High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species.
ABSTRACT: BACKGROUND:Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol. METHODS/PRINCIPAL FINDINGS:Exploring the High Resolution Melting (HRM) dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR) targeting heat-shock protein 70 coding gene (hsp70) revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania) infantum chagasi, L. (L.) amazonensis, L. (L.) mexicana, L. (Viannia) lainsoni, L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi and L. (V.) shawi, and three species found in Eurasia and Africa, including L. (L.) tropica, L. (L.) donovani and L. (L.) major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol. CONCLUSIONS/SIGNIFICANCE:HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.
Project description:BACKGROUND:The leishmaniases comprise a spectrum of clinical manifestations caused by different species of Leishmania. Identification of species is important for diagnosis, treatment and follow-up management. However, there is no gold standard for species identification. High resolution melting analysis (HRM) offers a possibility to differentiate Leishmania species without the need for processing of the PCR-product. The amino acid permease 3 (aap3) gene is an exclusive target for trypanosomatids and is conserved among Leishmania spp., thus it can be a valuable target for an HRM assay for diagnosis of the leishmaniases. RESULTS:The HRM dissociation profiles of three amplicons targeting the aap3-coding region allowed the discrimination of L. (Leishmania) donovani, L. (L.) infantum, L. (L.) major, L. (L.) tropica, L. (L.) mexicana, L. (L.) amazonensis, L. (Viannia) braziliensis, L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) naiffi and L. (V.) shawi using DNA from promastigote cultures. The protocol was validated with DNA samples from clinical infection in humans and a cat, naturally infected sand flies, and experimentally infected mice. CONCLUSIONS:HRM analysis using the aap3 coding sequence as target is a relatively cheap, fast and robust strategy to detect and discriminate Leishmania species from all the endemic regions worldwide. The target and method proved to be useful in clinical, field and experimental samples, thus it could be used as a tool in diagnosis as well as ecological and epidemiological studies.
Project description:<h4>Background/objectives</h4>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.<h4>Methodology/principal findings</h4>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.<h4>Conclusions/significance</h4>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.
Project description:In French Guiana, five species are associated with Cutaneous Leishmaniasis (CL). Though infections with Leishmania guyanensis, L. (V.) braziliensis and L. (L.) amazonensis have been extensively described, there are few available clinical and genetic data on L. (V.) lainsoni and L. (V.) naiffi. We determined the clinical and epidemiological features of all cases of CL due to L. (V.) naiffi and L. (V.) lainsoni diagnosed in French Guiana between 2003 and 2019. Phylogenetic analysis was performed by sequencing a portion of HSP70 and cyt b genes. Five cases of L. naiffi and 25 cases of L. lainsoni were reported. Patients infected by L. (V.) lainsoni were usually infected on gold camps, mostly along the Maroni river (60%), while L. naiffi was observed in French patients infected on the coast (100%). A high number of pediatric cases (n = 5; 20%) was observed for L. (V.) lainsoni. A mild clinical course was observed for all cases of L. (V.) naiffi. HSP70 and cyt b partial nucleotide sequence analysis revealed different geographical clusters within L. (V.) naiffi and L. (V.) lainsoni but no association were found between phylogenetic and clinical features. Our data suggest distinct socio-epidemiological features for these two Leishmania species. Patients seem to get infected with L. (V.) naiffi during leisure activities in anthropized coastal areas, while L. (V.) lainsoni shares common features with L. (V.) guyanensis and braziliensis and seems to be acquired during professional activities in primary forest regions. Phylogenetic analysis has provided information on the intraspecific genetic variability of L. (V.) naiffi and L. (V.) lainsoni and how these genotypes are distributed at the geographic level.
