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      High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species

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          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.

          Author Summary

          The different clinical forms of the Leishmaniases range from cutaneous to visceral infections and are caused by organisms belonging to the genus Leishmania. Controversy over the validity of different molecular methods to correctly identify a species hinders the association of a given species with different clinical forms, complicating the prognosis and the development of suitable treatment protocols. A correct identification leads to a better understanding of the action and consequent development of new drugs and immunological reactions. It also provides important information about the relationship of each species with its hosts (humans, animal reservoirs and sandflies) in different geographical areas and ecological situations, helping to design control strategies. Today, PCR is the most commonly used method for Leishmania identification, but even though several targets have been described, no simple and direct protocol has emerged. In this paper, we coupled hsp70 real-time PCR with the determination of amplicon melting profiles in order to explore polymorphic regions by HRM analysis. This methodology yielded discriminatory melting temperature (Tm) values for Brazilian and Eurasian/African Leishmania species. The protocol has proven to be 100% reliable with both clinical and experimental samples. The major advantage of the presently described method is that it is simple, less expensive, highly sensitive and easily automated.

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          Most cited references 23

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          Control of the leishmaniases.

            (2009)
          This report makes recommendations on new therapeutic regimens for visceral and cutaneous leishmaniasis, on the use of rapid diagnostic tests, details on the management of Leishmania-HIV coinfection and consideration of social factors and climate change as risk factors for increased spread. Recommendations for research include the furtherance of epidemiological knowledge of the disease and clinical studies to address the lack of an evidence-based therapeutic regimen for cutaneous and mucocutaneous leishmaniasis and post-kala-azar dermal leishmaniasis (PKDL). This report not only provides clear guidance on implementation but should also raise awareness about the global burden of leishmaniasis and its neglect. It puts forward directions for formulation of national control programmes and elaborates the strategic approaches in the fight against the leishmaniases. The committee's work reflects the latest scientific and other relevant developments in the field of leishmaniasis that can be considered by member states when setting national programmes and making public health decisions.
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            Molecular diagnosis of leishmaniasis: current status and future applications.

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              Phylogeny of Leishmania species based on the heat-shock protein 70 gene.

              The 70kDa heat-shock protein (HSP70) is conserved across prokaryotes and eukaryotes, and the protein as well as its encoding gene have been applied in phylogenetic studies of different parasites. In spite of the frequent use of New World Leishmania species identification on the basis of restriction fragment length polymorphisms (RFLP) in the hsp70 gene, it was never sequenced extensively for studying evolutionary relationships. To fill this void we determined the nucleotide sequence of an 1380bp fragment of the coding region commonly used in RFLP analysis, from 43 isolates and strains of different geographic origins. Combination with previously determined sequences amounted to a phylogenetic analysis including 52 hsp70 sequences representing 17 species commonly causing leishmaniasis both in the New and Old World. The genus Leishmania formed a monophyletic group with three distinct subgenera L. (Leishmania), L. (Viannia), and L. (Sauroleishmania). The obtained phylogeny supports the following eight species: L. (L.) donovani, L. (L.) major, L. (L.) tropica, L. (L.) mexicana, L. (V.) lainsoni, L. (V.) naiffi, L. (V.) guyanensis and L. (V.) braziliensis, in some of which subspecies can be recognized: L. (L.) donovani infantum, L. (V.) guyanensis panamensis, and L. (V.) braziliensis peruviana. The currently recognized L. (L.) aethiopica, L. (L.) garnhami, and L. (L.) amazonensis did not form monophyletic clusters. These findings are discussed in relation to results from other genes and proteins, which have to be integrated in order to build a genetically supported taxonomy for the entire genus.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                29 February 2016
                February 2016
                : 10
                : 2
                Affiliations
                [1 ]Physiology Department, Biosciences Institute, São Paulo University, São Paulo, São Paulo, Brazil
                [2 ]Pathology Department, Medical Faculty, São Paulo University, São Paulo, São Paulo, Brazil
                [3 ]Parasitology Department, Biomedical Institute, São Paulo University, São Paulo, São Paulo, Brazil
                US Food and Drug Administration, UNITED STATES
                Author notes

                The authors have declared that no competing interests exists.

                Conceived and designed the experiments: RAZ MFLS JJS LMFW. Performed the experiments: RAZ SMM ACSdL. Analyzed the data: RAZ MFLS SMM ACSdL JJS LMFW. Contributed reagents/materials/analysis tools: LMFW. Wrote the paper: RAZ MFLS SMM JJS LMFW.

                Article
                PNTD-D-15-01694
                10.1371/journal.pntd.0004485
                4771719
                26928050
                © 2016 Zampieri et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 6, Tables: 4, Pages: 18
                Product
                Funding
                This work was funded by: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) www.fapesp.br; and Conselho Nacional para o Desenvolvimento Científico e Tecnológico (CNPq) www.cnpq.br. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Protozoans
                Parasitic Protozoans
                Leishmania
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and Life Sciences
                Organisms
                Protozoans
                Parasitic Protozoans
                Leishmania
                Leishmania Infantum
                Physical Sciences
                Physics
                Condensed Matter Physics
                Phase Transitions
                Melting
                Biology and Life Sciences
                Organisms
                Protozoans
                Parasitic Protozoans
                Leishmania
                Leishmania Major
                Biology and Life Sciences
                Organisms
                Protozoans
                Parasitic Protozoans
                Leishmania
                Leishmania Donovani
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Alignment
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Alignment
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Sequencing techniques
                Sequence analysis
                DNA sequence analysis
                Research and analysis methods
                Molecular biology techniques
                Sequencing techniques
                Sequence analysis
                DNA sequence analysis
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

                Infectious disease & Microbiology

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