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Leishmaniasis is a transmissible disease caused by Leishmania protozoa. Spain is endemic for both visceral and cutaneous leishmaniasis, the autochthonous aetiological agent being Leishmania infantum. Around the world, the L. donovani complex is associated with visceral symptoms, while any species of the Leishmania or Viannia subgenera affecting human can produce tegumentary forms. In a context of growing numbers of imported cases, associated with globalisation, the aim of this study was to analyse the aetiological evolution of human tegumentary leishmaniasis in a region of Spain (Catalonia). Fifty-six Leishmania strains, isolated from 1981 to 2018, were analysed using MLEE, gene sequencing (hsp70, rpoIILS, fh and ITS2) and MALDI-TOF. The utility of these different analytical methods was compared. The results showed an increase in leishmaniasis over the two last decades, particularly imported cases, which represented 39% of all cases studied. Leishmania infantum, L. major, L. tropica, L. braziliensis, L. guyanensis and L. panamensis were identified. The combination of molecular and enzymatic methods allowed the identification of 29 different strain types (A to AC). Strain diversity was higher in L. (Viannia), whilst the different L. major types were relatable with geo-temporal data. Among the autochthonous cases, type C prevailed throughout the studied period (39%). Minor types generally appeared within a short time interval. While all the techniques provided identical identification at the species complex level, MALDI-TOF and rpoIILS or fh sequencing would be the most suitable identification tools for clinical practice, and the tandem hsp70-ITS2 could substitute MLEE in the epidemiological field ISGlobal members participate in the group GREPIMER (Grup de Recerca en Patologia Importada i Malaties Emergents i Re-emergents) which is supported by AGAUR (2017 SGR 00924), the Tropical Disease Cooperative Research Network (RICET) (RD12/0018/0010), and the Spanish Ministry of Science, Innovation and Universities through the “Centro de Excelencia Severo Ochoa 2019-2023” Program (CEX2018-000806-S). ISGlobal is member of the CERCA Program, Generalitat de Catalunya

Among the 20 or so Leishmania spp. described as pathogenic for humans, those of the Leishmania donovani complex are the exclusive causative agents of systemic and fatal visceral leishmaniasis. Although well studied, the complex is taxonomically controversial, which hampers clinical and epidemiological research. In this work, we analysed 56 Leishmania strains previously identified as L. donovani, Leishmania archibaldi or Leishmania infantum, isolated from humans, dogs and sandfly vectors throughout their distribution area. The strains were submitted to biochemical and genetic analyses and the resulting data were compared for congruence. Our results show: i) a partial concordance between biochemical and genetic-based data, ii) very limited genetic variability within the L. donovani complex, iii) footprints of frequent genetic exchange along an east-west gradient, marked by a widespread diffusion of alleles across the geographical range, and iv) a large-scale geographical spreading of a few genotypes. From a taxonomic point of view, considering the absence of relevant terminology in existing classes, the L. dono-vani complex could be treated as a single entity. (C) 2020 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is caused by Leishmania donovani. In addition to fatal VL, these parasites also cause skin diseases in immune-competent and -suppressed people, post-kala azar dermal leishmaniasis (PKDL) and HIV/VL co-infections, respectively. Genetic polymorphism in 36 Ethiopian and Sudanese L. donovani strains from VL, PKDL and HIV/VL patients was examined using Amplified Fragment Length Polymorphism (AFLP), kDNA minicircle sequencing and Southern blotting. Strains were isolated from different patient tissues: in VL from lymph node, spleen or bone marrow; and in HIV/VL from skin, spleen or bone marrow. When VL and PKDL strains from the same region in Sudan were examined by Southern blotting using a DNA probe to the L. donovani 28S rRNA gene only minor differences were observed. kDNA sequence analysis distributed the strains in no particular order among four clusters (A – D), while AFLP analysis grouped the strains according to geographical origin into two major clades, Southern Ethiopia (SE) and Sudan/Northern Ethiopia (SD/NE). Strains in the latter clade were further divided into subpopulations by zymodeme, geography and year of isolation, but not by clinical symptoms. However, skin isolates showed significantly (p, Graphical abstract Unlabelled Image, Highlights • Strains from VL, post kala-azar dermal leishmaniasis and HIV/VL co-infection patients examined • Bone marrow and spleen strain of same HIV/VL patient different • AFLP, kDNA sequencing and Southern blotting fail to group strains on basis of clinical symptoms • AFLP clades are divided by geography, not clinical symptoms • Skin isolates show fewer polymorphic AFLP fragments than visceral strains

