Heat-shock protein 70 gene sequencing for Leishmania species typing in European tropical infectious disease clinics

Auwera, Gert Van der; Maes, Ilse; Doncker, Simonne De; Ravel, Christophe; Cnops, Lieselotte; Esbroeck, Marjan Van; Gompel, Alfons Van; Clerinx, Jan; Dujardin, Jean‐Claude

doi:10.2807/1560-7917.es2013.18.30.20543

Cited by 83 publications

(94 citation statements)

References 44 publications

(66 reference statements)

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“…All PCRs from the individual markers used in this study have proven to be applicable directly on clinical specimens (4,8,(11)(12)(13)(14)16) and allow species identification on the basis of 1 PCR amplicon instead of the 7 amplicons used in MLST, without the need for parasite culturing, a technique that is time consuming and often not successful. The hsp70 target was identified as the best choice for high-resolution species discrimination, as it identifies the same species and species complexes as does the gold standard MLST applied here.…”

Section: Discussionmentioning

confidence: 99%

“…The 1,286-bp fragment PCR-F was amplified as described previously (14,16) and subsequently sequenced. A global sequence alignment was produced, which was facilitated because no size variation was observed.…”

Section: Strainsmentioning

confidence: 99%

“…Four of these targets are quite widespread in the literature: the miniexon (ME) or spliced leader (4, 5), the internal transcribed spacer (ITS1) of the ribosomal DNA (rDNA) array (6-10), the 7SL RNA gene (11,12), and the heat shock protein 70 gene (hsp70) (13)(14)(15)(16).…”

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confidence: 99%

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Evaluation of Four Single-Locus Markers for Leishmania Species Discrimination by Sequencing

et al. 2014

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f Several genetic markers have been described for discriminating Leishmania species. In most reported cases, one or a few polymorphisms are the basis of species identification, and the methods were validated on a limited number of strains from a particular geographical region. Therefore, most techniques may underestimate the global intraspecies variability and are applicable only in certain areas. In addition, interlaboratory standardization is mostly absent, complicating comparisons among different studies. Here, we compared species typing results from all sequence polymorphisms found in four popular markers that can be applied directly on clinical samples: the miniexon or spliced leader, the internal transcribed spacer of the ribosomal DNA array, the 7SL RNA gene, and the heat shock protein 70 gene. Clustering was evaluated among 74 Leishmania strains, selected to represent a wide geographic distribution and genetic variability of the medically relevant species of the genus. Results were compared with a multilocus sequence typing (MLST) approach using 7 single-copy household genes and with multilocus enzyme electrophoresis (MLEE), still considered the gold standard by some. We show that strain groupings are highly congruent across the four different single-locus markers, MLST, and MLEE. Overall, the heat shock protein 70 gene and the miniexon presented the best resolutions for separating medically relevant species. As gene sequence analysis is validated here on a global scale, it is advocated as the method of choice for use in genetic, clinical, and epidemiological studies and for managing patients with unknown origins of infection, especially in Western infectious disease clinics dealing with imported leishmaniasis.T he parasitic protozoa of the genus Leishmania cause a spectrum of diseases in humans, collectively called the leishmaniases. In its most benign form, referred to as cutaneous leishmaniasis, the disease manifests itself as a localized skin ulcer at the site of infection by the bite of a female infectious sandfly. Sometimes the parasites spread to other parts of the body, causing secondary lesions. In more severe cases, when the mucosa is infected, a condition known as mucosal leishmaniasis, the disease leads to disfiguring lesions of the nose and mouth. Finally, when the parasite colonizes internal organs such as the spleen, liver, and bone marrow, a condition referred to as visceral leishmaniasis, the disease becomes lethal. As the manifestation of disease depends, to a large extent, on the infecting species, so do the treatment options (1, 2).According to a recent estimate (3), leishmaniasis is endemic in 98 countries and 3 territories. Besides the indigenous population being at risk of infection, many active transmission areas are frequently visited by tourists, military personnel, expatriates, and people visiting friends and relatives. They can potentially import leishmaniasis into their home countries, and management of such cases calls for a globally applicable reliable species typing approa...

