Leishmaniasis epidemiology in endemic areas of metropolitan France and its overseas territories from 1998 to 2020.

BACKGROUND: In France, leishmaniasis is endemic in the Mediterranean region, in French Guiana and to a lesser extent, in the French West Indies. This study wanted to provide an updated picture of leishmaniasis epidemiology in metropolitan France and in its overseas territories. METHODOLOGY/PRINCIPAL FINDINGS: Leishmaniasis cases were collected by passive notification to the French National Reference Centre for Leishmaniases (NRCL) in Montpellier from 1998 to 2020 and at the associated Centre in Cayenne (French Guiana) from 2003 to 2020. In metropolitan France, 517 autochthonous leishmaniasis cases, mostly visceral forms due to Leishmania infantum (79%), and 1725 imported cases (French Guiana excluded), mainly cutaneous leishmaniasis from Maghreb, were recorded. A slight decrease of autochthonous cases was observed during the survey period, from 0.48 cases/100,000 inhabitants per year in 1999 (highest value) to 0.1 cases/100,000 inhabitants per year in 2017 (lowest value). Conversely, imported cases increased over time (from 59.7 in the 2000s to 94.5 in the 2010s). In French Guiana, 4126 cutaneous and mucocutaneous leishmaniasis cases were reported from 2003 to 2020. The mean incidence was 103.3 cases per 100,000 inhabitants/year but varied in function of the year (from 198 in 2004 to 54 in 2006). In Guadeloupe and Martinique (French West Indies), only sporadic cases were reported. CONCLUSIONS/SIGNIFICANCE: Because of concerns about disease expansion and outbreaks in other Southern Europe countries, and leishmaniasis monitoring by the NRCL should be continued and associated with a more active surveillance.

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.

The Montpellier Leishmania Collection, from a Laboratory Collection to a Biological Resource Center: A 39-Year-Long Story.

We report the development of a laboratory collection of Leishmania that was initiated in 1975 and, after 39 years, has become an international Biological Resource Center (BRC-Leish, Montpellier, France, BioBank No. BB-0033-00052), which includes 6353 strains belonging to 36 Leishmania taxa. This is a retrospective analysis of the technical and organizational changes that have been adopted over time to take into account the technological advances and related modifications in the collection management and quality system. The technical improvements concerned the culture and cryopreservation techniques, strain identification by isoenzymatic and molecular techniques, data computerization and quality management to meet the changes in international standards, and in the cryogenic and microbiological safety procedures. The BRC is working toward obtaining the NF-S 96-900 certification in the coming years. Our long-term expertise in Leishmania storage and typing and collection maintenance should encourage field epidemiologists and clinical practitioners in endemic countries to secure their own strain collection with the help of the French BRC-Leish.

Comparison of Leishmania killicki (syn. L. tropica) and Leishmania tropica Population Structure in Maghreb by Microsatellite Typing.

Leishmania (L.) killicki (syn. L. tropica), which causes cutaneous leishmaniasis in Maghreb, was recently described in this region and identified as a subpopulation of L. tropica. The present genetic analysis was conducted to explore the spatio-temporal distribution of L. killicki (syn. L. tropica) and its transmission dynamics. To better understand the evolution of this parasite, its population structure was then compared with that of L. tropica populations from Morocco. In total 198 samples including 85 L. killicki (syn. L. tropica) (from Tunisia, Algeria and Libya) and 113 L. tropica specimens (all from Morocco) were tested. Theses samples were composed of 168 Leishmania strains isolated from human skin lesions, 27 DNA samples from human skin lesion biopsies, two DNA samples from Ctenodactylus gundi bone marrow and one DNA sample from a Phlebotomus sergenti female. The sample was analyzed by using MultiLocus Enzyme Electrophoresis (MLEE) and MultiLocus Microsatellite Typing (MLMT) approaches. Analysis of the MLMT data support the hypothesis that L. killicki (syn. L. tropica) belongs to the L. tropica complex, despite its strong genetic differentiation, and that it emerged from this taxon by a founder effect. Moreover, it revealed a strong structuring in L. killicki (syn. L. tropica) between Tunisia and Algeria and within the different Tunisian regions, suggesting low dispersion of L. killicki (syn. L. tropica) in space and time. Comparison of the L. tropica (exclusively from Morocco) and L. killicki (syn. L. tropica) population structures revealed distinct genetic organizations, reflecting different epidemiological cycles.

