Whole-Genome Characterization of Rotavirus G9P[6] and G9P[4] Strains That Emerged after Rotavirus Vaccine Introduction in Mozambique.

Mozambique introduced the Rotarix® vaccine into the National Immunization Program in September 2015. Following vaccine introduction, rotavirus A (RVA) genotypes, G9P[4] and G9P[6], were detected for the first time since rotavirus surveillance programs were implemented in the country. To understand the emergence of these strains, the whole genomes of 47 ELISA RVA positive strains detected between 2015 and 2018 were characterized using an Illumina MiSeq-based sequencing pipeline. Of the 29 G9 strains characterized, 14 exhibited a typical Wa-like genome constellation and 15 a DS-1-like genome constellation. Mostly, the G9P[4] and G9P[6] strains clustered consistently for most of the genome segments, except the G- and P-genotypes. For the G9 genotype, the strains formed three different conserved clades, separated by the P type (P[4], P[6] and P[8]), suggesting different origins for this genotype. Analysis of the VP6-encoding gene revealed that seven G9P[6] strains clustered close to antelope and bovine strains. A rare E6 NSP4 genotype was detected for strain RVA/Human-wt/MOZ/HCN1595/2017/G9P[4] and a genetically distinct lineage IV or OP354-like P[8] was identified for RVA/Human-wt/MOZ/HGJM0644/2015/G9P[8] strain. These results highlight the need for genomic surveillance of RVA strains detected in Mozambique and the importance of following a One Health approach to identify and characterize potential zoonotic strains causing acute gastroenteritis in Mozambican children.

Zoonotic Threats: The (Re)emergence of Cercarial Dermatitis, Its Dynamics, and Impact in Europe.

Cercarial dermatitis (CD), or "Swimmer's itch" as it is also known, is a waterborne illness caused by a blood fluke from the family Schistosomatidae. It occurs when cercariae of trematode species that do not have humans as their definitive host accidentally penetrate human skin (in an aquatic environment) and trigger allergic symptoms at the site of contact. It is an emerging zoonosis that occurs through water and is often overlooked during differential diagnosis. Some of the factors contributing to the emergence of diseases like CD are related to global warming, which brings about climate change, water eutrophication, the colonization of ponds by snails susceptible to the parasite, and sunlight exposure in the summer, associated with migratory bird routes. Therefore, with the increase in tourism, especially at fluvial beaches, it is relevant to analyze the current epidemiological scenario of CD in European countries and the potential regions at risk.

Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiae.

Accurate genomic sequencing demands high-quality double-stranded RNA (dsRNA). Existing methods for dsRNA extraction from yeast, fungi, and plants primarily rely on cellulose, suitable only for small volume extractions, or the time-consuming lithium chloride precipitation. To streamline the traditional phenol-chloroform-based dsRNA extraction method, the main challenge is the reduction of mitochondrial DNA (mtDNA) and Single Stranded RNA (ssRNA) to no detectable levels after gel electrophoresis. This challenge is successfully addressed through the modified approach described here, involving phenol extraction at low pH, followed by the addition of ammonium sulfate to the aqueous buffer. The dsRNA isolated using this novel method exhibits comparable quality to that obtained through cellulose purification, and it is readily amenable to RT-PCR. Moreover, a single batch of yeast cell RNA isolation requires only 2-3 h of hands-on time, thus simplifying and expediting the process significantly.•Buffers were redesigned from [32,33,35].•No DNASE, Ribonuclease A or beads were used during the purification.•Simple and inexpensive dsRNA extraction and purification method is described.

First report of an Onchocercidae worm infecting Psychodopygus carrerai carrerai sandfly, a putative vector of Leishmania braziliensis in the Amazon.

Sandflies are insects of public health interest due to their role as vectors of parasites of the genus Leishmania, as well as other pathogens. Psychodopygus carrerai carrerai is considered an important sylvatic vector of Leishmania (Viannia) braziliensis in Amazonia. In this study, sandflies were collected in a forested area in the Xapuri municipality, in the State of Acre (Northern Brazil). Two Ps. carrerai carrerai females were found parasitized with a larval form of a filarial worm, one in the labium of the proboscis, the other after the head was squashed, suggesting they were infective larvae. Sandflies were identified through morphological characters as well as amplification and sequencing of the cytochrome oxidase gene (COI). This was the first sequence obtained for Ps. carrerai carrerai for this marker. The obtained nematodes were also characterized through direct sequencing of a fragment of COI and 12S genes, both mitochondrial, and ITS1, a nuclear marker. Phylogenetic analyses revealed that the filarial nematodes belong to a species without sequences for these markers in the database, part of family Onchocercidade and closely related to genus Onchocerca (12S tree). Although sandfly infection with nematodes including members of the Onchocercidae has been reported in the Old World, this is the first report of sandfly infection by a member of the Onchocercidae family in the New World, to the best of our knowledge. Considering that the phylogenetic relationships and location in the insect, it can be expected that this is a parasite of mammals and the transmission cycle should be clarified.

