Detection and Identification of Old World Leishmania by High Resolution Melt Analysis

Talmi-Frank, Dalit; Nasereddin, Abedelmajeed; Schnur, Lionel F.; Schönian, Gabriele; Töz, Seray; Jaffe, Charles L.; Baneth, Gad

doi:10.1371/journal.pntd.0000581

Cited by 110 publications

(101 citation statements)

References 19 publications

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“…Recently, high-resolution melt (HRM) analysis of a real-time PCR product from the ITS1 region was used to identify and quantify Old World Leishmania species (Talmi-Frank et al 2010). It is a closed tube assay that does not employ additional fluorescent probes but simply utilizes a DNA melting assay and computerized analysis of the results to produce a graphic output.…”

Section: O L E C U L a R E P I D E M I O L O G Y A N D M O L E C Umentioning

confidence: 99%

Molecular approaches for a better understanding of the epidemiology and population genetics ofLeishmania

2010

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S U M M A R YMolecular approaches are being used increasingly for epidemiological studies of visceral and cutaneous leishmaniases. Several molecular markers resolving genetic differences between Leishmania parasites at species and strain levels have been developed to address key epidemiological and population genetic questions. The current gold standard, multilocus enzyme typing (MLEE), needs cultured parasites and lacks discriminatory power. PCR assays identifying species directly with clinical samples have proven useful in numerous field studies. Multilocus sequence typing (MLST) is potentially the most powerful phylogenetic approach and will, most probably, replace MLEE in the future. Multilocus microsatellite typing (MLMT) is able to discriminate below the zymodeme level and seems to be the best candidate for becoming the gold standard for distinction of strains. Population genetic studies by MLMT revealed geographical and hierarchic population structure in L. tropica, L. major and the L. donovani complex. The existence of hybrids and gene flow between Leishmania populations suggests that sexual recombination is more frequent than previously thought. However, typing and analytical tools need to be further improved. Accessible databases should be created and sustained for integrating data obtained by different researchers. This would allow for global analyses and help to avoid biases in analyses due to small sample sizes.

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Section: O L E C U L a R E P I D E M I O L O G Y A N D M O L E C Umentioning

confidence: 99%

Molecular approaches for a better understanding of the epidemiology and population genetics ofLeishmania

2010

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“…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)(7)(8)(9)(10), the 7SL RNA gene (11,12), and the heat shock protein 70 gene (hsp70) (13)(14)(15)(16).…”

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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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“…These tests can also be used in studies of animal models, drug efficacy, and vectorial capacity (31). Among the various techniques available, some methods stand out for being applicable not only to diagnosis and differentiation of species, but also to treatment monitoring, and thus have become a promising tool to aid in clinical diagnosis.…”

Section: Resultsmentioning

confidence: 99%

Molecular techniques for the study and diagnosis of parasite infection

Tavares¹,

Staggemeier²,

Borges³

et al. 2011

J. Venom. Anim. Toxins incl. Trop. Dis

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Abstract:In parasitology, routine laboratory diagnosis involves conventional methods, such as optical microscopy, used for the morphological identification of parasites. Currently, molecular biology techniques are increasingly used to diagnose parasite structures in order to enhance the identification and characterization of parasites. The objective of the present study was to review the main current and new diagnostic techniques for confirmation of parasite infections, namely: polymerase chain reaction (PCR), real-time polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), Luminex xMAP, random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and restriction fragment length polymorphism (RFLP), in addition to microsatellites. Molecular assays have comprehensively assisted in the diagnosis, treatment and epidemiological studies of parasitic diseases that affect people worldwide, helping to control parasitic disease mortality.Key words: parasite infection, diagnosis, molecular techniques, molecular epidemiology. Review ARticle The Journal of Venomous Animals and Toxins including Tropical Diseases ISSN 1678-9199 | 2011 | volume 17 | issue 3 | pages 239-248 INTRODUCTIONIn parasitology, routine laboratory diagnosis involves conventional methods, such as optical microscopy, used for the morphological identification of parasites (1). However, the occasional difficulty of identifying these parasite structures may decrease the sensitivity of such methods. Currently, because of these difficulties, molecular biology has been employed to detect parasites responsible for parasitic diseases (2).Traditional parasitological analyses have the advantage of being less costly without requiring expensive reagents and equipment. Additionally, such analyses can be easily performed when a trained microscopist is available. On the other hand, molecular technology demonstrates the presence of parasites based on their antigenic components or DNA segments (3). These tests are not influenced by environmental factors that usually can interfere with the results of a stool test, for example, thus ensuring highly reliable results (4).Current laboratory diagnostic methods for the identification of parasites include: polymerase chain reaction (PCR), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), microsatellite marker method, Luminex xMAP-based technology (areas of multianalyte profiling), loop-mediated isothermal amplification (LAMP), and the recently added real-time PCR (5-12).The objective of the present article was to review the applications of molecular biology techniques in parasitology by evaluating and discussing their uses and benefits. MOLECULAR METHODS FOR DETECTION OF PARASITE STRUCTURESSeveral molecular tests to detect parasites have been developed in the last decade. Their specificity and sensitivity have gradually increased, and parasites that were previously difficult to diagnose usi...

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Detection and Identification of Old World Leishmania by High Resolution Melt Analysis

Cited by 110 publications

References 19 publications

Molecular approaches for a better understanding of the epidemiology and population genetics ofLeishmania

Molecular approaches for a better understanding of the epidemiology and population genetics ofLeishmania

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

Molecular techniques for the study and diagnosis of parasite infection

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