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Abstract Background The Namangan Region in the Pap District, located in Eastern Uzbekistan is the main focus of visceral leishmaniasis (VL) in Uzbekistan. In total, 28 cases of human VL were registered during 2006-2008 in this region. A study on the epidemiology of VL in this area was carried out in 2007-2008 in the villages of Chodak, Oltinkan, Gulistan and Chorkesar located at elevations of 900-1200 above sea level. Results A total of 162 dogs were tested for Leishmania infection. Blood was drawn for serology and PCR. When clinical signs of the disease were present, aspirates from lymph nodes and the spleen were taken. Forty-two dogs (25.9%) had clinical signs suggestive of VL and 51 (31.5%) were sero-positive. ITS-1 PCR was performed for 135 dogs using blood and tissue samples and 40 (29.6%) of them were PCR-positive. Leishmanial parasites were cultured from lymph node or spleen aspirates from 10 dogs. Eight Leishmania strains isolated from dogs were typed by multi-locus microsatellite typing (MLMT) and by multilocus enzyme electrophoretic analysis (MLEE), using a 15 enzyme system. These analyses revealed that the strains belong to the most common zymodeme of L. infantum, i.e., MON-1, and form a unique group when compared to MON-1 strains from other geographical regions. Conclusions The data obtained through this study confirm the existence of an active focus of VL in the Namangan region of Uzbekistan. The fact that L. infantum was the causative agent of canine infection with typical clinical signs, and also of human infection affecting only infants, suggests that a zoonotic form of VL similar in epidemiology to Mediterranean VL is present in Uzbekistan.

Abstract Background Leishmania (Leishmania) major, one of the agents causing cutaneous leishmaniasis (CL) in humans, is widely distributed in the Old World where different species of wild rodent and phlebotomine sand fly serve as animal reservoir hosts and vectors, respectively. Despite this, strains of L. (L.) major isolated from many different sources over many years have proved to be relatively uniform. To investigate the population structure of the species highly polymorphic microsatellite markers were employed for greater discrimination among it's otherwise closely related strains, an approach applied successfully to other species of Leishmania. Results Multilocus Microsatellite Typing (MLMT) based on 10 different microsatellite markers was applied to 106 strains of L. (L.) major from different regions where it is endemic. On applying a Bayesian model-based approach, three main populations were identified, corresponding to three separate geographical regions: Central Asia (CA); the Middle East (ME); and Africa (AF). This was congruent with phylogenetic reconstructions based on genetic distances. Re-analysis separated each of the populations into two sub-populations. The two African sub-populations did not correlate well with strains' geographical origin. Strains falling into the sub-populations CA and ME did mostly group according to their place of isolation although some anomalies were seen, probably, owing to human migration. Conclusion The model- and distance-based analyses of the microsatellite data exposed three main populations of L. (L.) major, Central Asia, the Middle East and Africa, each of which separated into two sub-populations. This probably correlates with the different species of rodent host.

Background: Cutaneous leishmaniasis (CL) is a major public health problem in Libya. In this paper, we describe the eco-epidemiological parameters of CL during the armed conflict period from January 2011 till December 2012. Current spatiotemporal distributions of CL cases were explored and projected to the future using a correlative modelling approach. In addition the present results were compared with our previous data obtained for the time period 1995–2008. Methodology/Principal findings: We investigated 312 CL patients who presented to the Dermatology Department at the Tripoli Central Hospital and came from 81 endemic areas distributed in 10 districts. The patients presented with typical localized lesions which appeared commonly on the face, arms and legs. Molecular identification of parasites by a PCR-RFLP approach targeting the ITS1 region of the rDNA was successful for 81 patients with two causative species identified: L. major and L. tropica comprised 59 (72.8%) and 22 (27.2%) cases, respectively. Around 77.3% of L. tropica CL and 57.7% of L. major CL caused single lesions. Five CL patients among our data set were seropositive for HIV. L. tropica was found mainly in three districts, Murqub (27.3%), Jabal al Gharbi (27.3%) and Misrata (13.7%) while L. major was found in two districts, in Jabal al Gharbi (61%) and Jafara (20.3%). Seasonal occurrence of CL cases showed that most cases (74.2%) admitted to the hospital between November and March, L. major cases from November till January (69.4%), and L. tropica cases mainly in January and February (41%). Two risk factors were identified for the two species; the presence of previously infected household members, and the presence of rodents and sandflies in patient’s neighborhoods. Spatiotemporal projections using correlative distribution models based on current case data and climatic conditions showed that coastal regions have a higher level of risk due to more favourable conditions for the transmitting vectors. Conclusion: Future projection of CL until 2060 showed a trend of increasing incidence of CL in the north-western part of Libya, a spread along the coastal region and a possible emergence of new endemics in the north-eastern districts of Libya. These results should be considered for control programs to prevent the emergence of new endemic areas taking also into consideration changes in socio-economical factors such as migration, conflicts, urbanization, land use and access to health care. [ABSTRACT FROM AUTHOR]