International audience; Internal transcribed spacer regions of ribosomal DNA were sequenced, and species-specific primerswere designed to simplify the identification of two morphologically similar species of the Detrituscomplex,Ochlerotatus detritusandOchlerotatus coluzzii. Each newly designed primer was able toamplify a species-specific fragment with a different size. Samples from France and Germany were suc-cessfully tested. This new tool prompts for bio-ecological studies to refine basic knowledge on thebionomics of this species complex, towards a better control and prevention of ensuing nuisances.Moreover, ITS2 sequencing revealed the existence of (1) two distinct haplotypes ofOc. detritusthatare sympatric and widely distributed along the French Atlantic and Mediterranean littorals and (2)a specific haplotype in mosquitoes sampled from Tunisia, raising the question of the taxonomic statusof this North-African population.

We studied the development of antimony-resistant Leishmania infantum in natural vectors Lutzomyia longipalpis and Phlebotomus perniciosus to ascertain the risk of parasite transmission by sand flies. All three resistant strains produced fully mature late-stage infections in sand flies; moreover, the resistant phenotype was maintained after the passage through the vector. These results highlight the risk of circulation of resistant Leishmania strains and question the use of human drugs for treatment of dogs as Leishmania reservoirs.

International audience; Matrix-assisted laser desorption ionization time-of-flightMALDI-TOF mass spectrometry (MS) is now widely recognized as a powerful tool with which to identify bacteria and fungi at the species level, and sometimes in a rapid and accurate manner. We report herein an approach to identify, at the species level, Leishmania promastigotes from in vitro culture. We first constructed a reference database of spectra including the main Leishmania species known to cause human leishmaniasis. Then, the performance of the reference database in identifying Leishmania promastigotes was tested on a panel of 69 isolates obtained from patients. Our approach correctly identified 66 of the 69 isolates tested at the species level with log (score) values superior to 2. Two Leishmania isolates yielded non-interpretable MALDI-TOF MS patterns, owing to low log (score) values. Only one Leishmania isolate of Leishmania peruviana was misidentified as the closely related species Leishmania braziliensis, with a log (score) of 2.399. MALDI-TOF MS is a promising approach, providing rapid and accurate identification of Leishmania from in vitro culture at the species level.

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.

International audience; 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.Keywords

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.

International audience; 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.

International audience; BACKGROUND:Recently, there has been a re-emergence of cutaneous leishmaniasis in endemic countries and an increase in imported cases in non-endemic countries by travelers, workers, expatriates, immigrants, and military force personnel. Old World cutaneous leishmaniasis is caused primarily by Leishmania major, L. tropica and L. aethiopica. Despite their low sensitivity, diagnosis traditionally includes microscopic and histopathological examinations, and in vitro cultivation. Several conventional PCR techniques have been developed for species identification, which are time-consuming and labour-intensive. Real-time PCR using SYBR green dye, although provides rapid detection, may generate false positive signals. Therefore, a rapid and easy method such as a FRET-based real-time PCR would improve not only the turn-around time of diagnosing Old World cutaneous Leishmania species but will also increase its specificity and sensitivity.METHODS:A FRET-based real-time PCR assay which amplifies the cathepsin L-like cysteine protease B gene encoding a major Leishmania antigen was developed to differentiate L. major, L. tropica, and L. aethiopica in one single step using one set of primers and probes. Assay performance was tested on cutaneous and visceral strains of Leishmania parasite cultures and isolates of other protozoan parasites as well as human biopsy specimen.RESULTS:The assay readily differentiates between the three Old World cutaneous leishmaniasis species based on their melting curve characteristics. A single Tm at 55.2 ± 0.5 °C for L. aethiopica strains was distinguished from a single Tm at 57.4 ± 0.2 °C for L. major strains. A double curve with melting peaks at 66.6 ± 0.1 °C and 48.1 ± 0.5 °C or 55.8 ± 0.6 °C was observed for all L. tropica strains. The assay was further tested on biopsy specimens, which showed 100% agreement with results obtained from isoenzyme electrophoresis and Sanger sequencing.CONCLUSION:Currently, there are no published data on real-time PCR using FRET technology to differentiate between Old World cutaneous Leishmania species. In summary, our assay based on specific hybridization addresses the limitations of previous PCR technology and provides a single step, reliable method of species identification and rapid diagnostic applications.

