica and L. m. mexicana. All seven Jordan isolates showed enzyme electromorphs identical to L. major, confirming our ecological/epidemiological studies that P. obesus is a major reservoir for human cutaneous leishmaniasis in Jordan.     

Climate change and potential distribution of zoonotic cutaneous leishmaniasis in Central Iran: Horizon 2030 and 2050

Objective: To investigate and predict the effects of climate change on the potential distribution of the main vector and reservoir hosts of the disease in Yazd province in the future.Methods: Distribution data for vector and reservoir hosts of zoonotic cutaneous leishmaniasis in Yazd province were obtained from earlier studies conducted in the area.MaxEnt ecological niche modeling was used to predict environmental suitability.BCC-CSM1-1(m) model and two climate change scenarios, RCP 4.5 and RCP 8.5 were used for horizons 2030 and 2050 climate projections.Future projections were based on data of a regional climate change model.Results: With both scenarios in 2030 and 2050, the results of jackknife test indicated that the mean temperature of wettest quarter and temperature annual range had the greatest effect on the model for the vector and the reservoir hosts, respectively.Conclusions: The climate conditions are the major determinants of zoonotic cutaneous leishmaniasis incidence rate in Yazd Province.These climate conditions provide favorable habitats for ease transmission of zoonotic cutaneous leishmaniasis in this endemic area.Habitats suitability for the vector and reservoir will be expanding in the coming years compared with the current conditions, such that, in horizon 2030 2050, the probability of the presence of the vector and reservoir within 38 580 and 37 949 km~2, respectively, from Yazd province is above 60%.Moreover, an increase is predicted in the presence of the vector in the western parts and the reservoir in the northern and central parts of the province in the future.Understanding the role of environmental and bioclimatic factors in zoonotic cutaneous leishmaniasis occurrence can provide a guide for policy-makers in the creation and implementation of more effective policies for prevention and control.     

Immunoenzyme analysis with corpuscular antigen in the study of zoonotic cutaneous leishmaniasis

Comparative study of sera from patients suffering from zoonotic cutaneous leishmaniasis by indirect immunofluorescence (IIF) and by enzyme immunoassay (ELISA) gave 72.4 and 89.6% positive results, correspondingly, with homologous antigen L. major. Good correlation of these methods was revealed--coincidence in 83% cases. Authors used ELISA with corpuscular Leishmania antigen. The test was superior to IIF in sensitivity and specificity. Creation of type-specific Leishmania test systems for ELISA opens new vistas in studies of the population immune structure in combined zoonotic cutaneous leishmaniasis and visceral leishmaniasis foci.     

Leishmania (Viannia) infection in the domestic dog in Chaparral, Colombia

Peridomestic transmission of American cutaneous leishmaniasis is increasingly reported and dogs may be a reservoir of Leishmania (Viannia) in this setting. We investigated the prevalence of infection in dogs in Chaparral County, Colombia, the focus of an epidemic of human cutaneous leishmaniasis caused by Leishmania (Viannia) guyanensis. Two (0.72%) of 279 dogs had lesions typical of cutaneous leishmaniasis that were biopsy positive by kinetoplast DNA polymerase chain reaction-Southern blotting. Seroprevalence was 2.2% (6 of 279) by enzyme-linked immunosorbent assay. Buffy coat and ear skin biopsy specimens were positive by polymerase chain reaction-Southern blotting in 7.3% (10 of 137) and 11.4% (12 of 105) of dogs, respectively. Overall 20% of dogs (21 of 105) showed positive results for one or more tests. Amplification and sequencing of the Leishmania 7SL RNA gene identified L. guyanensis in one dog and L. braziliensis in two dogs. No association was identified between the risk factors evaluated and canine infection. Dogs may contribute to transmission but their role in this focus appears to be limited.     

Isoenzymatic variability of Leishmania infantum in Tunisia concerning 254 human strains

The different clinical forms of leishmaniasis are the result of both the immunological status of individuals and the species of the parasite causing the infection.

In Mediterranean countries, the Leishmania infantum complex groups zymodemes which are responsible for visceral, cutaneous and exceptionally cutaneomucosal or mucosal leishmaniasis.

We report in this study a synthesis concerning 254 cases of L. infantum that have been characterized at the “Laboratoire de Parasitologie” of the Rabta Hospital. The strains were isolated from human cases of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) by culture on NNN medium: 156 VL cases and 98 CL cases.

The isoenzymatic characterization revealed three zymodemes of L. infantum.

• L. infantum MON 1, a common zymodeme of VL, occurred in 154 cases (61%): 147 VL (95%) and 7 CL (5%). All CL cases were from the northern provinces, six of them occurring during an epidemic disease in 2001.

• L. infantum MON 24, a common zymodeme of CL in the north, occurred in 98 cases (38.5%): 91 CL (93%) and 7 VL (7%). The seven VL cases were immunocompetent children aged from 8 months to 9 years and native of northern Tunisia. Two of the CL cases were from central regions of the country. This is the first time that cases from these regions are reported.