Background: Leishmania (Leishmania) aethiopica and L. (L.) tropica cause cutaneous leishmaniases and appear to be related. L. aethiopica is geographically restricted to Ethiopia and Kenya; L. tropica is widely dispersed from the Eastern Mediterranean, through the Middle East into eastern India and in north, east and south Africa. Their phylogenetic inter-relationship is only partially revealed. Some studies indicate a close relationship. Here, eight strains of L. aethiopica were characterized genetically and compared with 156 strains of L. tropica from most of the latter species' geographical range to discern the closeness. Methodology/Principal Findings: Twelve unlinked microsatellite markers previously used to genotype strains of L. tropica were successfully applied to the eight strains of L. aethiopica and their microsatellite profiles were compared to those of 156 strains of L. tropica from various geographical locations that were isolated from human cases of cutaneous and visceral leishmaniasis, hyraxes and sand fly vectors. All the microsatellite profiles were subjected to various analytical algorithms: Bayesian statistics, distance-based and factorial correspondence analysis, revealing: (i) the species L. aethiopica, though geographically restricted, is genetically very heterogeneous; (ii) the strains of L. aethiopica formed a distinct genetic cluster; and (iii) strains of L. aethiopica are closely related to strains of L. tropica and more so to the African ones, although, by factorial correspondence analysis, clearly separate from them. Conclusions/Significance: The successful application of the 12 microsatellite markers, originally considered species-specific for the species L. tropica, to strains of L. aethiopica confirmed the close relationship between these two species. The Bayesian and distance-based methods clustered the strains of L. aethiopica among African strains of L. tropica, while the factorial correspondence analysis indicated a clear separation between the two species. There was no correlation between microsatellite profiles of the eight strains of L. aethiopica and the type of leishmaniasis, localized (LCL) versus diffuse cutaneous leishmaniasis (DCL), displayed by the human cases. [ABSTRACT FROM AUTHOR]

There is an extensive body of medical and scientific research literature on visceral leishmaniasis (VL) in the Caucasus, Central Asia, the Crimean Peninsula and the southern part of The Russian Federation that is written in Russian, making it inaccessible to the majority of people who are interested in the leishmaniases in general and VL in particular. This review and summary in English of VL in what was Imperial Russia, which then became the Soviet Union and later a number of different independent states intends to give access to that majority. There are numerous publications in Russian on VL and, mostly, those published in books and the main scientific journals have been included here. The vast geographical area encompassed has been subdivided into four main parts: the southern Caucasus, covering Armenia, Azerbaijan and Georgia; Central Asia, covering Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan and Uzbekistan; the Crimean Peninsula and the northern Caucasus, which is part of The Russian Federation. Only rare cases of VL have been recorded in the northern Caucasus and Crimean Peninsula. In the other countries mentioned, human VL has been more intense but epidemics like those associated with L. donovani in India and East Africa have not occurred. For most of the countries, there are sections on the distribution, clinical aspects, the causative agent, the reservoirs and the vectors. Serological surveys and research into therapy are also covered. Recent studies on VL in Uzbekistan covered the application of serological, biochemical and molecular biological methods to diagnose human and canine VL, to identify the leishmanial parasites causing them in Uzbekistan and neighbouring Tajikistan and the epidemiology of VL in the Namangan Region of the Pap District, Eastern Uzbekistan. More recently, two studies were carried out in Georgia investigating the prevalence of human and canine VL, and the species composition of phlebotomine sand flies and their rates of infection with what was probably L. infantum in Tbilisi, eastern Georgia and Kutaisi, a new focus, in western Georgia. Though published in English, summaries of this information have been included where relevant to update the parts on VL in Uzbekistan and Georgia. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL), caused by the members of the Leishmania donovani complex, has been responsible for devastating VL epidemics in the Sudan. Multilocus microsatellite and sequence typing studies can provide valuable insights into the molecular epidemiology of leishmaniasis, when applied at local scales. Here we present population genetic data for a large panel of strains and clones collected in endemic Sudan between 1993 and 2001. Methods: Genetic diversity was evaluated at fourteen microsatellite markers and eleven nuclear sequence loci across 124 strains and clones. Results: Microsatellite data defined six genetic subpopulations with which the nuclear sequence data were broadly congruent. Pairwise estimates of FST (microsatellite) and KST (sequence) indicated small but significant shifts among the allelic repertoires of circulating strains year on year. Furthermore, we noted the co-occurrence of human and canine L. donovani strains in three of the six clusters defined. Finally, we identified widespread deficit in heterozygosity in all four years tested but strong deviation from inter-locus linkage equilibrium in two years. Conclusions: Significant genetic diversity is present among L. donovani in Sudan, and minor population structuring between years is characteristic of entrenched, endemic disease transmission. Seasonality in vector abundance and transmission may, to an extent, explain the shallow temporal clines in allelic frequency that we observed. Genetically similar canine and human strains highlight the role of dogs as important local reservoirs of visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