International audience; 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 parasitological 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 substantial extension of this zoonotic form to the North. The isolation of L.killicki, whose presence was unsuspected, reiterated 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.

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.

This bibliographic review reports the isoenzyme polymorphism of 1023 Leishmania infantum strains isolated from dogs that have been characterized by multilocus enzyme electrophoresis in the Leishmania Reference Centre of Montpellier, or in other laboratories, to which this typification technique has already been transferred. Between 1981 and 2010, a total of 12 zymodemes were identified around the Mediterranean basin: MON-1, MON-24, MON-34, MON-72, MON-77, MON-80, MON-98, MON-105, MON-108, MON-199, MON-199 var NP1130 and MON-281, of which 6 were present in Algeria. The zymodeme MON-1 was predominant (86.5% of the strains). The dog was confirmed as the main reservoir of L. infantum MON-1, while the reservoir of the other zymodemes has not yet been identified. The enzymatic polymorphism is relatively high in Algeria and in Spain in contrast to other Mediterranean countries. The reasons for this polymorphism are discussed.

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.

International audience; Multilocus microsatellite typing (MLMT) was employed to compare strains of Leishmania donovani belonging to the MON-37 zymodeme (MON-37 strains) from Cyprus and Israel to MON-37 strains from the Indian subcontinent, the Middle East, China and East Africa as well as strains of other zymodemes. The MLMT data were processed with a distance-based method for construction of phylogenetic trees, factorial correspondence analysis and a Bayesian model-based clustering algorithm. All three approaches assigned the MON-37 strains to different distantly related genetically defined subgroups, corresponding to their geographical origin. Specifically, the Kenyan, Sri Lankan and Indian MON-37 strains were genetically closer to strains of other zymodemes from the same regions than to MON-37 strains from other areas. MON-37 strains from Cyprus and Israel were clearly different not only among themselves, but also compared to all the other MON-37 strains studied and could, therefore, be autochthonous. This study showed that the zymodeme MON-37 is paraphyletic and does not reflect the genetic relationship between strains of different geographical origin.

Resume Depuis 1979, le nombre croissant de transplantations d’organes s’est accompagne d’une augmentation de leishmanioses associees et notamment de leishmanioses viscerales. Les aspects epidemiologiques, cliniques, biologiques, et therapeutiques sont analyses a partir de 66 cas releves dans la litterature. Les leishmanioses tegumentaires, beaucoup plus rares, sont au nombre de sept. Exceptionnellement, elles ont ete trouvees associees a des leishmanioses viscerales.

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.

In the south of France, Leishmania infantum is responsible for numerous cases of canine leishmaniasis (CanL), sporadic cases of human visceral leishmaniasis (VL) and rare cases of cutaneous and muco-cutaneous leishmaniasis (CL and MCL, respectively). Several endemic areas have been clearly identified in the south of France including the Pyrénées-Orientales, Cévennes (CE), Provence (P), Alpes-Maritimes (AM) and Corsica (CO). Within these endemic areas, the two cities of Nice (AM) and Marseille (P), which are located 150 km apart, and their surroundings, concentrate the greatest number of French autochthonous leishmaniasis cases. In this study, 270 L. infantum isolates from an extended time period (1978–2011) from four endemic areas, AM, P, CE and CO, were assessed using Multi-Locus Microsatellite Typing (MLMT). MLMT revealed a total of 121 different genotypes with 91 unique genotypes and 30 repeated genotypes. Substantial genetic diversity was found with a strong genetic differentiation between the Leishmania populations from AM and P. However, exchanges were observed between these two endemic areas in which it seems that strains spread from AM to P. The genetic differentiations in these areas suggest strong epidemiological structuring. A model-based analysis using STRUCTURE revealed two main populations: population A (consisting of samples primarily from the P and AM endemic areas with MON-1 and non-MON-1 strains) and population B consisting of only MON-1 strains essentially from the AM endemic area. For four patients, we observed several isolates from different biological samples which provided insight into disease relapse and re-infection. These findings shed light on the transmission dynamics of parasites in humans. However, further data are required to confirm this hypothesis based on a limited sample set. This study represents the most extensive population analysis of L. infantum strains using MLMT conducted in France., Author Summary In the south of France, the parasite Leishmania infantum is responsible for diseases that primarily affect dogs but can also impact humans. Several endemic areas have been clearly identified in the south of France including the Pyrénées-Orientales, Cévennes (CE), Provence (P), Alpes-Maritimes (AM) and Corsica (CO). In this study, 270 L. infantum isolates from four endemic areas, AM, P, CE and CO, were assessed using Multi-Locus Microsatellite Typing (MLMT), a tool applied for population genetic studies. MLMT revealed a strong genetic differentiation between the Leishmania populations from AM and P with exchanges observed between these two endemic areas. For four patients, the occurrence of disease relapses and re-infections was examined. These findings shed light on the transmission dynamics of parasites in humans. This study represents the most extensive population analysis of L. infantum isolates using MLMT conducted in France.