Project description:The aims of the study were to determine the blood feeding preferences of sandflies and to identify species of Leishmania that infected phlebotomines in Caxias, Maranhão, Brazil, an area that is highly endemic for leishmaniasis. Sandflies were captured in light traps located in the peridomiciliary environments of randomly selected houses in urban and rural settings between 1800 and 0600 hours on new moon days between March 2013 and February 2015. DNA extracts from 982 engorged female sandflies were submitted to fragment length polymorphism analysis to identify infecting species of Leishmania, and blood sources were identified for 778 of these specimens. Infection by Leishmania infantum was detected in Lutzomyia longipalpis, Lu. whitmani and Lu. termitophila; L. infantum/L. braziliensis in Lu. longipalpis, Lu. whitmani and Lu. trinidadensis; L. shawi in Lu. longipalpis; L. mexicana in Lu. longipalpis; L. braziliensis in Lu. longipalpis and Lu. whitmani; L. guyanensis in Lu. longipalpis and Lu. termitophila; L. amazonensis in Lu. longipalpis and L. lainsoni or L. naiffi in Lu. longipalpis, while Lu. longipalpis and Lu. trinidadensis were infected with unidentified Leishmania sp. Blood sources were identified in 573 individual phlebotomines and the preferred hosts were, in decreasing order, chicken, dog, rodent and human with lower preferences for pig, horse, opossum and cattle. Lu. longipalpis and Lu. whitmani performed mixed feeding on man, dog and rodent, while Lu. longipalpis was the most opportunistic species, feeding on the blood of all hosts surveyed, but preferably on dog/chicken, dog/rodent and rodent/chicken. Our findings reveal the concomitant circulation of Leishmania species that cause visceral leishmaniasis and tegumentary leishmaniasis in the study area, and explain the occurrence of autochthonous human cases of both clinical forms of leishmaniasis in Caxias, Maranhão. The results support our hypothesis that, in the municipality of Caxias, transmission of Leishmania occurs in close proximity to humans.
Project description:<h4>Background</h4>Caviomorph rodents, some of the oldest Leishmania spp. hosts, are widely dispersed in Brazil. Despite both experimental and field studies having suggested that these rodents are potential reservoirs of Leishmania parasites, not more than 88 specimens were analyzed in the few studies of natural infection. Our hypothesis was that caviomorph rodents are inserted in the transmission cycles of Leishmania in different regions, more so than is currently recognized.<h4>Methodology</h4>We investigated the Leishmania infection in spleen fragments of 373 caviomorph rodents from 20 different species collected in five Brazilian biomes in a period of 13 years. PCR reactions targeting kDNA of Leishmania sp. were used to diagnose infection, while Leishmania species identification was performed by DNA sequencing of the amplified products obtained in the HSP70 (234) targeting. Serology by IFAT was performed on the available serum of these rodents.<h4>Principal findings</h4>In 13 caviomorph rodents, DNA sequencing analyses allowed the identification of 4 species of the subgenus L. (Viannia): L. shawi, L. guyanensis, L. naiffi, and L. braziliensis; and 1 species of the subgenus L. (Leishmania): L. infantum. These include the description of parasite species in areas not previously included in their known distribution: L. shawi in Thrichomys inermis from Northeastern Brazil and L. naiffi in T. fosteri from Western Brazil. From the four other positive rodents, two were positive for HSP70 (234) targeting but did not generate sequences that enabled the species identification, and another two were positive only in kDNA targeting.<h4>Conclusions/significance</h4>The infection rate demonstrated by the serology (51.3%) points out that the natural Leishmania infection in caviomorph rodents is much higher than that observed in the molecular diagnosis (4.6%), highlighting that, in terms of the host species responsible for maintaining Leishmania species in the wild, our current knowledge represents only the "tip of the iceberg."