Background Zoonotic cutaneous leishmaniasis (ZCL) is a polymorphic disease which may show various symptoms. Genetic diversity of the parasite is suggested to be one of the factors influencing the clinical manifestation of the disease. Methods This study used PCR for the detection and identification of leishmanial parasites at the species level and applied a multilocus microsatellite typing approach for investigating the genetic diversity of Leishmania major isolated from captured rodents in two foci of ZCL in Iran: Turkemen Sahara and Fars province. Results ITS1-rDNA amplification and subsequent RFLP analyses were performed using DNA extracted from the rodents' ears. Approximately one third of the rodents tested positive for Leishmania; in all rodents L. major was the predominating infecting agent. Seven Rhombomys opimus were positive for L. turanica DNA and one for both L. major and L. turanica. DNA of L. infantum was identified in one Rh. opimus. Seventeen strains of L. major, 15 from Turkemen Sahara and two from Fars province, isolated from different rodents were tested for variation at nine polymorphic microsatellite loci. Ten different MLMT genotypes were observed. They were compared to 89 previously published microsatellite profiles obtained for strains of L. major of different geographical origin. Bayesian model-based and genetic distance based approaches confirmed that strains from Turkemen Sahara and from Fars are genetically different and belong to different genetic groups, largely corresponding to their geographical origins. Discussion The considerable genetic variability of L. major might be related to differences in reservoir host and/or to the existence of different populations of the vector, Phlebotomus papatasi. [ABSTRACT FROM PUBLISHER]

Background: Cutaneous leishmaniasis (CL) is a major and fast increasing public health problem, both among the local Pakistani populations and the Afghan refugees in camps. Leishmania (Leishmania) major is one of the etiological agents responsible for CL in Pakistan. Genetic variability and population structure have been investigated for 66 DNA samples of L. (L.) major isolated from skin biopsy of CL patients. Methods: Multilocus microsatellite typing (MLMT), employing 10 independent genetic markers specific to L. (L.) major, was used to investigate the genetic polymorphisms and population structures of Pakistani L. (L.) major DNA isolated from CL human cases. Their microsatellite profiles were compared to those of 130 previously typed strains of L. (L.) major from various geographical localities. Results: All the markers were polymorphic and fifty-one MLMT profiles were recognized among the 66 L. (L.) major DNA samples. The data displayed significant microsatellite polymorphisms with rare allelic heterozygosities. A Bayesian model-based approach and phylogenetic analysis inferred two L. (L.) major populations in Pakistan. Thirty-four samples belonged to one population and the remaining 32 L. (L.) major samples grouped together into another population. The two Pakistani L. (L.) major populations formed separate clusters, which differ genetically from the populations of L. (L.) major from Central Asia, Iran, Middle East and Africa. Conclusions: The considerable genetic variability of L. (L.) major might be related to the existence of different species of sand fly and/or rodent reservoir host in Sindh province, Pakistan. A comprehensive study of the epidemiology of CL including the situation or spreading of reservoirs and sand fly vectors in these foci is, therefore, warranted. [ABSTRACT FROM AUTHOR]

Background: Leishmaniases are divided into cutaneous (CL) and visceral leishmaniasis (VL). In the Old World, CL is caused by Leishmania (L.) major, L. tropica and L. aethiopica. L. tropica can also visceralize and cause VL. In India, the large epidemics of VL are caused by L. donovani and cases of CL are caused by L. major and L. tropica. However, strains of L. tropica have also been isolated from Indian cases of VL. This study was done to see if Indian strains of L. tropica isolated from human cases of CL are genetically identical to or different from Indian strains of L. tropica isolated from human cases of VL and to see if any genetic differences found correlated with clinical outcome presenting as either CL or VL. Methods: Multilocus microsatellite typing (MLMT), employing 12 independent genetic markers specific to L. tropica, was used to characterize and identify eight strains of L. tropica isolated from human cases of CL examined in clinics in Bikaner City, Rajasthan State, north-west India. Their microsatellite profiles were compared to those of 156 previously typed strains of L. tropica from various geographical locations that were isolated from human cases of CL and VL, hyraxes and sand fly vectors. Results: Bayesian, distance-based and factorial correspondence analyses revealed two confirmed populations: India/Asia and Israel/Palestine that subdivided, respectively, into two and three subpopulations. A third population, Africa/Galilee, as proposed by Bayesian analysis was not supported by the other applied methods. The strains of L. tropica from Bikaner isolated from human cases of CL fell into one of the subpopulations in the population India/Asia together with strains from other Asian foci. Indian strains isolated from human cases of VL fell into the same sub-population but were not genetically identical to the Bikaner strains of L. tropica. Conclusions: It seems that the genetic diversity encountered between the two groups of Indian strains is mainly owing to their geographical origins rather than their different times of isolation. Also, the genetic differences seen between the dermatotropic and viscerotropic strains might be connected with the difference in pathogenicity. [ABSTRACT FROM AUTHOR]