International audience; Treatment failure and symptomatic relapse are major concerns in American tegumentary leishmaniasis (TL). Such complications are seen frequently in Leishmania guyanensis infections, in which patients respond variously to first-line antileishmanials and are more prone to develop chronic cutaneous leishmaniasis. The factors underlying this pathology, however, are unknown. Recently, we reported that a double-stranded RNA virus, Leishmania RNA virus 1 (LRV1), nested within L. guyanensis parasites is able to exacerbate experimental murine leishmaniasis by inducing a hyperinflammatory response. This report investigates the prevalence of LRV1 in human L. guyanensis infection and its effect on treatment efficacy, as well as its correlation to symptomatic relapses after the completion of first-line treatment. In our cohort of 75 patients with a diagnosis of primary localized American TL, the prevalence of LRV1-positive L. guyanensis infection was elevated to 58%. All patients infected with LRV1-negative L. guyanensis were cured after 1 dose (22 of 31 [71%]) or 2 doses (31 of 31 [100%]) of pentamidine. In contrast, 12 of 44 LRV1-positive patients (27%) presented with persistent infection and symptomatic relapse that required extended therapy and the use of second-line drugs. Finally, LRV1 presence was associated with a significant increase in levels of intra-lesional inflammatory markers. In conclusion, LRV1 status in L. guyanensis infection is significantly predictive (P = .0009) of first-line treatment failure and symptomatic relapse and has the potential to guide therapeutic choices in American TL.

As in the countries edging the Mediterranean basin, Leishmania infantum zymodeme MON-1 is the main causative agent of visceral leishmaniasis in Morocco, where visceral leishmaniasis is most active in the North-Eastern slopes of the Rif mountains. The dog was confirmed to be the main reservoir of L. infantum MON-1, while the reservoir of L. infantum MON-24 causative agent of both infantile visceral leishmaniasis and cutaneous leishmaniasis has not yet been identified. Here we report the first detection of this last zymodeme in a dog in Morocco. The isolated strain was first identified by the use of genotyping markers and confirmed by isoenzyme analysis. Phylogenetic analysis with the use of concatenated sequences from 26 Leishmania donovani complex strains revealed strong geographical correlation with the MON-24 strain from Morocco clustering with other East African strains whereas two other MON-24 strains clustered with L. infantum strains. Interestingly, the two distinct populations of MON-24 identified with the use of genotyping markers cannot be distinguished by multilocus enzyme electrophoresis.

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., Author Summary Leishmania killicki (syn. L. tropica) was discovered in 1986. Few studies have been conducted on this parasite exclusively described in Maghreb. Consequently, many elements on its epidemiology, transmission, population structure and dynamics remain unknown. To better understand the evolution of this parasite, its population structure has been compared with that of L. tropica populations from Morocco using Multilocus Enzyme Electrophoresis (MLEE) and MultiLocus Microsatellite Typing (MLMT) typing. MLMT data support the hypothesis that L. killicki (syn. L. tropica) belongs to the L. tropica complex despite the strong genetic differentiation between them. Despite the probable recent divergence between L. killicki (syn. L. tropica) and L. tropica, they seem to evolve differently. Indeed, L. killicki (syn. L. tropica) appears slightly polymorphic and highly structured in space and time, while L. tropica was genetically heterogeneous, slightly structured geographically and temporally. The different population structures revealed distinct genetic organizations, reflecting different epidemiological cycles. Several parameters could explain these opposite epidemiological and genetic patterns such as ecosystems, vectors and reservoirs.

International audience; 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.