Molecular Epidemiology of Rotavirus A Strains Pre- and Post-Vaccine (Rotarix®) Introduction in Mozambique, 2012-2019: Emergence of Genotypes G3P[4] and G3P[8].

Group A rotavirus (RVA) remains the most important etiological agent associated with severe acute diarrhea in children. Rotarix® monovalent vaccine was introduced into Mozambique's Expanded Program on Immunization in September 2015. In the present study, we report the diversity and prevalence of rotavirus genotypes, pre- (2012-2015) and post-vaccine (2016-2019) introduction in Mozambique, among diarrheic children less than five years of age. Genotyping data were analyzed for five sentinel sites for the periods indicated. The primary sentinel site, Mavalane General Hospital (HGM), was analyzed for the period 2012-2019, and for all five sites (country-wide analyses), 2015-2019. During the pre-vaccine period, G9P[8] was the most predominant genotype for both HGM (28.5%) and the country-wide analysis (46.0%). However, in the post-vaccine period, G9P[8] was significantly reduced. Instead, G3P[8] was the most common genotype at HGM, while G1P[8] predominated country-wide. Genotypes G9P[4] and G9P[6] were detected for the first time, and the emergence of G3P[8] and G3P[4] genotypes were observed during the post-vaccine period. The distribution and prevalence of rotavirus genotypes were distinct in pre- and post-vaccination periods, while uncommon genotypes were also detected in the post-vaccine period. These observations support the need for continued country-wide surveillance to monitor changes in strain diversity, due to possible vaccine pressure, and consequently, the effect on vaccine effectiveness.

Molecular epidemiology of rotavirus a strains pre- and post-vaccine (rotarix®) introduction in mozambique, 2012­2019: emergence of genotypes g3p[4] and g3p[8]

Group A rotavirus (RVA) remains the most important etiological agent associated with severe acute diarrhea in children. Rotarix® monovalent vaccine was introduced into Mozambique's Expanded Program on Immunization in September 2015. In the present study, we report the diversity and prevalence of rotavirus genotypes, pre- (2012­2015) and post-vaccine (2016­2019) introduction in Mozambique, among diarrheic children less than five years of age. Genotyping data were analyzed for five sentinel sites for the periods indicated. The primary sentinel site, Mavalane General Hospital (HGM), was analyzed for the period 2012­2019, and for all five sites (country-wide analyses), 2015­2019. During the pre-vaccine period, G9P[8] was the most predominant genotype for both HGM (28.5%) and the country-wide analysis (46.0%). However, in the post-vaccine period, G9P[8] was significantly reduced. Instead, G3P[8] was the most common genotype at HGM, while G1P[8] predominated country-wide. Genotypes G9P[4] and G9P[6] were detected for the first time, and the emergence of G3P[8] and G3P[4] genotypes were observed during the post-vaccine period. The distribution and prevalence of rotavirus genotypes were distinct in pre- and post-vaccination periods, while uncommon genotypes were also detected in the post-vaccine period. These observations support the need for continued country-wide surveillance to monitor changes in strain diversity, due to possible vaccine pressure, and consequently, the effect on vaccine effectiveness.

Global genome diversity of the Leishmania donovani complex.

Protozoan parasites of the Leishmania donovani complex - L. donovani and L. infantum - cause the fatal disease visceral leishmaniasis. We present the first comprehensive genome-wide global study, with 151 cultured field isolates representing most of the geographical distribution. L. donovani isolates separated into five groups that largely coincide with geographical origin but vary greatly in diversity. In contrast, the majority of L. infantum samples fell into one globally-distributed group with little diversity. This picture is complicated by several hybrid lineages. Identified genetic groups vary in heterozygosity and levels of linkage, suggesting different recombination histories. We characterise chromosome-specific patterns of aneuploidy and identified extensive structural variation, including known and suspected drug resistance loci. This study reveals greater genetic diversity than suggested by geographically-focused studies, provides a resource of genomic variation for future work and sets the scene for a new understanding of the evolution and genetics of the Leishmania donovani complex.