Background/Objectives: Parasites of the subgenus Leishmania (Viannia) cause varying clinical symptoms ranging from cutaneous leishmaniases (CL) with single or few lesions, disseminated CL (DL) with multiple lesions to disfiguring forms of mucocutaneous leishmaniasis (MCL). In this population genetics study, 37 strains of L. (V.) guyanensis, 63 of L. (V.) braziliensis, four of L. (V.) shawi, six of L. (V.) lainsoni, seven of L. (V.) naiffi, one each of L. (V.) utingensis and L. (V.) lindenbergi, and one L. (V.) lainsoni/L. naiffi hybrid from different endemic foci in Brazil were examined for variation at 15 hyper-variable microsatellite markers. Methodology/Principal findings: The multilocus microsatellite profiles obtained for the 120 strains were analysed using both model- and distance-based methods. Significant genetic diversity was observed for all L. (Viannia) strains studied. The two cluster analysis approaches identified two principal genetic groups or populations, one consisting of strains of L. (V.) guyanensis from the Amazon region and the other of strains of L. (V.) braziliensis isolated along the Atlantic coast of Brazil. A third group comprised a heterogeneous assembly of species, including other strains of L. braziliensis isolated from the north of Brazil, which were extremely polymorphic. The latter strains seemed to be more closely related to those of L. (V.) shawi, L. (V.) naiffi, and L. (V.) lainsoni, also isolated in northern Brazilian foci. The MLMT approach identified an epidemic clone consisting of 13 strains of L. braziliensis from Minas Gerais, but evidence for recombination was obtained for the populations of L. (V.) braziliensis from the Atlantic coast and for L. (V.) guyanensis. Conclusions/Significance: Different levels of recombination versus clonality seem to occur within the subgenus L. (Viannia). Though clearly departing from panmixia, sporadic, but long-term sustained recombination might explain the tremendous genetic diversity and limited population structure found for such L. (Viannia) strains. Author Summary: Cutaneous leishmaniasis (CL) constitutes a significant public health problem in all federal states of Brazil. Most cases are caused by parasites of the subgenus Leishmania (Viannia) which can cause a variety of clinical symptoms ranging from single or few lesions, disseminated CL with multiple lesions, to disfiguring forms of mucocutaneous leishmaniasis. This study has used a multilocus microsatellite typing approach for exploring the genetic diversity and population structure among 120 strains representing different subgenus L. (Viannia) species and different Brazilian CL foci. Genetic diversity within the subgenus was much higher than expected, especially within L. (V.) braziliensis, L. (V.) shawi, L. (V.) naiffi, and L. (V.) lainsoni which were all from the north of Brazil. These strains could not be assigned to well-defined populations, but presented a rather loosely associated group. Strains of L. (V.) braziliensis isolated along the Atlantic coast of Brazil and strains of L. (V.) guyanensis formed, however, two clearly separated populations exhibiting remarkable levels of sexual exchange. The latter finding is in contrast to previous studies suggesting clonal modes of propagation or inbreeding for natural populations of Leishmania parasites and might explain the genetic heterogeneity and limited population structure for Brazilian strains of subgenus L. (Viannia) observed in this study. [ABSTRACT FROM AUTHOR]

Background/Objectives: Parasites of the subgenus Leishmania (Viannia) cause varying clinical symptoms ranging from cutaneous leishmaniases (CL) with single or few lesions, disseminated CL (DL) with multiple lesions to disfiguring forms of mucocutaneous leishmaniasis (MCL). In this population genetics study, 37 strains of L. (V.) guyanensis, 63 of L. (V.) braziliensis, four of L. (V.) shawi, six of L. (V.) lainsoni, seven of L. (V.) naiffi, one each of L. (V.) utingensis and L. (V.) lindenbergi, and one L. (V.) lainsoni/L. naiffi hybrid from different endemic foci in Brazil were examined for variation at 15 hyper-variable microsatellite markers. Methodology/Principal findings: The multilocus microsatellite profiles obtained for the 120 strains were analysed using both model- and distance-based methods. Significant genetic diversity was observed for all L. (Viannia) strains studied. The two cluster analysis approaches identified two principal genetic groups or populations, one consisting of strains of L. (V.) guyanensis from the Amazon region and the other of strains of L. (V.) braziliensis isolated along the Atlantic coast of Brazil. A third group comprised a heterogeneous assembly of species, including other strains of L. braziliensis isolated from the north of Brazil, which were extremely polymorphic. The latter strains seemed to be more closely related to those of L. (V.) shawi, L. (V.) naiffi, and L. (V.) lainsoni, also isolated in northern Brazilian foci. The MLMT approach identified an epidemic clone consisting of 13 strains of L. braziliensis from Minas Gerais, but evidence for recombination was obtained for the populations of L. (V.) braziliensis from the Atlantic coast and for L. (V.) guyanensis. Conclusions/Significance: Different levels of recombination versus clonality seem to occur within the subgenus L. (Viannia). Though clearly departing from panmixia, sporadic, but long-term sustained recombination might explain the tremendous genetic diversity and limited population structure found for such L. (Viannia) strains. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: During 2002–2009, 466 cases of cutaneous leishmaniasis (CL) were reported from Jenin District, Palestine, affecting both genders. The average annual incidence was 23 cases per 100000 inhabitants, increasing with age in children. Most cases presented a single lesion, generally on the face. Diagnosis and species identification was done by applying internal transcribed spacer 1 (ITS1) RFLP analysis to 47 isolates, of which 44 (93.6%) were Leishmania tropica and 3 (6.4%) were L. major. RFLP analysis was also performed on 256 skin tissue scrapings spotted onto filter papers, showing that 138 (53.9%) were positive, of which 50.7% were infected with L. tropica, 17.4% with L. major and 2.9% with L. donovani s.l., and 29.0% could not be identified. This is the first report from Palestine on human CL caused by L. infantum. Nine of the strains of L. tropica were subjected to multilocus enzyme electrophoresis, six of which belonged to the zymodeme MON-137 and three to a new zymodeme (MON-307). This separation was corroborated by excreted factor serotyping. This observation modifies the classical epidemiological view of CL in Palestine. Jenin District is an active focus of CL caused by L. tropica, where Phlebotomus sergenti, the putative vector, is abundant. These data suggest that CL is a zoonotic infection, but an animal reservoir has not been found. [Copyright &y& Elsevier]