Background The Leishmania (Viannia) braziliensis complex is responsible for most cases of New World tegumentary leishmaniasis. This complex includes two closely related species but with different geographic distribution and disease phenotypes, L. (V.) peruviana and L. (V.) braziliensis. However, the genetic basis of these differences is not well understood and the status of L. (V.) peruviana as distinct species has been questioned by some. Here we sequenced the genomes of two L. (V.) peruviana isolates (LEM1537 and PAB-4377) using Illumina high throughput sequencing and performed comparative analyses against the L. (V.) braziliensis M2904 reference genome. Comparisons were focused on the detection of Single Nucleotide Polymorphisms (SNPs), insertions and deletions (INDELs), aneuploidy and gene copy number variations. Results We found 94,070 variants shared by both L. (V.) peruviana isolates (144,079 in PAB-4377 and 136,946 in LEM1537) against the L. (V.) braziliensis M2904 reference genome while only 26,853 variants separated both L. (V.) peruviana genomes. Analysis in coding sequences detected 26,750 SNPs and 1,513 indels shared by both L. (V.) peruviana isolates against L. (V.) braziliensis M2904 and revealed two L. (V.) braziliensis pseudogenes that are likely to have coding potential in L. (V.) peruviana. Chromosomal read density and allele frequency profiling showed a heterogeneous pattern of aneuploidy with an overall disomic tendency in both L. (V.) peruviana isolates, in contrast with a trisomic pattern in the L. (V.) braziliensis M2904 reference. Read depth analysis allowed us to detect more than 368 gene expansions and 14 expanded gene arrays in L. (V.) peruviana, and the likely absence of expanded amastin gene arrays. Conclusions The greater numbers of interspecific SNP/indel differences between L. (V.) peruviana and L. (V.) braziliensis and the presence of different gene and chromosome copy number variations support the classification of both organisms as closely related but distinct species. The extensive nucleotide polymorphisms and differences in gene and chromosome copy numbers in L. (V.) peruviana suggests the possibility that these may contribute to some of the unique features of its biology, including a lower pathology and lack of mucosal development. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1928-z) contains supplementary material, which is available to authorized users.

Around the Mediterranean basin Leishmania infantum is an important parasite causing canine leishmaniasis and visceral and cutaneous clinical forms in both immunocompetent and immunocompromised humans. Efficient monitoring and evaluation of epidemiology with discriminatory molecular markers are required. We investigated the genetic diversity of L. infantum in Portugal by polymerase chain amplification and restriction fragment length polymorphism analysis of kinetoplastid DNA, as molecular marker. We analysed 120 Portuguese isolates of L. infantum plus 16 other non-Portuguese isolates (as a reference group) from humans, dogs and sand flies. The Portuguese population showed a high degree of polymorphism with a total of 13 profiles identified. The predominant profile was A, which was only detected in the Portuguese samples. The kinetoplastid DNA PCR-RFLP assay described here was suitable for use directly with biological samples and the profiles obtained were stable during long-term growth in vitro and in laboratory animals.

At least 13 characterizedLeishmaniaspecies are known to infect humans in South America. Five of these parasites are transmitted in the sylvatic ecotopes of the whole French Guianan territory and responsible for cutaneous leishmaniasis. For the diagnosis of cutaneous leishmaniasis, restriction fragment length polymorphism (RFLP) analyses have shown promising results. Thus, the end of the small subunit and internal transcribed spacer 1 of the rRNA genes were sequenced and targeted by PCR-RFLP analysis in the 10 main New World (NW)Leishmaniaspecies from the two subgenera. Then, the procedure was tested on 40 samples from patients with cutaneous leishmaniasis, and its results were compared with those of conventional methods. (i) The results of this simple genus-specific method were in agreement with those of previous isoenzyme analyses. (ii) This method distinguished the most medically relevantLeishmaniaspecies with only one enzyme (RsaI). (iii) This method could be performed directly on human biopsy specimens (sensitivity of 85.7%). Performing NWLeishmaniaspecies typing rapidly and easily in the field constitutes a very valuable improvement for detection ofLeishmaniaspp. Revealing great diversity with several enzymes, this method could also be useful for taxonomic, ecological, and epidemiological studies in space and time.