• L. infantum MON 80, an uncommon zymodeme in Tunisia, occurred in two VL cases (0.5%): two children aged 7 and 5. The small number of strains of this zymodeme does not allow understanding of its epidemiological role.

The results of this study indicate a low enzymatic variability of L. infantum in the country. However, our study includes only human strains and should be extended to animal ones (dogs, rodents and sand flies). This would lead to a better understanding of the epidemiology of leishmaniasis in Tunisia.     

Ecology of the leishmania species in the Guianan ecoregion complex

In the Guianan ecoregion complex, at least seven Leishmania species belonging to both the L. (Leishmania) Saf' janova, 1982 and L. (Viannia) Lainson and Shaw, 1987 sub-genera are causative agents of American visceral leishmaniasis, mucocutaneous leishmaniasis, and cutaneous leishmaniases, including localized and diffuse forms. They are mostly sylvatic species subjugated to their own specific Lutzomyia sand fly vectors and to particular wild mammalian reservoir hosts of the dense rain forest. Originally implicated strictly in wild zoonoses and developing in forested ecotopes, Leishmania show a particular adaptive plasticity to face environmental modifications following deforestation and anthropization. This review presents the different pathogenic complexes of these parasites, emphasizes their implication in human diseases, and considers the influence of humans on their ecology.     

Leishmaniasis in Greece I. Isolation and identification of the parasite causing human and canine visceral leishmaniasis

Three human and 19 canine leishmanial stocks were typed according to their excreted factor serotype and the electrophoretic mobility of their MDH, GPI, G6PDH and 6PGDH and shown to be identical with regard to these characters and, thus with Leishmania donovani infantum. This verifies the opinion of earlier researchers, who suggested that the parasites which cause human and canine visceral leishmaniasis in Greece are the same organism and that dogs are the reservoir for the human infection. The complexities raised by the co-existence of human cutaneous leishmaniasis in Greece caused by L. tropica (formerly L. t. minor) are stressed. A comparison was made of the clinical symptomatology, serological diagnosis by IFA and ELISA tests and parasitological diagnosis of the human cases and canine infections.     

Herbicides to curb human parasitic infections: in vitro and in vivo effects of trifluralin on the trypanosomatid protozoans.

Leishmaniasis is a major tropical disease for which current chemotherapies, pentavalent antimonials, are inadequate and cause severe side effects. It has been reported that trifluralin, a microtubule-disrupting herbicide, is inhibitory to Leishmania amazonensis. In this study, the in vitro effect of trifluralin on different species of trypanosomatid protozoans was determined. In addition to L. amazonensis, trifluralin is effective against Leishmania major and Leishmania tropica, which cause cutaneous infections, Leishmania donovani, which causes visceral disease, Leishmania panamensis, which may cause mucocutaneous infection, and Trypanosoma brucei, an important human and veterinary pathogen. Moreover, most encouragingly, trifluralin is effective in vivo as a topical ointment against L. major and Leishmania mexicana murine cutaneous leishmaniasis. Thus, trifluralin is a promising lead drug for several related, prevalent tropical diseases: leishmaniasis, trypanosomiasis of animals, and, possibly, African trypanosomiasis in humans.     

Molecular detection of Leishmania species in human and animals from cutaneous leishmaniasis endemic areas of Waziristan,Khyber Pakhtunkhwa,Pakistan

Objectives: To detect Leishmania species in human patients, animal reservoirs and Phlebotomus sandflies in Waziristan, Pakistan. Methods: Tissue smears and aspirates from 448 cutaneous leishmaniasis(CL) suspected patients were analyzed. To sort out role of the reservoir hosts, skin scrapings, spleen and liver samples from 104 rodents were collected. Furthermore, buffy coat samples were obtained from 60 domestic animals. Sandflies were also trapped. All human, animals and sandfly samples were tested by microscopy, kinetoplastic PCR and internal transcribed spacer 1(ITS1) PCR followed by restriction fragment length polymorphism for detection of Leishmania species. Results: An overall prevalence of 3.83% and 5.21% through microscopy and ITS1 PCR respectively was found. However, the statistically non-significant correlation was found between area, gender, and number of lesions. The presence of rodents, sandflies, domestic animals and internally displaced people increased the risk of CL. Using ITS1-PCR-RFLP, Leishmania tropica(L. tropica) was confirmed in 106 samples while 25 of the isolates were diagnosed as Leishmania major(L. major). Similarly, 3/104 rodents were positive for L. major and 14 pools of DNA samples containing Phlebotomus sergenti sandflies were positive for L. tropica. None of samples from domestic animals were positive for leishmaniasis. Conclusions: In the present study, L. tropica and L. major are found to be the main causative agents of CL in study area. Movement of internally displaced people from CL endemic areas presents a risk for nearby CL free areas. To the best of our knowledge, we report for the first time L. major infection in rodents(Rattus rattus) and L. tropica in Phlebotomus sergenti sandflies trapped in Waziristan, Pakistan.