Phylogenetic Studies.

Phylogenetics is an important component of the systems biology approach. Knowledge about evolution of the genus Leishmania is essential to understand various aspects of basic biology of these parasites, such as parasite-host or parasite-vector relationships, biogeography, or epidemiology. Here, we present a comprehensive guideline for performing phylogenetic studies based on DNA sequence data, but with principles that can be adapted to protein sequences or other molecular markers. It is presented as a compilation of the most commonly used genetic targets for phylogenetic studies of Leishmania, including their respective primers for amplification and references, as well as details of PCR assays. Guidelines are, then, presented to choose the best targets in relation to the types of samples under study. Finally, and importantly, instructions are given to obtain optimal sequences, alignments, and datasets for the subsequent data analysis and phylogenetic inference. Different bioinformatics methods and software for phylogenetic inference are presented and explained. This chapter aims to provide a compilation of methods and generic guidelines to conduct phylogenetics of Leishmania for nonspecialists.

Comparative efficacy, toxicity and biodistribution of the liposomal amphotericin B formulations Fungisome® and AmBisome® in murine cutaneous leishmaniasis.

Fungisome® (F), a liposomal amphotericin B (AmB) product, is marketed in India as a safe and effective therapeutic for the parasitic infection visceral leishmaniasis. Its potential in the treatment of cutaneous leishmaniasis (CL), a disfiguring form of the disease affecting the skin, is currently unknown. Here, we report the evaluation of the efficacy of F in the Leishmania major BALB/c murine model of CL, including a head-to-head comparison with the standard liposomal AmB formulation AmBisome® (A). Upon intravenous administration at dose levels of 5, 10 and 15 mg/kg of body weight (on days 0, 2, 4, 6 and 8), F showed clear signs of toxicity (at 15 mg/kg), while A did not. After complete treatment (day 10), the tolerated doses of 5 and 10 mg/kg F had significant antileishmanial activity (ED50 = 4.0 and 12.8 mg/kg for qPCR-based parasite load and lesion size, respectively), although less than that of A at identical doses (ED50 = 3.0 and 8.8 mg/kg). The efficacy of F was inferior compared to A because lower levels of the active agent AmB accumulated within the infected lesion. In conclusion, despite possibly being less safe and efficacious than A at equivalent doses, the moderate in vivo activity of F could indicate a role in the systemic pharmacotherapy of CL.

Molecular characterization of Dirofilaria spp. circulating in Portugal.

BACKGROUND: Dirofilariosis is a potentially zoonotic parasitic disease, mainly transmitted by mosquito vectors in many parts of the world. Data concerning the canine Dirofilaria species currently circulating in Portugal is scarce. Thereby, a large-scale study was conducted to determine the Dirofilaria spp. present in Portugal, based on a molecular approach, and also to optimize a reliable and highly sensitive species-specific polymerase chain reaction (PCR) assay that could be used for the simultaneous detection and differentiation of Dirofilaria immitis, Dirofilaria repens, and other concurrent filarial species in animal reservoirs. METHODS: Blood samples were collected from three districts of Portugal (Coimbra, Santarém and Setúbal) between 2011 and 2013. Samples were tested using rapid immunomigration tests (Witness® Dirofilaria), modified Knott's technique and acid phosphatase histochemical staining. In addition, molecular analysis was performed by amplification of the internal transcribed spacer (ITS) region using two different PCR protocols, specific for molecular screening of canine filarial species. RESULTS: Of the 878 dogs sampled, 8.8% (n = 77) were positive for D. immitis circulating antigen and 13.1% (n = 115) positive for microfilariae by the modified Knott's technique. Of the 134 samples tested by acid phosphatase histochemical staining, 100 (74.6%) were positive for D. immitis. Overall, 13.7% (n = 120) were positive by PCR for D. immitis by ITS2, of which 9.3% (67/720) were also positive by ITS1. ITS2 PCR was the most sensitive and specific method, capable of detecting mixed D. immitis and A. reconditum infections. Heterozygosity, in the form of double peaks, was detected by sequencing of both ITS regions. No D. repens was detected by any of the diagnostic methods. CONCLUSIONS: The present study confirmed D. immitis as the dominant species of the genus Dirofilaria infecting Portuguese dogs, based on sequencing of ITS1 and ITS2 PCR fragments. Additionally, ITS2 PCR was the most adequate method for diagnosis and prevalence estimation.