Project description:The unicellular protozoan parasite Leishmania causes the neglected tropical disease leishmaniasis, affecting 12 million people in 98 countries. In South America, where the Viannia subgenus predominates, so far only L. (Viannia) braziliensis and L. (V.) panamensis have been sequenced, assembled and annotated as reference genomes. Addressing this deficit in molecular information can inform species typing, epidemiological monitoring and clinical treatment. Here, L. (V.) naiffi and L. (V.) guyanensis genomic DNA was sequenced to assemble these two genomes as draft references from short sequence reads. The methods used were tested using short sequence reads for L. braziliensis M2904 against its published reference as a comparison. This assembly and annotation pipeline identified 70 additional genes not annotated on the original M2904 reference. Phylogenetic and evolutionary comparisons of L. guyanensis and L. naiffi with 10 other Viannia genomes revealed four traits common to all Viannia: aneuploidy, 22 orthologous groups of genes absent in other Leishmania subgenera, elevated TATE transposon copies and a high NADH-dependent fumarate reductase gene copy number. Within the Viannia, there were limited structural changes in genome architecture specific to individual species: a 45?Kb amplification on chromosome 34 was present in all bar L. lainsoni, L. naiffi had a higher copy number of the virulence factor leishmanolysin, and laboratory isolate L. shawi M8408 had a possible minichromosome derived from the 3' end of chromosome 34. This combination of genome assembly, phylogenetics and comparative analysis across an extended panel of diverse Viannia has uncovered new insights into the origin and evolution of this subgenus and can help improve diagnostics for leishmaniasis surveillance.
Project description:A countrywide epidemiological study was performed to elucidate the current geographic distribution of causative species of cutaneous leishmaniasis (CL) in Ecuador by using FTA card-spotted samples and smear slides as DNA sources. Putative Leishmania in 165 samples collected from patients with CL in 16 provinces of Ecuador were examined at the species level based on the cytochrome b gene sequence analysis. Of these, 125 samples were successfully identified as Leishmania (Viannia) guyanensis, L. (V.) braziliensis, L. (V.) naiffi, L. (V.) lainsoni, and L. (Leishmania) mexicana. Two dominant species, L. (V.) guyanensis and L. (V.) braziliensis, were widely distributed in Pacific coast subtropical and Amazonian tropical areas, respectively. Recently reported L. (V.) naiffi and L. (V.) lainsoni were identified in Amazonian areas, and L. (L.) mexicana was identified in an Andean highland area. Importantly, the present study demonstrated that cases of L. (V.) braziliensis infection are increasing in Pacific coast areas.
Project description:<h4>Background</h4>Leishmaniasis is endemic in Tunisia and presents with different clinical forms, caused by the species Leishmania infantum, Leishmania major, and Leishmania tropica. The life cycle of Leishmania is complex and involves several phlebotomine sand fly vectors and mammalian reservoir hosts. The aim of this work is the development and evaluation of a high-resolution melting PCR (PCR-HRM) tool to detect and identify Leishmania parasites in wild and domestic hosts, constituting confirmed (dogs and Meriones rodents) or potential (hedgehogs) reservoirs in Tunisia.<h4>Methods</h4>Using in vitro-cultured Leishmania isolates, PCR-HRM reactions were developed targeting the 7SL RNA and HSP70 genes. Animals were captured or sampled in El Kef Governorate, North West Tunisia. DNA was extracted from the liver, spleen, kidney, and heart from hedgehogs (Atelerix algirus) (n = 3) and rodents (Meriones shawi) (n = 7) and from whole blood of dogs (n = 12) that did not present any symptoms of canine leishmaniasis. In total, 52 DNA samples were processed by PCR-HRM using both pairs of primers.<h4>Results</h4>The results showed melting curves enabling discrimination of the three Leishmania species present in Tunisia, and were further confirmed by Sanger sequencing. Application of PCR-HRM assays on reservoir host samples showed that overall among the examined samples, 45 were positive, while seven were negative, with no Leishmania infection. Meriones shawi were found infected with L. major, while dogs were infected with L. infantum. However, co-infections with L. major/L. infantum species were detected in four Meriones specimens and in all tested hedgehogs. In addition, multiple infections with the three Leishmania species were found in one hedgehog specimen. Sequence analyses of PCR-HRM products corroborated the Leishmania species found in analyzed samples.<h4>Conclusions</h4>The results of PCR-HRM assays applied to field specimens further support the possibility of hedgehogs as reservoir hosts of Leishmania. In addition, we showed their usefulness in the diagnosis of canine leishmaniasis, specifically in asymptomatic dogs, which will ensure a better evaluation of infection extent, thus improving elaboration of control programs. This PCR-HRM method is a robust and reliable tool for molecular detection and identification of Leishmania and can be easily implemented in epidemiological surveys in endemic regions.