Background: Cutaneous leishmaniasis (CL) is a major public health problem in Libya. The objective of this study was to investigate, for the first time, epidemiological features of CL outbreaks in Libya including molecular identification of parasites, the geographical distribution of cases and possible scenarios of parasite transmission. Methodology/Principal Findings: We studied 450 patients that came from 49 areas distributed in 12 districts in north-west Libya. The patients' ages ranged from 9 months to 87 years (median age 25 years); 54% of the cases were males. Skin scrapings spotted on glass slides were collected for molecular identification of causative agent. The ribosomal internal transcribed spacer 1 (ITS1) was amplified and subsequently characterized by restriction fragment length polymorphism (RFLP) analysis. In total, 195 samples were successfully identified of which 148 (75.9%) were Leishmania major, and 47 (24.1%) Leishmania tropica. CL cases infected with L. major were found in all CL areas whereas L. tropica cases came mainly from Al Jabal Al Gharbi (46.4%), Misrata (17.8%) and Tarhuna districts (10.7%). A trend of seasonality was noticed for the infections with L. major which showed a clear peak between November and January, but was less pronounced for infections by L. tropica. Conclusion: The first molecular study on CL in Libya revealed that the disease is caused by L. major and L. tropica and the epidemiological patterns in the different foci were the same as in other Mediterranean foci of CL. Author Summary: Cutaneous leishmaniasis (CL) is caused by protozoan parasites of the genus Leishmania. The disease is characterized by the formation of chronic skin lesions followed by permanent scars and deformation of the infected area. It is distributed in many tropical and subtropical countries with more than 2 million cases every year. During the past few years CL has emerged as a major public health problem in Libya. So far, diagnosis was based on clinical symptoms and microscopic observation of parasites. Disease outbreaks were not investigated and the causative leishmanial species of CL were not identified so far. Our study indicates the presence of two coexisting species: Leishmania major and Leishmania tropica. These results are crucial in order to provide accurate treatment, precise prognosis and appropriate public health control measures. The recent armed conflict in Libya that ended with the Gadhafi regime collapse on October 2011 has affected all aspects of the life in the country. In this study we discussed multiple risk factors that could be associated with this conflict and present major challenges that should be considered by local and national health authorities for evaluating the CL burden and highlighting priority actions for disease control. [ABSTRACT FROM AUTHOR]

Background: New foci of human CL caused by strains of the Leishmania donovani (L. donovani) complex have been recently described in Cyprus and the Çukurova region in Turkey (L. infantum) situated 150 km north of Cyprus. Cypriot strains were typed by Multilocus Enzyme Electrophoresis (MLEE) using the Montpellier (MON) system as L. donovani zymodeme MON-37. However, multilocus microsatellite typing (MLMT) has shown that this zymodeme is paraphyletic; composed of distantly related genetic subgroups of different geographical origin. Consequently the origin of the Cypriot strains remained enigmatic. Methodology/Principal Findings: The Cypriot strains were compared with a set of Turkish isolates obtained from a CL patient and sand fly vectors in south-east Turkey (Çukurova region; CUK strains) and from a VL patient in the south-west (Kuşadasi; EP59 strain). These Turkish strains were initially analyzed using the K26-PCR assay that discriminates MON-1 strains by their amplicon size. In line with previous DNA-based data, the strains were inferred to the L. donovani complex and characterized as non MON-1. For these strains MLEE typing revealed two novel zymodemes; L. donovani MON-309 (CUK strains) and MON-308 (EP59). A population genetic analysis of the Turkish isolates was performed using 14 hyper-variable microsatellite loci. The genotypic profiles of 68 previously analyzed L. donovani complex strains from major endemic regions were included for comparison. Population structures were inferred by combination of Bayesian model-based and distance-based approaches. MLMT placed the Turkish and Cypriot strains in a subclade of a newly discovered, genetically distinct L. infantum monophyletic group, suggesting that the Cypriot strains may originate from Turkey. Conclusion: The discovery of a genetically distinct L. infantum monophyletic group in the south-eastern Mediterranean stresses the importance of species genetic characterization towards better understanding, monitoring and controlling the spread of leishmaniasis in this region. Author Summary: In eastern Mediterranean, leishmaniasis represents a major public health problem with considerable impact on morbidity and potential to spread. Cutaneous leishmaniasis (CL) caused by L. major or L. tropica accounts for most cases in this region although visceral leishmaniasis (VL) caused by L. infantum is also common. New foci of human CL caused by L. donovani complex strains were recently described in Cyprus and Turkey. Herein we analyzed Turkish strains from human CL foci in Çukurova region (north of Cyprus) and a human VL case in Kuşadasi. These were compared to Cypriot strains that were previously typed by Multilocus Enzyme Electrophoresis (MLEE) as L. donovani MON-37. Nevertheless, they were found genetically distinct from MON-37 strains of other regions and therefore their origin remained enigmatic. A population study was performed by Multilocus Microsatellite Typing (MLMT) and the profile of the Turkish strains was compared to previously analyzed L. donovani complex strains. Our results revealed close genetic relationship between Turkish and Cypriot strains, which form a genetically distinct L. infantum monophyletic group, suggesting that Cypriot strains may originate from Turkey. Our analysis indicates that the epidemiology of leishmaniasis in this region is more complicated than originally thought. [ABSTRACT FROM AUTHOR]