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Section: Discussionmentioning

confidence: 99%

Section: Strainsmentioning

confidence: 99%

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Evaluation of Four Single-Locus Markers for Leishmania Species Discrimination by Sequencing

et al. 2014

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“…The hsp70 gene is probably the molecular marker more widely used for Leishmania species identification, and currently the hsp70-RFLP approach has become a reference molecular method in many laboratories for these purposes. 10 In this regard, an almost perfect agreement was observed between our calmodulin-RFLP methodology and the hsp70-RFLP protocol previously described. 9 All field samples were typed as L. (V.) panamensis by both molecular methods.…”

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confidence: 54%

Calmodulin Polymerase Chain Reaction–Restriction Fragment Length Polymorphism for Leishmania Identification and Typing

Miranda

Samudio

González

et al. 2016

The American Society of Tropical Medicine and Hygiene

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Abstract. A precise identification of Leishmania species involved in human infections has epidemiological and clinical importance. Herein, we describe a preliminary validation of a restriction fragment length polymorphism assay, based on the calmodulin intergenic spacer region, as a tool for detecting and typing Leishmania species. After calmodulin amplification, the enzyme HaeIII yielded a clear distinction between reference strains of Leishmania mexicana, Leishmania amazonensis, Leishmania infantum, Leishmania lainsoni, and the rest of the Viannia reference species analyzed. The closely related Viannia species: Leishmania braziliensis, Leishmania panamensis, and Leishmania guyanensis, are separated in a subsequent digestion step with different restriction enzymes. We have developed a more accessible molecular protocol for Leishmania identification/typing based on the exploitation of part of the calmodulin gene. This methodology has the potential to become an additional tool for Leishmania species characterization and taxonomy.Leishmaniasis is a major neglected tropical disease affecting around 2 million people worldwide every year. 1 The infections tend to cluster among the poorest and most isolated increasing the socioeconomic burden upon these vulnerable populations. There are more than 20 species of Leishmania capable of infecting humans and each having distinct epidemiological/demographic patterns, and nearly all have a zoonotic transmission with one main sylvatic reservoir.2 In the absence of vaccines and other effective preventive measures, the control of leishmaniasis is currently based on accurate diagnosis and prompt treatment.A precise identification of the species has epidemiological, and particularly clinical importance, since the Leishmania species involved in human infections can influence the treatment decision and outcome.3 Furthermore, species identification is particularly relevant now that the disease appears to be currently underestimated and on the rise in several countries.

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“…(Folgueira e Requena, 2007;Montalvo et al, 2012;Odiwuor et al , 2012). É importante ressaltar que esse gene já tinha sido utilizado para diferenciação de espécies de Leishmania, porém em estudos utilizando a técnica de PCR-RFLP (Garcia et al, 2004;Graca et al, 2012), sequenciamento (Van der Auwera et al , 2013) e mais recentemente com a técnica de HRM (Hernandez et al , 2014 Além disso, também sugerimos a continuidade dessa linha de pesquisa na validação desses ensaios em amostras de lesão tegumentar do paciente.…”

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Utilização do alvo hsp70 em técnicas de biologia molecular para diferenciação de subgêneros de Leishmania spp

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Heat-shock protein 70 gene sequencing for Leishmania species typing in European tropical infectious disease clinics

Cited by 83 publications

References 44 publications

Evaluation of Four Single-Locus Markers for Leishmania Species Discrimination by Sequencing

Evaluation of Four Single-Locus Markers for Leishmania Species Discrimination by Sequencing

Calmodulin Polymerase Chain Reaction–Restriction Fragment Length Polymorphism for Leishmania Identification and Typing

Utilização do alvo hsp70 em técnicas de biologia molecular para diferenciação de subgêneros de Leishmania spp

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