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Three new sensitive and specific heat-shock protein 70 PCRs for global Leishmania species identification

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Abstract

The heat-shock protein 70 gene (hsp70) has been exploited for Leishmania species identification in the New and Old World, using PCR followed by restriction fragment length polymorphism (RFLP) analysis. Current PCR presents limitations in terms of sensitivity, which hampers its use for analyzing clinical and biological samples, and specificity, which makes it inappropriate to discriminate between Leishmania and other trypanosomatids. The aim of the study was to improve the sensitivity and specificity of a previously reported hsp70 PCR using alternative PCR primers and RFLPs. Following in silico analysis of available sequences, three new PCR primer sets and restriction digest schemes were tested on a globally representative panel of 114 Leishmania strains, various other infectious agents, and clinical samples. The largest new PCR fragment retained the discriminatory power from RFLP, while two smaller fragments discriminated less species. The detection limit of the new PCRs was between 0.05 and 0.5 parasite genomes, they amplified clinical samples more efficiently, and were Leishmania specific. We succeeded in significantly improving the specificity and sensitivity of the PCRs for hsp70 Leishmania species typing. The improved PCR-RFLP assays can impact diagnosis, treatment, and epidemiological studies of leishmaniasis in any setting worldwide.

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Acknowledgements

The authors would like to thank all colleagues and institutes who kindly donated the Leishmania reference strains or DNA: J. Arévalo (Instituto de Medicina Tropical Alexander von Humboldt, Lima, Peru); L. García (Centro Universitario de Medicina Tropical, Cochabamba, Bolivia); E. Cupolillo (Instituto Oswaldo Cruz, Rio de Janeiro, Brazil); G. Schönian (Institut für Mikrobiologie und Hygiene, Berlin, Germany); I. Mauricio and D. Evans (London School of Hygiene and Tropical Medicine, London, UK); P. Desjeux (Instituto Boliviano de Biología de Altura, La Paz, Bolivia); J.-P. Dedet and J.A. Rioux (Centre National de Référence des Leishmania, Montpellier, France); J.J. Shaw (University of São Paulo, São Paulo, Brazil); G. Schoone and A. El Harith (Royal Tropical Institute, Amsterdam, The Netherlands); Laboratorio de Biología Molecular (Departamento de Parasitología, Bacteriología y Virología, IPK, Havana, Cuba); and the LeishEpinetSA consortium (EU contract INCO-CT2005-015407). Clinical samples were provided by M. Gramiccia (Istituto Superiore di Sanità, Rome, Italy); A. Llanos (Instituto de Medicina Tropical Alexander von Humboldt, Lima, Peru); L. Campino (Instituto de Higiene e Medicina Tropical, Lisbon, Portugal); S. El Safi (University of Khartoum, Khartoum, Sudan); and A. Ben Salah (Institut Pasteur de Tunis, Tunis, Tunisia). We acknowledge Simonne De Doncker (ITM) for technical assistance. This work was funded by the third framework program of the Belgian Development Cooperation between ITM and IPK, which also provided salary support for GVDA.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Parasitology Department, Institute of Tropical Medicine “Pedro Kourí” (IPK), Havana, Cuba

    A. M. Montalvo & J. Fraga

  2. Department of Biomedical Sciences, Institute of Tropical Medicine (ITM), Nationalestraat 155, 2000, Antwerp, Belgium

    I. Maes, J.-C. Dujardin & G. Van der Auwera

  3. Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium

    J.-C. Dujardin

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  1. A. M. Montalvo
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  2. J. Fraga
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  4. J.-C. Dujardin
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  5. G. Van der Auwera
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Corresponding author

Correspondence to G. Van der Auwera.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Table ESM-1

Lists all reference strains and isolates used for the study, with WHO codes. Strains used for the wet and in silico analyses are indicated, as well as accession numbers of the hsp70 sequences (PDF 498 kb)

Table ESM-2

Gives a schematic overview of the recommended RFLP scheme for all identified groups (PDF 152 kb)

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Montalvo, A.M., Fraga, J., Maes, I. et al. Three new sensitive and specific heat-shock protein 70 PCRs for global Leishmania species identification. Eur J Clin Microbiol Infect Dis 31, 1453–1461 (2012). https://doi.org/10.1007/s10096-011-1463-z

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