Leishmania infantum (syn. L. chagasi) is the causative agent of visceral leishmaniasis (VL) in the New World (NW) with endemic regions extending from southern USA to northern Argentina. The two hypotheses about the origin of VL in the NW suggest (1) recent importation of L. infantum from the Old World (OW), or (2) an indigenous origin and a distinct taxonomic rank for the NW parasite. Multilocus microsatellite typing was applied in a survey of 98 L. infantum isolates from different NW foci. The microsatellite profiles obtained were compared to those of 308 L. infantum and 20 L. donovani strains from OW countries previously assigned to well-defined populations. Two main populations were identified for both NW and OW L. infantum. Most of the NW strains belonged to population 1, which corresponded to the OW MON-1 population. However, the NW population was much more homogeneous. A second, more heterogeneous, population comprised most Caribbean strains and corresponded to the OW non-MON-1 population. All Brazilian L. infantum strains belonged to population 1, although they represented 61% of the sample and originated from 9 states. Population analysis including the OW L. infantum populations indicated that the NW strains were more similar to MON-1 and non-MON-1 sub-populations of L. infantum from southwest Europe, than to any other OW sub-population. Moreover, similarity between NW and Southwest European L. infantum was higher than between OW L. infantum from distinct parts of the Mediterranean region, Middle East and Central Asia. No correlation was found between NW L. infantum genotypes and clinical picture or host background. This study represents the first continent-wide analysis of NW L. infantum population structure. It confirmed that the agent of VL in the NW is L. infantum and that the parasite has been recently imported multiple times to the NW from southwest Europe. Author Summary: Leishmaniasis is a vector borne disease with a broad spectrum of clinical forms caused by protozoan parasites of the genus Leishmania. Visceral leishmaniasis is the most severe, systemic form of the disease. It is caused by parasites belonging to the Leishmania donovani complex, which includes L. infantum and L. donovani in the Old World (OW) and L. infantum (syn. L. chagasi) in the New World (NW). The identity and origin of the causative agent of VL in the Americas have been the subjects of much debate for decades. Different scientific approaches led to different conclusions, either favouring the hypothesis of indigenous origin of this parasite and its status as distinct species, or a recent importation of L. infantum by European colonists and synonymy of L. infantum and L. chagasi. We performed the first broad population study of these parasites from the NW using highly variable microsatellite markers. The level of heterogeneity and population structure was very low in contrast to the OW. Using a combined data analysis of NW and OW strains we have provided conclusive evidence of recent multiple introductions of L. infantum from Southwest Europe into the New World and for synonymy of L. infantum and L. chagasi. [ABSTRACT FROM AUTHOR]

Background: The Namangan Region in the Pap District, located in Eastern Uzbekistan is the main focus of visceral leishmaniasis (VL) in Uzbekistan. In total, 28 cases of human VL were registered during 2006-2008 in this region. A study on the epidemiology of VL in this area was carried out in 2007-2008 in the villages of Chodak, Oltinkan, Gulistan and Chorkesar located at elevations of 900-1200 above sea level. Results: A total of 162 dogs were tested for Leishmania infection. Blood was drawn for serology and PCR. When clinical signs of the disease were present, aspirates from lymph nodes and the spleen were taken. Forty-two dogs (25.9%) had clinical signs suggestive of VL and 51 (31.5%) were sero-positive. ITS-1 PCR was performed for 135 dogs using blood and tissue samples and 40 (29.6%) of them were PCR-positive. Leishmanial parasites were cultured from lymph node or spleen aspirates from 10 dogs. Eight Leishmania strains isolated from dogs were typed by multi-locus microsatellite typing (MLMT) and by multilocus enzyme electrophoretic analysis (MLEE), using a 15 enzyme system. These analyses revealed that the strains belong to the most common zymodeme of L. infantum, i.e., MON-1, and form a unique group when compared to MON-1 strains from other geographical regions. Conclusions: The data obtained through this study confirm the existence of an active focus of VL in the Namangan region of Uzbekistan. The fact that L. infantum was the causative agent of canine infection with typical clinical signs, and also of human infection affecting only infants, suggests that a zoonotic form of VL similar in epidemiology to Mediterranean VL is present in Uzbekistan. [ABSTRACT FROM AUTHOR]