Project description:To obtain further insight into geographic distribution of Leishmania species in Peru, a countrywide survey, including central to southern rainforest areas where information on causative parasite species is limited, was performed based on cytochrome b (cyt b) and mannose phosphate isomerase (mpi) gene analyses. A total of 262 clinical samples were collected from patients suspected of cutaneous leishmaniasis (CL) in 28 provinces of 13 departments, of which 99 samples were impregnated on FTA (Flinders Technology Associates) cards and 163 samples were Giemsa-stained smears. Leishmania species were successfully identified in 83 (83.8%) of FTA-spotted samples and 59 (36.2%) of Giemsa-stained smear samples. Among the 142 samples identified, the most dominant species was Leishmania (Viannia) braziliensis (47.2%), followed by L. (V.) peruviana (26.1%), and others were L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) shawi, a hybrid of L. (V.) braziliensis and L. (V.) peruviana, and Leishmania (Leishmania) amazonensis. Besides the present epidemiological observations, the current study provided the following findings: 1) A hybrid of L. (V.) braziliensis and L. (V.) peruviana is present outside the Department of Huanuco, the only place reported, 2) Many cases of CL due to L. (V.) lainsoni, an uncommon causative species in Peru, were observed, and 3) L. (V.) shawi is widely circulating in southern Amazonian areas in Peru.
Project description:Tegumentary Leishmaniasis (TL) is endemic in Latin America, and Brazil contributes approximately 20 thousand cases per year. The pathogenesis of TL, however, is still not fully understood. Clinical manifestations vary from cutaneous leishmaniasis (CL) to more severe outcomes, such as disseminated leishmaniasis (DL), mucosal leishmaniasis (ML) and diffuse cutaneous leishmaniasis (DCL). Many factors have been associated with the severity of the disease and the development of lesions. Recent studies have reported that the presence of Leishmania RNA virus 1 infecting Leishmania (Leishmania RNA virus 1, LRV1) is an important factor associated with the severity of ML in experimental animal models. In the present study, 156 patients who attended Rondonia's Hospital of Tropical Medicine with both leishmaniasis clinical diagnoses (109 CL; 38 ML; 5 CL+ML; 3 DL and 1 DCL) and molecular diagnoses were investigated. The clinical diagnosis were confirmed by PCR by targeting hsp70 and kDNA DNA sequences and the species causing the infection were determined by HSP70 PCR-RFPL. The presence of LVR1 was tested by RT-PCR. Five Leishmania species were detected: 121 (77.6%) samples were positive for Leishmania (Viannia) braziliensis, 18 (11.5%) were positive for Leishmania (V.) guyanensis, 3 (1.8%) for Leishmania (V.) lainsoni, 2 (1.3%) for Leishmania (Leishmania) amazonensis and 2 (1.3%) for Leishmania (V.) shawi. Six (3.9%) samples were positive for Leishmania sp. but the species could not be determined, and 4 (2.6%) samples were suggestive of mixed infection by L. (V.) braziliensis and L. (V.) guyanensis. The virus was detected in L. braziliensis (N = 54), L. guyanensis (N = 5), L. amazonensis (N = 2), L. lainsoni (N = 1) and inconclusive samples (N = 6). Patients presenting with CL+ML, DL and DCL were excluded from further analysis. Association between the presence of the virus and the disease outcome were tested among the remaining 147 patients (CL = 109 and ML = 38). Of them, 71.1% (n = 27) mucosal lesions were positive for LRV1, and 28.9% (n = 11) were negative. In cutaneous lesions, 36.7% (n = 40) were positive and 63.3% (n = 69) were negative for LRV1. The ratio P(ML|LRV1+)/P(ML|LRV1-) was 2.93 (CI95% 1.57...5.46; p<0.001), thus corroborating the hypothesis of the association between LRV1 and the occurrence of mucosal leishmaniasis, as previously described in animal models; it also indicates that LRV1 is not the only factor contributing to the disease outcome.