Characterization of Leishmania (Leishmania) waltoni n.sp. (Kinetoplastida: Trypanosomatidae), the Parasite Responsible for Diffuse Cutaneous Leishmaniasis in the Dominican Republic.

Leishmania parasites isolated, between 1979 and 1988 by the late Bryce Walton, from Dominican Republic (DR) patients with diffuse cutaneous leishmaniasis, were characterized using a panel of 12 isoenzymes, 23 monoclonal antibodies, small subunit ribosomal DNA (SSu rDNA), and multilocus sequence analysis (MLSA). The isoenzyme and monoclonal antibody profiles and the MLSA results showed that the Dominican Republic parasites were distinct from other described Leishmania species. This new species belongs to the mexicana complex, which is distributed in central and parts of northern South America. It is suggested that the parasites uniqueness from other members of the mexicana complex is related to it being isolated on an island for millions of years. If Leishmania (Leishmania) waltoni fails to adapt to some imported mammal, such as the house rat, it will be the only Leishmania to be classified as an endangered species. The excessive destruction of habitats on Hispaniola threatens the survival of its vectors and presumed natural reservoirs, such as the rodent hutias and the small insectivorous mammal solenodon. The concept of Leishmania species is discussed in the light of recent evaluations on criteria for defining bacterial species.

Epidemiological characteristics of a new focus of cutaneous leishmaniasis caused by Leishmania tropica in Settat, Morocco.

A new emerging focus of human cutaneous leishmaniasis (HCL) caused by Leishmania tropica was identified within the province of Settat. This study was performed in order to analyze the reasons of the extension of CL in this area, and to describe the clinico-epidemiological characteristic of this emerging focus during 2007-2012. A total of 553 suspected cases of CL were diagnosed in laboratory of Settat, controlled and confirmed in reference national laboratory of leishmaniasis in Rabat. Leishmania parasite is found in 356 cases. Most of them (33.89%) were recorded in localities of Ouled Ghalem (110 cases) and Laamarcha (102 cases) of El Borouj sector. The lesions were typically small, dry and mostly located on the face and extremities. Majority of infection (25%) was recorded among children under 11 years old, and female (72%). Strains of L. tropica were identified by PCR ITS1 from positive slides and zymodeme MON-102 was typed using isoenzyme technique on starch gel electrophoresis.

Evolutionary history of Leishmania killicki (synonymous Leishmania tropica) and taxonomic implications.

BACKGROUND: The taxonomic status of Leishmania (L.) killicki, a parasite that causes chronic cutaneous leishmaniasis, is not well defined yet. Indeed, some researchers suggested that this taxon could be included in the L. tropica complex, whereas others considered it as a distinct phylogenetic complex. To try to solve this taxonomic issue we carried out a detailed study on the evolutionary history of L. killicki relative to L. tropica. METHODS: Thirty-five L. killicki and 25 L. tropica strains isolated from humans and originating from several countries were characterized using the MultiLocus Enzyme Electrophoresis (MLEE) and the MultiLocus Sequence Typing (MLST) approaches. RESULTS: The results of the genetic and phylogenetic analyses strongly support the hypothesis that L. killicki belongs to the L. tropica complex. Our data suggest that L. killicki emerged from a single founder event and that it evolved independently from L. tropica. However, they do not validate the hypothesis that L. killicki is a distinct complex. Therefore, we suggest naming this taxon L. killicki (synonymous L. tropica) until further epidemiological and phylogenetic studies justify the L. killicki denomination. CONCLUSIONS: This study provides taxonomic and phylogenetic information on L. killicki and improves our knowledge on the evolutionary history of this taxon.

Leishmania (Leishmania) martiniquensis n. sp. (Kinetoplastida: Trypanosomatidae), description of the parasite responsible for cutaneous leishmaniasis in Martinique Island (French West Indies).