Background: Parasites' evolution in response to parasite-targeted control strategies, such as vaccines and drugs, is known to be influenced by their population genetic structure. The aim of this study was to describe the population structure of Ethiopian strains of Leishmania donovani derived from different areas endemic for visceral leishmaniasis (VL) as a prerequisite for the design of effective control strategies against the disease. Methodology/Principal Findings: Sixty-three strains of L. donovani newly isolated from VL cases in the two main Ethiopian foci, in the north Ethiopia (NE) and south Ethiopia (SE) of the country were investigated by using 14 highly polymorphic microsatellite markers. The microsatellite profiles of 60 previously analysed L. donovani strains from Sudan, Kenya and India were included for comparison. Multilocus microsatellite typing placed strains from SE and Kenya (n = 30) in one population and strains from NE and Sudan (n = 65) in another. These two East African populations corresponded to the areas of distribution of two different sand fly vectors. In NE and Sudan Phlebotomus orientalis has been implicated to transmit the parasites and in SE and Kenya P. martini. The genetic differences between parasites from NE and SE are also congruent with some phenotypic differences. Each of these populations was further divided into two subpopulations. Interestingly, in one of the subpopulations of the population NE we observed predominance of strains isolated from HIV-VL co-infected patients and of strains with putative hybrid genotypes. Furthermore, high inbreeding irreconcilable from strict clonal reproduction was found for strains from SE and Kenya indicating a mixed-mating system. Conclusions/Significance: This study identified a hierarchical population structure of L. donovani in East Africa. The existence of two main, genetically and geographically separated, populations could reflect different parasite-vector associations, different ecologies and varying host backgrounds and should be further investigated. Author Summary: In the Horn of Africa, visceral leishmaniasis, caused by protozoan parasites of the Leishmania donovani complex, continues to pose a major health problem affecting the poorest of the poor. Population genetic studies are crucial for the development of drugs and vaccines against microorganisms. However, our knowledge about the population structure of L. donovani parasites in this region is still very limited. Using a highly discriminatory multilocus microsatellite typing approach, we found a remarkably high genetic diversity among the East African strains of L. donovani studied which grouped into two genetically and geographically distinct populations comprising parasites from SE and Kenya, and those from NE and Sudan. Despite Leishmania being widely regarded as a clonal organism, our results suggest a possible co-existence of clonal and sexually reproducing strains of L. donovani from SE. The information obtained by the present study is helpful for future design of parasite-targeted control measures in East Africa. [ABSTRACT FROM AUTHOR]

Background: Three major forms of human disease, cutaneous leishmaniasis, visceral leishmaniasis and mucocutaneous leishmaniasis, are caused by several leishmanial species whose geographic distribution frequently overlaps. These Leishmania species have diverse reservoir hosts, sand fly vectors and transmission patterns. In the Old World, the main parasite species responsible for leishmaniasis are Leishmania infantum, L. donovani, L. tropica, L. aethiopica and L. major. Accurate, rapid and sensitive diagnostic and identification procedures are crucial for the detection of infection and characterization of the causative leishmanial species, in order to provide accurate treatment, precise prognosis and appropriate public health control measures. Methods/Principal Findings: High resolution melt analysis of a real time PCR product from the Internal Transcribed Spacer-1 rRNA region was used to identify and quantify Old World Leishmania in 300 samples from human patients, reservoir hosts and sand flies. Different characteristic high resolution melt analysis patterns were exhibited by L. major, L. tropica, L. aethiopica, and L. infantum. Genotyping by high resolution melt analysis was verified by DNA sequencing or restriction fragment length polymorphism. This new assay was able to detect as little as 2-4 ITS1 gene copies in a 5 µl DNA sample, i.e., less than a single parasite per reaction. Conclusions/Significance: This new technique is useful for rapid diagnosis of leishmaniasis and simultaneous identification and quantification of the infecting Leishmania species. It can be used for diagnostic purposes directly from clinical samples, as well as epidemiological studies, reservoir host investigations and vector surveys. Author Summary: Protozoal parasites of the genus Leishmania are transmitted by sand fly bites to humans and animals. Three major forms of disease are caused by these parasites: cutaneous leishmaniasis, responsible for disfiguring skin wounds; mucocutaneous leishmaniasis, causing non-healing ulceration around the mouth and nose; and the potentially fatal visceral leishmaniasis, involving internal organs such as the spleen and liver. More than 2 million new human infections are caused annually by leishmaniasis globally, it is endemic in more than 88 countries and prevalent also as an imported disease in non-endemic regions due to travel and tourism. Most species of Leishmania that infect humans are zoonotic and transmitted from animal reservoir hosts. As various leishmanial parasites cause disease with similar symptoms, but require different therapeutic regimens and have dissimilar prognoses, reliable, sensitive and rapid diagnostic assays are needed. This study focuses on the five main species that cause leishmaniasis in the Old World. It presents a new assay for rapid detection, species identification and quantification of leishmanial parasites in clinical samples, reservoir hosts and sand flies. This technique could be especially valuable in regions where several leishmanial species exist, in non-endemic regions where infected patients require a rapid diagnosis, and for epidemiological host and vector studies leading to prevention programs. [ABSTRACT FROM AUTHOR]

