Jean Pierre Dedet

Evolutionary and geographical history of the Leishmania donovani complex with a revision of current taxonomy

Leishmaniasis is a geographically widespread severe disease, with an increasing incidence of two million cases per year and 350 million people from 88 countries at risk. The causative agents are species of Leishmania, a protozoan flagellate. Visceral leishmaniasis, the most severe form of the disease, lethal if untreated, is caused by species of the Leishmania donovani complex. These species are morphologically indistinguishable but have been identified by molecular methods, predominantly multilocus enzyme electrophoresis. We have conducted a multifactorial genetic analysis that includes DNA sequences of protein-coding genes as well as noncoding segments, microsatellites, restriction-fragment length polymorphisms, and randomly amplified polymorphic DNAs, for a total of ≈18,000 characters for each of 25 geographically representative strains. Genotype is strongly correlated with geographical (continental) origin, but not with current taxonomy or clinical outcome. We propose a new taxonomy, in which Leishmania infantum and L. donovani are the only recognized species of the L. donovani complex, and we present an evolutionary hypothesis for the origin and dispersal of the species. The genus Leishmania may have originated in South America, but diversified after migration into Asia. L. donovani and L. infantum diverged ≈1 Mya, with further divergence of infraspecific genetic groups between 0.4 and 0.8 Mya. The prevailing mode of reproduction is clonal, but there is evidence of genetic exchange between strains, particularly in Africa.

Antigen Genes for Molecular Epidemiology of Leishmaniasis: Polymorphism of Cysteine Proteinase B and Surface Metalloprotease Glycoprotein 63 in the Leishmania donovani Complex

BACKGROUND Efficient monitoring of endemic and resurgent visceral leishmaniasis (VL) requires discriminatory molecular tools that allow direct characterization of etiological agents (i.e., the Leishmania donovani complex) in host tissues. This characterization is possible through restriction fragment-length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified sequences (PCR-RFLP). METHODS We present 2 new PCR-RFLP assays that target the gene locus of cysteine proteinase B (cpb), an important Leishmania antigen. The assays were applied to the characterization of 15 reference strains of the L. donovani complex, and their discriminatory power was compared with that of PCR-RFLP analysis of the gp63 gene, another Leishmania antigen, and with that of multilocus enzyme electrophoresis (MLEE), which is the reference standard for parasite typing. RESULTS Restriction patterns of the cpb locus were polymorphic, but less so than gp63 patterns. When data for both loci were combined, differences between PCR-RFLP and MLEE results were encountered. Antigen gene analysis was more discriminatory and supported a different classification of parasites, one that fitted with their geographic origin. PCR-RFLP analysis of cpb also allowed direct genotyping of parasites in bone marrow aspirate and venous blood samples obtained from patients with VL. CONCLUSION Antigen genes constitute valid targets for PCR-based Leishmania typing without the need for isolation of parasites.

Leishmaniases and the Cyprus Paradox

In Cyprus, leishmaniasis has been considered exclusively a veterinary problem. It was prevalent before 1945, and until its recent reemergence, it was nearly eradicated by 1996 as a consequence of the destruction of reservoir hosts and vectors. A survey carried out to provide an unbiased estimate of current transmission rates in dogs and humans showed a 9-fold increase in dog seroprevalence (reaching 14.9%) compared with 10 years ago. However, no human cases caused by Leishmania infantum were detected, although L. donovani cases were reported recently. The 62 strains isolated from dogs were typed as L. infantum MON-1 (98.4%), which is the predominating zymodeme in the Mediterranean region, and MON-98 (1.6%). The Phlebotomus species P. tobbi (vector of L. infantum in Cyprus), P. galilaeus, and P. papatasi were the predominant species captured. Two transmission cycles seem to run in parallel in Cyprus: in dogs with L. infantum and in humans with L. donovani.

Description of a dermatropic Leishmania close to L. killicki (Rioux, Lanotte & Pratlong 1986) in Algeria.

Cutaneous leishmaniasis (CL) is endemic in Algeria where two forms have been previously described, the sporadic form caused by Leishmania infantum in the north and the cutaneous form caused by L. major in central and southern parts of the country. During 2005, a CL outbreak occurred in the province of Ghardaïa, located in the north of Sahara, where 2040 cases were recorded, of which several were from urban areas. Six strains isolated from patients with active lesions were identified by isoenzyme electrophoresis and by molecular typing using systematic sequencing of a large subunit of the RNA polymerase. Four of the strains belonged to a new zymodeme, MON-301, close to L. killicki MON-8. The two other isolates were identified as L. major zymodeme MON-25. The new dermatropic Leishmania close to L. killicki is reported for the first time in Algeria and coexists sympatrically with L. major MON-25 in the region of Ghardaïa where they occur in their usual vectors of Phlebotomus papatasi (L. major) and P. sergenti (L. tropica). This new parasite demonstrates the need for further investigations to elucidate the life cycle and transmission of the emergent disease and to evaluate its phylogenetic position in the taxonomy of Leishmania.

Leishmaniases in Maghreb: An endemic neglected disease

Maghreb is known to be one of the most endemic areas of leishmaniases where both visceral and cutaneous forms are reported. Cutaneous leishmaniasis (CL) is older and has a higher prevalence than visceral one (VL). It is caused by four taxa (Leishmania (L.) major, L. infantum, L. tropica and L. killicki) which are responsible for a large clinical spectrum of lesions. Most transmission cycles of these taxa are known and many phlebotomine sandflies vectors and reservoir hosts are identified. The zoonotic transmission is well established for L. major. However, for L. infantum and L. killicki it needs more investigations to be proven. Regarding L. tropica, studies suggest it to be of both zoonotic and anthroponotic types. The isoenzymatic characterization of these four taxa showed a large enzymatic polymorphism varying from two zymodemes for L. major to 10 zymodemes for L. tropica. Cutaneous leishmaniasis is widely distributed and covers all bioclimatic stages with the coexistence of more than one taxon in the same foci. Visceral leishmaniasis is the second form of leishmaniases in Maghreb. Only L. infantum is known to cause this disease. The transmission cycle of this parasite is zoonotic but still not well known. The isoenzymatic identification of L. infantum causing VL showed the presence of six zymodemes. Geographically, VL is distributed in all bioclimatic stages of Maghreb countries. Despite all the previous studies realized on leishmaniases in Maghreb, they are still considered as neglected diseases because of the rarity or the absence of efficient control strategies.

The First Isoenzymatic Characterizations of the Leishmania Strains Responsible for Cutaneous Leishmaniasis in the Area of Annaba (Eastern Algeria)

The epidemiological situation of cutaneous leishmaniasis in the region of Annaba (North Eastern Algeria) is explored for the first time. During a clinical survey carried out in the Annaba Hospital between 2004 and 2008, the parasi- tological study of lesions has revealed 259 positive cases (43.31%). Isoenzymatic identification of 16 strains isolated showed the presence of three Leishmania species and four zymodemes. The hypothesis of the presence of L. infantum is confirmed with two zymodemes, MON-1 and MON-24. The unexpected presence of L. major MON-25 certifies the sub- stantial extension of this zoonotic form to the North. The isolation of L.killicki, whose presence was unsuspected, reiter- ated the interest of eco-epidemiological analysis of households affected by the disease. Moreover, it is a new zymodeme never isolated so far, the MON-306.