The parasite responsible for autochthonous cutaneous leishmaniasis in Martinique island (French West Indies) was first isolated in 1995; its taxonomical position was established only in 2002, but it remained unnamed. In the present paper, the authors name this parasite Leishmania (Leishmania) martiniquensis Desbois, Pratlong & Dedet n. sp. and describe the type strain of this taxon, including its biological characteristics, biochemical and molecular identification, and pathogenicity. This parasite, clearly distinct from all other Euleishmania, and placed at the base of the Leishmania phylogenetic tree, is included in the subgenus Leishmania.

Frequency of MDR1-related p-gp overexpression in Greek Leishmania isolates.

In this work, we investigated Greek Leishmania isolates (n = 70) for their individual MDR1-gene-related p-gp (belonging to the ABC-B subfamily of permeases) expression levels by means of flow cytometric analysis of Rhodamine 123 extrusion kinetics. Of all used isolates, 5.71% express this drug-extruding ABC-transporter at alarming levels and are distributed widely over the country. Some 33% of all examined isolates originated on the island of Crete though none of the strains showed vastly elevated p-gp extrusion activity, indicating a reasonable implementation of anti-leishmanial compounds in this part of the country. Compared to isolates obtained from canine tissue, human Leishmania isolates were superior both in size and in subcellular differentiation in flow cytometry. Furthermore, a specific t test confirmed verapamil hydrochloride to be a highly potent p-gp reversal agent with p < 0.0001. In a second test series, the loading of Leishmania with Rhodamine 123 was moreover reduced when occurring under influence of verapamil hydrochloride, a known p-gp reversal agent, indicating an ATP-dependant influx of the fluorescent dye and therewith the drug itself. In a final, third experiment series, it was shown that Sb(V) does not act upon the promastigote form 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.

Will the introduction of Leishmania tropica MON-58, in the island of Crete, lead to the settlement and spread of this rare zymodeme?

The rare zymodeme, Leishmania tropica MON-58, was isolated from a young Afghan refugee with a facial cutaneous lesion who had come to live in Crete early 2008. The same zymodeme variant was isolated from a local dog that had never travelled outside the island, with symptoms of visceral leishmaniasis, which stayed in the area where the patient worked during the summer months. This is the first record of L. tropica in a host, other than human, in Greece and another example of introduction of a vector borne pathogen in a focus where local vector/s can sustain it, with the risk of initiation of new transmission cycle/s.

Genetic diversity and structure in Leishmania infantum populations from southeastern Europe revealed by microsatellite analysis.

BACKGROUND: The dynamic re-emergence of visceral leishmaniasis (VL) in south Europe and the northward shift to Leishmania-free European countries are well-documented. However, the epidemiology of VL due to Leishmania infantum in southeastern (SE) Europe and the Balkans is inadequately examined. Herein, we aim to re-evaluate and compare the population structure of L. infantum in SE and southwestern (SW) Europe. METHODS: Leishmania strains collected from humans and canines in Turkey, Cyprus, Bulgaria, Greece, Albania and Croatia, were characterized by the K26-PCR assay and multilocus enzyme electrophoresis (MLEE). Genetic diversity was assessed by multilocus microsatellite typing (MLMT) and MLM Types were analyzed by model- and distance- based algorithms to infer the population structure of 128 L. infantum strains. RESULTS: L. infantum MON-1 was found predominant in SE Europe, whilst 16.8% of strains were MON-98. Distinct genetic populations revealed clear differentiation between SE and SW European strains. Interestingly, Cypriot canine isolates were genetically isolated and formed a monophyletic group, suggesting the constitution of a clonal MON-1 population circulating among dogs. In contrast, two highly heterogeneous populations enclosed all MON-1 and MON-98 strains from the other SE European countries. Structure sub-clustering, phylogenetic and Splitstree analysis also revealed two distinct Croatian subpopulations. A mosaic of evolutionary effects resulted in consecutive sub-structuring, which indicated substantial differentiation and gene flow among strains of both zymodemes. CONCLUSIONS: This is the first population genetic study of L. infantum in SE Europe and the Balkans. Our findings demonstrate the differentiation between SE and SW European strains; revealing the partition of Croatian strains between these populations and the genetic isolation of Cypriot strains. This mirrors the geographic position of Croatia located in central Europe and the natural isolation of the island of Cyprus. We have analysed the largest number of MON-98 strains so far. Our results indicate extensive gene flow, recombination and no differentiation between MON-1 and MON-98 zymodemes. No correlation either to host specificity or place and year of strain isolation was identified. Our findings may be associated with intensive host migration and common eco-epidemiological characteristics in these countries and give valuable insight into the dynamics of VL.