Summary: Seventy-six cases of visceral leishmaniasis (VL) were reported from the Hebron district of the West Bank, Palestine between 1993 and 2007. All cases were in children less than 9 years old (median age 2 years). The average number of cases was 5.06/year and the average annual incidence was 3.02/100000 children. Ribosomal internal transcribed spacer 1 (ITS1) PCR-RFLP was performed using DNA extracted from two cultures and 36 archived Giemsa-stained slides from VL patients. Leishmania infantum was revealed as the causative agent of VL in the focus. Isoenzyme analysis identified two isolates as zymodeme MON-1. A serological survey of 455 children screened for serum anti-Leishmania antibodies revealed 8.4% seropositivity. Seropositivity was highest for children in households of previous VL cases [odds ratio (OR) 7.5; 95% CI 3.17–17.61; P <0.001] and among people who had domestic dogs and/or other animals (OR 2.4; 95% CI 1.19–4.68; P =0.017). No difference was seen between males and females (P =0.073). A preliminary survey of sand fly distribution showed the abundance of two putative vector species: Phlebotomus syriacus (45%) and Ph. tobbi (10%). The focus of VL in Hebron district was shown to follow the epidemiological pattern of paediatric disease characteristic of the Mediterranean region. [Copyright &y& Elsevier]

Abstract: Multilocus microsatellite typing (MLMT) was used to investigate the genetic variation among 44 Israeli and Palestinian strains of L. infantum isolated from infected dogs and human cases to determine their population structure and to compare them with strains isolated from different European countries. Most of the Israeli and Palestinian strains had their own individual MLMT profiles; a few shared the same profile. A Bayesian model-based approach and phylogenetic reconstructions based on genetic distances inferred two main populations that were significantly different from the European strains: population A, containing 16 strains from places in the West Bank and 11 strains from central Israel; and population B, containing 7 strains from northern Israel, 9 from central Israel, and one Palestinian strain from the Jenin District. Geographically distributed sub-populations were detected within population B. These results demonstrate similar disease dynamics in Israel and the Palestinian Authority. The re-emergence of VL in the case of population A is more likely owing to increased dog and human contact with sylvatic cycles of parasitic infection rather than to recent introduction from the older foci of northern Israel. The latter scenario could be true for population B found in few foci of Central Israel. [Copyright &y& Elsevier]

Background: Leishmania (Leishmania) major, one of the agents causing cutaneous leishmaniasis (CL) in humans, is widely distributed in the Old World where different species of wild rodent and phlebotomine sand fly serve as animal reservoir hosts and vectors, respectively. Despite this, strains of L. (L.) major isolated from many different sources over many years have proved to be relatively uniform. To investigate the population structure of the species highly polymorphic microsatellite markers were employed for greater discrimination among it's otherwise closely related strains, an approach applied successfully to other species of Leishmania. Results: Multilocus Microsatellite Typing (MLMT) based on 10 different microsatellite markers was applied to 106 strains of L. (L.) major from different regions where it is endemic. On applying a Bayesian model-based approach, three main populations were identified, corresponding to three separate geographical regions: Central Asia (CA); the Middle East (ME); and Africa (AF). This was congruent with phylogenetic reconstructions based on genetic distances. Re-analysis separated each of the populations into two sub-populations. The two African sub-populations did not correlate well with strains' geographical origin. Strains falling into the sub-populations CA and ME did mostly group according to their place of isolation although some anomalies were seen, probably, owing to human migration. Conclusion: The model- and distance-based analyses of the microsatellite data exposed three main populations of L. (L.) major, Central Asia, the Middle East and Africa, each of which separated into two subpopulations. This probably correlates with the different species of rodent host. [ABSTRACT FROM AUTHOR]

Leishmaniasis diagnosis in regions where multiple species exist should identify each species directly in the clinical sample without parasite culturing. The sensitivity of two PCR approaches which amplify part of the ssu rRNA gene and the ribosomal internal transcribed spacer (ITS), respectively, was determined using human and dog blood seeded with Leishmania promastigotes. ssu-rDNA-PCR was more sensitive than ITS1-PCR, however species identification was not possible by the former approach. When a nested ITS1-PCR was used its sensitivity equaled the ssu-rDNA-PCR. Digestion of ITS1 amplicon with the restriction enzyme HaeIII distinguished all medically relevant Leishmania species. ITS1-PCR was used to diagnose 162 local and imported suspected cases of leishmaniasis in Israel, the Palestinian Authority and Germany. 113 cases (69.7%) were positive by PCR and species identification was possible in 110 samples. Leishmania DNA was also amplified and identified at the species level from archived non-stained and Giemsa stained microscope slides. [Copyright &y& Elsevier]

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