Dirofilariasis by Dirofilaria repens: an imported case and a brief review.

Human infections caused by Dirofilaria repens, a cosmopolitan zoonotic parasitosis endemic in Southern and Eastern Europe and Asia still is an underdiagnosed infection due to parasite identification difficulties. Here, we report the first human case of subcutaneous dirofilariasis by D. repens diagnosed in Portugal. This was probably an imported case from India, as judged by epidemiological and clinical data. With this presentation we aim to alert clinicians for the emergence of vector-borne zoonoses associated with global warming and international travel. This case showed that differential diagnosis of D. repens in subcutaneous nodules is needed, in order to avoid further complications.

Leishmania donovani populations in Eastern Sudan: temporal structuring and a link between human and canine transmission.

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.

Detection of Wolbachia in Dirofilaria infected dogs in Portugal.

Wolbachia pipiens, an intracellular endosymbiont bacteria of filarial nematodes, has been implicated in the pathogenesis of filarial diseases, in particular in heavy Dirofilaria spp. infections. Antibiotic therapy (doxycycline) against Wolbachia has been proven to be suitable adjunct therapy, prior to adulticide treatment of canine dirofilariosis. Despite its importance, investigation on the Wolbachia/Dirofilaria complex in Portugal had not been undertaken so far. This study reports the first detection of Wolbachia in Dirofilaria spp. infected dogs in the context of an ongoing epidemiological survey in central-south regions in the country. Wolbachia DNA was detected by PCR in 52.6% (20/38) of canine blood samples positive for Dirofilaria immitis based on parasitological (Knott's and Acid Phosphatase) and serological (Witness(®)Dirofilaria) methods. No Wolbachia DNA could be detected in samples from dogs with occult infections (parasite negative but antigen positive). The lack of Wolbachia detection in some microfilaremic dogs was somewhat unexpected and needs to be elucidated in further studies, as the presence or absence of these bacteria in association with microfilaria is of importance for veterinarians in the management and control of canine dirofilariosis.

Genetic diversity evaluation on Portuguese Leishmania infantum strains by multilocus microsatellite typing.

Leishmania infantum is the main etiological agent of zoonotic visceral leishmaniasis in the Mediterranean region, including Portugal, but, given its low isoenzyme diversity in this country, the population structure is poorly known. A set of 14 polymorphic microsatellite markers was studied on 136 Portuguese Leishmania strains isolated from different hosts, geographic regions and different clinical forms. A total of 108 different genotypes were found, which is a degree of genetic diversity comparable to other regions, even within zymodeme MON-1. A single most common genotype was detected in 1:5 of all strains, which, with a greater number of multi-strain genotypes found in the Lisbon Metropolitan Region, particularly for human strains, was suggestive of the occurrence of clonal transmission. In addition, a high re-infection rate was found among HIV+ patients. Model based analysis by STRUCTURE uncovered two main populations (populations A and B, composed of MON-1 and non-MON-1 strains, respectively), with great genetic diversity between them, and two MON-1 sub-populations (A1 and A2). High inbreeding coefficients were found in these populations, although strains with mixed ancestry were identified, suggesting that recombination also plays a role in the epidemiology of this species in Portugal. Some but limited geographical differentiation was observed, with groups of strains from the same regions clustering together, particularly those from canine origin. Our results show that L. infantum isolates from Portugal present microsatellite diversity comparable to other regions and that different transmission models play a role in its epidemiology, from clonal transmission to recombination. In addition, although Portugal is a small country, mobility of people and animals is high and Leishmania can be probably easily disseminated between infected hosts throughout the country, two instances of seemingly local restricted transmission were identified.

Population structure and evidence for both clonality and recombination among Brazilian strains of the subgenus Leishmania (Viannia).

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.

New insights on taxonomy, phylogeny and population genetics of Leishmania (Viannia) parasites based on multilocus sequence analysis.