[Epidemiology of autochthonous leishmaniases in France]. / Épidémiologie des leishmanioses autochtones en France métropolitaine et d'outre-mer.

Leishmania infantum is the only species occurring in metropolitan France; located in the Mediterranean part of the country, it is responsible for a highly enzootic canine disease, while the human endemicity is low, with about 23 cases yearly reported to the National Reference Centre of Leishmaniases, mainly visceral forms. In French Guyana, five Leishmania species occur in the Amazonian forest, of which L. guyanensis is the predominant species, and L. braziliensis is responsible for the most critical forms. The most frequent clinical feature is cutaneous leishmaniasis, with a mean annual incidence reaching 2 p. 1000, with some inter-annual fluctuations. In Martinique Island, recent studies have confirmed the presence of an ancestral Leishmania species, responsible for small cutaneous lesions, of mild evolution; the life cycle of this species remains unknown. In Guadeloupe Island, a few autochthonous visceral leishmaniasis cases have been reported, needing a prospective study.

A newly emerged cutaneous leishmaniasis focus in northern Israel and two new reservoir hosts of Leishmania major.

In 2006/7, 18 cases of cutaneous leishmaniasis (CL) were reported for the first time from Sde Eliyahu (pop. 650), a village in the Beit She'an valley of Israel. Between 2007-2011, a further 88 CL cases were diagnosed bringing the total to 106 (16.3% of the population of Sde Eliyahu). The majority of cases resided in the south-western part of the village along the perimeter fence. The causative parasite was identified as Leishmania major Yakimoff & Schokhor, 1914 (Kinetoplastida: Trypanosomatidae). Phlebotomus papatasi (Scopoli), 1786 (Diptera: Psychodidae) was found to be the most abundant phlebotomine species comprising 97% of the sand flies trapped inside the village, and an average of 7.9% of the females were positive for Leishmania ITS1 DNA. Parasite isolates from CL cases and a sand fly were characterized using several methods and shown to be L. major. During a comprehensive survey of rodents 164 Levant voles Microtus guentheri Danford & Alston, 1880 (Rodentia: Cricetidae) were captured in alfalfa fields bordering the village. Of these 27 (16.5%) tested positive for Leishmania ITS1 DNA and shown to be L. major by reverse line blotting. A very high percentage (58.3%-21/36) of Tristram's jirds Meriones tristrami Thomas, 1892 (Rodentia: Muridae), found further away from the village also tested positive for ITS1 by PCR. Isolates of L. major were successfully cultured from the ear of a wild jird found positive by ITS1 PCR. Although none of the wild PCR-positive voles exhibited external pathology, laboratory-reared voles that were infected by intradermal L. major inoculation, developed patent lesions and sand flies became infected by feeding on the ears of these laboratory-infected voles. This is the first report implicating M. guentheri and M. tristrami as reservoirs of Leishmania. The widespread co-distribution of M. guentheri and P. papatasi, suggests a significant threat from the spread of CL caused by L. major in the Middle East, central Asia and southern Europe.

Geographical distribution and epidemiological features of Old World Leishmania infantum and Leishmania donovani foci, based on the isoenzyme analysis of 2277 strains.

A series of 2277 Leishmania strains from Old World visceral leishmaniasis foci, isolated between 1973 and 2008, were studied by isoenzyme analysis. The strains were obtained from humans, domestic and wild carnivores, rodents and phlebotomine sandflies, and came from 36 countries. In all, 60 different zymodemes were identified and clustered by a phenetic analysis into 3 different groups corresponding to the typically visceralizing species L. donovani (20 zymodemes, 169 strains), L. archibaldi (3 zymodemes, 46 strains) and L. infantum (37 zymodemes, 2,062 strains). The taxonomic position of these isoenzymatic groups is discussed in view of contradictory results obtained from recent molecular studies.

