ITS1 and cpb genetic polymorphisms in Algerian and Tunisian Leishmania infantum isolates from humans and dogs.

Leishmania (L.) infantum strains, isolated from varying hosts and clinical manifestations (cutaneous, visceral and canine leishmaniasis), were investigated in order to understand the genetic polymorphisms within this species in Algeria and Tunisia. Two DNA-based typing methods were tested in order to evaluate their effectiveness against Multilocus enzyme electrophoresis (MLEE), widely considered as the reference method for Leishmania parasite typing. On the other hand, MLEE is cumbersome, high-cost, time consuming and frequently does not detect intra-species genetic polymorphisms. In this work, we used two molecular target regions to discriminate L. infantum strains, Internal transcribed spacer 1 (ITS1) and the cysteine proteinase B (cpb). The ITS1 region offers good resolution for Leishmania discrimination but does not spotlight intra-species polymorphisms. In contrast, cpbE and cpbF PCR-Sequencing demonstrated a certain variability within CL and VL Algerian and Tunisian L. infantum isolates. Following phylogenetic analyses of 44 L. infantum isolates, two main groups were identified, a group with 39 bp deletion in the cpb sequence, composed of cutaneous, visceral and canine isolates from both countries with no significant clinical or geographic distribution; these samples were typed as MON-1, MON-24, and MON-80 zymodemes. A second group which presents a clear clusterization of Tunisian cutaneous strains belonging to the L. infantum MON-24. This group, with no deletion in the mature domain of the cpb gene sequence, should be further explored with a higher number of samples.

Antimony susceptibility of Leishmania isolates collected over a 30-year period in Algeria.

BACKGROUND: In Algeria, the treatment of visceral and cutaneous leishmanioses (VL and CL) has been and continues to be based on antimony-containing drugs. It is suspected that high drug selective pressure might favor the emergence of chemoresistant parasites. Although treatment failure is frequently reported during antimonial therapy of both CL and VL, antimonial resistance has never been thoroughly investigated in Algeria. Determining the level of antimonial susceptibility, amongst Leishmania transmitted in Algeria, is of great importance for the development of public health policies. METHODOLOGY/PRINCIPAL FINDINGS: Within the framework of the knowledge about the epidemiology of VL and CL amassed during the last 30 years, we sampled Leishmania isolates to determine their susceptibility to antimony. We analyzed a total of 106 isolates including 88 isolates collected between 1976 and 2013 in Algeria from humans, dogs, rodents, and phlebotomines and 18 collected from dogs in France. All the Algerian isolates were collected in 14 localities where leishmaniasis is endemic. The 50% inhibitory concentrations (IC50) of potassium antimony tartrate (the trivalent form of antimony, Sb(III)) and sodium stibogluconate (the pentavalent form of antimony, Sb(V)) were determined in promastigotes and intramacrophage amastigotes, respectively. The epidemiological cutoff (ECOFF) that allowed us to differentiate between Leishmania species causing cutaneous or visceral leishmaniases that were susceptible (S+) or insusceptible (S-) to the trivalent form of antimony was determined. The computed IC50 cutoff values were 23.83 µg/mL and 15.91 µg/mL for VL and CL, respectively. We report a trend of increasing antimony susceptibility in VL isolates during the 30-year period. In contrast, an increase in the frequency of S- phenotypes in isolates causing CL was observed during the same period. In our study, the emergence of S- phenotypes correlates with the inclusion of L. killicki (syn: L. tropica) isolates that cause cutaneous leishmaniasis and that have emerged in Algeria during the last decade. CONCLUSION/SIGNIFICANCE: Our results provide insight into the spatiotemporal dynamics of Leishmania antimony susceptibility in Algeria. We highlight the need for the future implementation of an effective methodology to determine the antimony susceptibility status of Leishmania isolates to detect the emergence of and prevent the dissemination of drug-resistant strains.

Presence of Phlebotomus perniciosus Atypical Form in Algeria.

BACKGROUND: Phlebotomus perniciosus and Phlebotomus longicuspis are two phlebotomine sand fly species morphologically similar and differing in males only by the shape of the copulatory valves which are bifurcated in P. perniciosus, tip long and tapered in P. longicuspis. METHODS: A count of the median coxite setae was carried out on 208 specimens from the collections of Dedet and of Parrot, identified previously as P. longicuspis and on 38 P. perniciosus male sand flies captured during the year 2012-2013, in order to seek the presence of atypical P. perniciosus form. RESULTS: The analysis revealed the presence of 33/246 (13%) atypical P. perniciosus previously confused with P. longicuspis species and whose distribution is mainly located in the semi-arid and arid bioclimatic regions. CONCLUSION: This study proved for the first time the presence of atypical form of P. perniciosus in Algeria.

Development of a Murine Infection Model with Leishmania killicki, Responsible for Cutaneous Leishmaniosis in Algeria: Application in Pharmacology.

In Algeria, Leishmania infantum, Leishmania major, and Leishmania killicki (Leishmania tropica) are responsible for cutaneous leishmaniosis. We established a murine model of L. killicki infection to investigate its infective capacity, some immunophysiopathological aspects, and its suitability for pharmacological purposes. Following the injection of L. major or L. killicki metacyclic promastigotes in the ear dermis of BALB/c mice, the course of infection was followed. The infection with L. killicki caused slower lesion formation than with L. major. The presence of L. killicki or L. major DNA and parasites was detected in the ear dermis and in lymph nodes, spleen, and liver. Lesions induced by L. killicki were nonulcerative in their aspect, whereas those caused by L. major were highly ulcerative and necrotic, which matches well with the lesion phenotype reported in humans for L. killicki and L. major, respectively. The treatment of L. killicki lesions by injection of Glucantime® significantly reduced the lesion thickness and parasite burden. Ear dermal injection of BALB/c mice constitutes a model to study lesions physiopathology caused by L. killicki and presents interest for in vivo screening of new compounds against this pathogen, emerging in Algeria.

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.