The current rapid spread of leishmaniases caused by Leishmania tropica and the complexity of its clinical spectrum call for this parasite's epidemiological and evolutionary investigation. Evaluation of its population structure by isoenzyme electrophoresis and previous molecular biological analysis has proved difficult. In this study, we used 21 microsatellite loci to type 117 strains from different African and Asian locations. Eighty-one different genotypes were found. A genetic bottleneck supported by a gradient in the number of alleles and consistent with the geographical structure of the Middle East suggests an African origin of this species. A Bayesian approach identified 10 genetic clusters that correlated predominantly with geographical origin. The strains in the 'Asia' cluster form a very heterogeneous sub-population, with a varied but inter-related genotype that is geographically very widely dispersed and consistent with anthroponotic transmission of the parasite. The other nine clusters were more homogenous. The propagation of L. tropica appears to be predominantly clonal. In Africa and the Middle East, anthroponotic and zoonotic systems of distribution may contribute to the development of overlapping, genetically distinct populations of L. tropica.

Eleven new cases of visceral leishmaniasis (VL) are reported in organ transplant patients in France. The epidemiological, clinical, biological, diagnostic and therapeutic features are reviewed, based on these cases and 46 cases reported in the literature. VL was most commonly associated with renal transplantation (77% of the cases). Most patients were from Southern European countries. The main clinical symptom was fever. Leucopoenia and anaemia were the most frequent haematological disorders. Diagnosis was by direct finding of the parasite in smears of bone marrow (85.2%) or, by positive serology (90.9%). Without antileishmanial treatment, VL in transplant recipients was fatal. Treatment using either antimonials or amphotericine B gave similar cure rates of around 80% of the cases. But toxicity was higher for antimonials. Relapses occurred in 14.3%.

To understand phylogenetic relationships of species and strains within the Leishmania donovani complex, we have analyzed the ribosomal DNA internal transcribed spacer (ITS) sequences of 27 Leishmania infantum, 2 Leishmania chagasi, 18 L. donovani and 5 Leishmania archibaldi strains of different zymodemes and geographical origin. Eight ITS sequence types were found. All detected sequence variation within ITS1 and ITS2 was based on 12 polymorphic microsatellites. The L. infantum strains from the Mediterranean region, China and L. chagasi from the New World formed a phylogenetic group well separated from the second main group including all strains from East Africa and India. Within the latter group three distinct phylogenetic subgroups could be differentiated: (1) L. donovani (Sudan/Ethiopia, China) + L. archibaldi (Sudan), (2) L. donovani (Sudan/Ethiopia) + L. infantum (Sudan) + L. archibaldi (Sudan/Ethiopia), and (3) L. donovani (Kenya, India). These groups are not consistent with previous species definitions based on isoenzyme analyses, e.g. L. infantum is polyphyletic and L. archibaldi is not supported as a distinct species. Two groups of Indian strains could be differentiated, one of which has an identical sequence type to the strains from Kenya. Three main lineages of strains can thus be differentiated in East Africa: two quite distantly related groups of strains from Sudan/Ethiopia, and a third group including all strains from Kenya, which is more closely related to part of the Indian strains than to any of the Sudanese/Ethiopian groups. The ITS sequence analysis presented here supports the need for revision of the taxonomy of the L. donovani complex.

The first identification of the Leishmania species responsible for visceral leishmaniasis in Djibouti is described. Four strains, obtained from three autochthonous cases, were identified by starch-gel electrophoresis and iso-enzyme analysis of 15 enzymatic systems. The strains were found to belong to two newly recognized zymodemes of L. donovani: MON-268 and MON-287.

The predominant sand fly species collected inside houses in Kfar Adumim, an Israeli village in the Judean Desert that is a focus of cutaneous leishmaniasis, was Phlebotomus papatasi, which was also caught attempting to bite humans. Phlebotomus sergenti, which is rarely seen inside houses, constituted the predominant sand fly species in caves near the village. Leishmania isolates from Ph. sergenti and humans typed as Leishmania tropica. Sand fly and human isolates produced similar small nodular cutaneous lesions in hamsters. Isolates produced excreted factor (EF) of subserotypes A(9) or A(9)B(2), characteristic of L. tropica and reacted with L. tropica-specific monoclonal antibodies. Isoenzyme analysis consigned the strains to the L. tropica zymodemes MON-137 and MON-275. Molecular genetic analyses confirmed the strains were L. tropica and intraspecific microheterogeneity was observed. Genomic fingerprinting using a mini-satellite probe separated the L. tropica strains into two clusters that were not entirely congruent with geographic distribution. These results support the heterogeneous nature of L. tropica and incriminate Ph. sergenti as its vector in this Judean Desert focus.