Genetic, serological and biochemical characterization of Leishmania tropica from foci in northern Palestine and discovery of zymodeme MON-307.

BACKGROUND: Many cases of cutaneous leishmaniasis (CL) have been recorded in the Jenin District based on their clinical appearance. Here, their parasites have been characterized in depth. METHODS: Leishmanial parasites isolated from 12 human cases of CL from the Jenin District were cultured as promastigotes, whose DNA was extracted. The ITS1 sequence and the 7SL RNA gene were analysed as was the kinetoplast minicircle DNA (kDNA) sequence. Excreted factor (EF) serotyping and multilocus enzyme electrophoresis (MLEE) were also applied. RESULTS: This extensive characterization identified the strains as Leishmania tropica of two very distinct sub-types that parallel the two sub-groups discerned by multilocus microsatellite typing (MLMT) done previously. A high degree of congruity was displayed among the results generated by the different analytical methods that had examined various cellular components and exposed intra-specific heterogeneity among the 12 strains.Three of the ten strains subjected to MLEE constituted a new zymodeme, zymodeme MON-307, and seven belonged to the known zymodeme MON-137. Ten of the 15 enzymes in the profile of zymodeme MON-307 displayed different electrophoretic mobilities compared with the enzyme profile of the zymodeme MON-137. The closest profile to that of zymodeme MON-307 was that of the zymodeme MON-76 known from Syria.Strains of the zymodeme MON-307 were EF sub-serotype A2 and those of the zymodeme MON-137 were either A9 or A9B4. The sub-serotype B4 component appears, so far, to be unique to some strains of L. tropica of zymodeme MON-137. Strains of the zymodeme MON-137 displayed a distinctive fragment of 417 bp that was absent in those of zymodeme MON-307 when their kDNA was digested with the endonuclease RsaI. kDNA-RFLP after digestion with the endonuclease MboI facilitated a further level of differentiation that partially coincided with the geographical distribution of the human cases from which the strains came. CONCLUSIONS: The Palestinian strains that were assigned to different genetic groups differed in their MLEE profiles and their EF types. A new zymodeme, zymodeme MON-307 was discovered that seems to be unique to the northern part of the Palestinian West Bank. What seemed to be a straight forward classical situation of L. tropica causing anthroponotic CL in the Jenin District might be a more complex situation, owing to the presence of two separate sub-types of L. tropica that, possibly, indicates two separate transmission cycles involving two separate types of phlebotomine sand fly vector.

Vector-borne diseases--constant challenge for practicing veterinarians: recommendations from the CVBD World Forum.

The human-animal bond has been a fundamental feature of mankind's history for millennia. The first, and strongest of these, man's relationship with the dog, is believed to pre-date even agriculture, going back as far as 30,000 years. It remains at least as powerful today. Fed by the changing nature of the interactions between people and their dogs worldwide and the increasing tendency towards close domesticity, the health of dogs has never played a more important role in family life. Thanks to developments in scientific understanding and diagnostic techniques, as well as changing priorities of pet owners, veterinarians are now able, and indeed expected, to play a fundamental role in the prevention and treatment of canine disease, including canine vector-borne diseases (CVBDs).The CVBDs represent a varied and complex group of diseases, including anaplasmosis, babesiosis, bartonellosis, borreliosis, dirofilariosis, ehrlichiosis, leishmaniosis, rickettsiosis and thelaziosis, with new syndromes being uncovered every year. Many of these diseases can cause serious, even life-threatening clinical conditions in dogs, with a number having zoonotic potential, affecting the human population.Today, CVBDs pose a growing global threat as they continue their spread far from their traditional geographical and temporal restraints as a result of changes in both climatic conditions and pet dog travel patterns, exposing new populations to previously unknown infectious agents and posing unprecedented challenges to veterinarians.In response to this growing threat, the CVBD World Forum, a multidisciplinary group of experts in CVBDs from around the world which meets on an annual basis, gathered in Nice (France) in 2011 to share the latest research on CVBDs and discuss the best approaches to managing these diseases around the world.As a result of these discussions, we, the members of the CVBD Forum have developed the following recommendations to veterinarians for the management of CVBDs.