The Leishmania genus comprises up to 35 species, some with status still under discussion. The multilocus sequence typing (MLST)--extensively used for bacteria--has been proposed for pathogenic trypanosomatids. For Leishmania, however, a detailed analysis and revision on the taxonomy is still required. We have partially sequenced four housekeeping genes--glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), mannose phosphate isomerase (MPI) and isocitrate dehydrogenase (ICD)--from 96 Leishmania (Viannia) strains and assessed their discriminatory typing capacity. The fragments had different degrees of diversity, and are thus suitable to be used in combination for intra- and inter-specific inferences. Species-specific single nucleotide polymorphisms were detected, but not for all species; ambiguous sites indicating heterozygosis were observed, as well as the putative homozygous donor. A large number of haplotypes were detected for each marker; for 6PGD a possible ancestral allele for L. (Viannia) was found. Maximum parsimony-based haplotype networks were built. Strains of different species, as identified by multilocus enzyme electrophoresis (MLEE), formed separated clusters in each network, with exceptions. NeighborNet of concatenated sequences confirmed species-specific clusters, suggesting recombination occurring in L. braziliensis and L. guyanensis. Phylogenetic analysis indicates L. lainsoni and L. naiffi as the most divergent species and does not support L. shawi as a distinct species, placing it in the L. guyanensis cluster. BURST analysis resulted in six clonal complexes (CC), corresponding to distinct species. The L. braziliensis strains evaluated correspond to one widely geographically distributed CC and another restricted to one endemic area. This study demonstrates the value of systematic multilocus sequence analysis (MLSA) for determining intra- and inter-species relationships and presents an approach to validate the species status of some entities. Furthermore, it contributes to the phylogeny of L. (Viannia) and might be helpful for epidemiological and population genetics analysis based on haplotype/diplotype determinations and inferences.

In vitro and in vivo behaviour of sympatric Leishmania (V.) braziliensis, L. (V.) peruviana and their hybrids.

Leishmania (Viannia) braziliensis is the main cause of highly disfiguring mucocutaneous leishmaniasis (MCL) in South America. The related species L. (V.) peruviana has only been identified in simple cutaneous lesions (CL). Hybrids between L. braziliensis and L. peruviana have been reported although genetic exchange in Leishmania is considered to be rare. Here we compared growth in vitro, adaptive capacity under thermal and oxidative stress and behaviour in a hamster model, of L. braziliensis, L. peruviana, and their putative hybrids. At 24°C, the optimal temperature for in vitro growth, L. braziliensis had the highest growth rate. In in vitro studies hybrid clones presented heterogeneous phenotypes, from slower growth rates, similar to L. peruviana, to higher growth rates, as observed in L. braziliensis. Hamsters infected with hybrid strains, presented the highest parasite densities and aggressive relapses at a later stage of infection. Hybrids generally presented higher plasticity and phenotypic diversity than the putative parental species, with potential eco-epidemiological implications, including an impact on the success of disease control.

Multilocus sequence typing (MLST) for lineage assignment and high resolution diversity studies in Trypanosoma cruzi.

BACKGROUND: Multilocus sequence typing (MLST) is a powerful and highly discriminatory method for analysing pathogen population structure and epidemiology. Trypanosoma cruzi, the protozoan agent of American trypanosomiasis (Chagas disease), has remarkable genetic and ecological diversity. A standardised MLST protocol that is suitable for assignment of T. cruzi isolates to genetic lineage and for higher resolution diversity studies has not been developed. METHODOLOGY/PRINCIPAL FINDINGS: We have sequenced and diplotyped nine single copy housekeeping genes and assessed their value as part of a systematic MLST scheme for T. cruzi. A minimum panel of four MLST targets (Met-III, RB19, TcGPXII, and DHFR-TS) was shown to provide unambiguous assignment of isolates to the six known T. cruzi lineages (Discrete Typing Units, DTUs TcI-TcVI). In addition, we recommend six MLST targets (Met-II, Met-III, RB19, TcMPX, DHFR-TS, and TR) for more in depth diversity studies on the basis that diploid sequence typing (DST) with this expanded panel distinguished 38 out of 39 reference isolates. Phylogenetic analysis implies a subdivision between North and South American TcIV isolates. Single Nucleotide Polymorphism (SNP) data revealed high levels of heterozygosity among DTUs TcI, TcIII, TcIV and, for three targets, putative corresponding homozygous and heterozygous loci within DTUs TcI and TcIII. Furthermore, individual gene trees gave incongruent topologies at inter- and intra-DTU levels, inconsistent with a model of strict clonality. CONCLUSIONS/SIGNIFICANCE: We demonstrate the value of systematic MLST diplotyping for describing inter-DTU relationships and for higher resolution diversity studies of T. cruzi, including presence of recombination events. The high levels of heterozygosity will facilitate future population genetics analysis based on MLST haplotypes.

Comparative microsatellite typing of new world leishmania infantum reveals low heterogeneity among populations and its recent old world origin.

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.