This study describes a new focus of cutaneous leishmaniasis (CL) due to Leishmania tropica, in the Galilee region of northern Israel. Thirty-three cases from 4 villages (northern part) and from the city of Tiberias (southern part) have been clinically diagnosed since 1996. Parasites from 13 patients and from 6 sand flies were characterized by isoenzyme electrophoresis, 2 immunological methods, and 3 polymerase chain reaction (PCR)-based methods. Isolates from the northern part were antigenically similar to Leishmania major and were different from other L. tropica isolates, including those from the southern part of the focus. They belonged to a newly reported zymodeme and were separable from all known Israeli L. tropica isolates, by use of 2 different PCR-based methods. Five (5.2%) of 97 Phlebotomus (Adlerius) arabicus and 2 (1.2%) of 162 Phlebotomus (Paraphlebotomus) sergenti females from the northern part of the focus were found to be infected with L. tropica. Three of 29 hyraxes (Procavia capensis) were positive for Leishmania ribosomal DNA. Thus, the northern part of this emerging focus of CL in Israel is distinct from all known L. tropica foci. P. arabicus is the main vector, and it transmits parasites that are different from other L. tropica isolates, with respect to antigenic, molecular, and biochemical parameters.

In 1996, an epidemic outbreak of visceral leishmaniasis (VL) started in Barbar el Fugara, a village in Gedarif State (eastern Sudan). From 1997 to 2000, regular epidemiological studies were carried out in the human population, as well as in mammals and sand flies. In symptomatic patients, 46/69 lymph node, 6/20 post kala-azar dermal leishmaniasis (PKDL) and 1/4 cutaneous cultures in NNN medium were positive. In 69 dogs, 23/79 lymph node cultures were positive. In other mammals (47 rodents, five donkeys, one mongoose and one monkey) spleen and/or blood cultures were negative. Characterization of isolated strains (by starch gel electrophoresis and isoelectrofocusing) identified three zymodemes of Leishmania donovani, two of L. infantum and two of L. archibaldi complexes from patient samples and three zymodemes of L. donovani, three of L. infantum and two of L. archibaldi complexes from dog samples. Five of them were present in both man and dog. For the first time, a strain from a PKDL case was identified as L. infantum, and a child had the same L. infantum zymodeme in VL and in subsequent PKDL. Blood samples from dogs were studied by immunofluorescent antibody test (IFAT). The seroprevalence in dogs was 72.5%, 74.3% and 42.9% in 1998, 1999 and 2000, respectively. By using CDC miniature light traps 12 745 sand flies were collected and then identified. Phlebotomus papatasi (7%) and P. orientalis (5%) were sympatric, mainly inside homes (85% and 75%, respectively). These results, the relative stability of seroprevalence in dogs and the intradomiciliar presence of P. orientalis, known as a vector of VL in Sudan, suggest several hypotheses: (i) man is responsible for the disease in dogs, (ii) the dog is the reservoir of VL, (iii) the dog is an intermediate host between a possible sylvatic cycle and the anthroponotic cycle. More extensive studies are needed to assess the transmission cycle of VL in this area of Sudan.

To the Editor: During 2000–2010, serial Leishmania isolates obtained from an HIV-infected patient who was not responding to treatment showed a gradual decrease in in vitro miltefosine susceptibility. We performed L. donovani miltefosine transporter (Ldmt) gene analysis to identify an association between miltefosine resistance of reference L. donovani lines and variability in miltefosine response of L. infantum isolates. A new single-nucleotide polymorphism (SNP), L832F, was identified, which might be a marker of miltefosine resistance in leishmaniasis. The patient, a 46-year-old woman, had lived in France since 1994 but regularly returned to Algeria, her country of birth. HIV-1 infection was diagnosed in 1991. Antiretroviral therapy was initiated in 1993, leading to undetectable viral load and a CD4+ T-cell count of 185 cells/mm3 (reference >450/mm3). Concurrent conditions were thoracic herpes zoster in 1996, hairy leukoplakia of tongue, oropharyngeal candidiasis, and chronic renal failure of unknown cause since 2000. Visceral leishmaniasis was diagnosed in 1998 by culture of a bone marrow smear, which showed intracellular amastigotes. Use of meglumine antimonate (Glucantime; Sanofi, Paris, France), a drug of choice for the treatment of leishmaniasis, was contraindicated because of pancreatitis in the patient and in vitro isolate susceptibility variation; therefore, induction therapy consisted of liposomal amphotericin B (AmpB [AmBisome; Astellas Pharma US, Deerfield, IL, USA]) at a dose of 3 mg/kg/d for 5 consecutive days, then 1× week for 5 weeks (total dose 30 mg/kg) during 1998–2000 (Table). The same medication was administered for relapses at 4 mg/kg/d for 5 days, then 4 mg/kg 1× week for 5 weeks (total dose 40 mg/kg) during 2001–2010. Given the adverse effects of AmpB and the availability of oral miltefosine (Impavido; AEterna Zentaris Inc., Quebec City, Quebec, Canada), the latter drug was used for maintenance treatment during 2001–2007 at 50 mg 2×/d. Leishmaniasis was monitored by leukocytoconcentration and culture of blood samples on Novy-Nicolle-McNeal medium. Table Comparions of IC50 for AmpB and miltefosine against promastigotes and axenic amastigotes and distribution of LdMT SNPs in Leishmania infantum isolates and reference strains* When signs of biological and clinical relapse appeared, bone marrow was aspirated for parasite detection. After culture of the aspirate and isoenzyme determination, the strain was identified as L. infantum, zymodeme MON-24. Eleven relapses were documented; all were confirmed by positive direct examination of bone marrow or blood, but cultures of only 7 samples yielded positive results (Table). The susceptibility of 4 cryopreserved isolates (S1, S3, S4, and S6; Table) to AmpB and to miltefosine was studied in the in vitro promastigote and axenic amastigote form by determining the concentrations inhibiting parasite growth by 50% (1,2). The 50% inhibitory concentration (IC50) was determined in parallel for the following reference L. donovani lines: a wild-type L. donovani LV9 (MHOM/ET/67/HU3) line (LV9 WT), a wild-type L. donovani DD8 (MHOM/IN/80/DD8) line (DD8 WT), a laboratory miltefosine-resistant line obtained from LV9 WT (LV9 miltefosine-R, resistant to 90 μmol/L miltefosine), and the laboratory AmB-resistant line obtained from DD8 WT (DD8 AmB-R, resistant to 1.4 μmol/L AmB) on promastigote and axenic amastigote forms (3,4). The AmB susceptibility of the isolates did not change notably over time; IC50 values ranged from 0.09 µmol/L to 0.24 µmol/L, regardless of parasite form, similar to those of wild-type reference strains (Table). In contrast, the IC50 values of miltefosine increased greatly over time, from 5.00 µmol/L to 50.10 μmol/L. During the 6 years of follow-up with miltefosine maintenance therapy, the susceptibility of the isolate (S6) obtained 6 months after miltefosine treatment withdrawal in 2008 was 6-fold higher than that of the first isolate (S1) obtained in 2000. The L. donovani miltefosine transporter protein (LdMT) promotes miltefosine translocation (5), and LdMT inactivation in L. donovani promastigotes leads to miltefosine resistance at the promastigote and amastigote stages (6). In 2003 and 2006 studies, several mutations were linked to the inability of parasites to take up miltefosine and to miltefosine resistance (5,7). In a 2009 study, the weak expression of LdMT and its β subunit LdROS3 in L. braziliensis isolates was linked to diminished sensitivity (8). We sequenced the entire Ldmt gene (3,294 bp) in the reference strains and the clinical isolates for SNP analysis (5,7). Only 1 new SNP, L832F, was found in the miltefosine-resistant reference strain (LV9 miltefosine-R) and in clinical isolate S6. The L832 wild-type allele was found in isolate S1 and in the miltefosine-sensitive reference lines (LV9, DD8, and DD8 AmpB-R), whereas both alleles were found in isolates S3 and S4, with a decrease in the wild-type allele (Table). The last isolate, which was obtained 3 years after miltefosine withdrawal and could not be subcultured, had reverted to the wild-type allele (L832). These results point to a relation between the 832F allele and diminished susceptibility to miltefosine. Analysis of this case of miltefosine resistance in a patient co-infected with Leishmania sp. and HIV strongly suggests that an SNP (L832F) in the Ldmt gene could represent a molecular marker of miltefosine resistance in L. infantum and L. donovani.