Advertisement

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

  • A. M. Montalvo
  • J. Fraga
  • I. Maes
  • J.-C. Dujardin
  • G. Van der Auwera
Article

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.

Keywords

Restriction Fragment Length Polymorphism Herpes Zoster Leishmaniasis Restriction Fragment Length Polymorphism Analysis Trypanosoma Cruzi 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

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.

Supplementary material

10096_2011_1463_MOESM1_ESM.pdf (499 kb)
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)
10096_2011_1463_MOESM2_ESM.pdf (153 kb)
Table ESM-2 Gives a schematic overview of the recommended RFLP scheme for all identified groups (PDF 152 kb)

References

  1. 1.
    Yamey G, Torreele E (2002) The world’s most neglected diseases. British Med J 325:176–177, Erratum British Med J 325:352CrossRefGoogle Scholar
  2. 2.
    Alvar J, Yactayo S, Bern C (2006) Leishmaniasis and poverty. Trends Parasitol 22:552–557PubMedCrossRefGoogle Scholar
  3. 3.
    Herwaldt BL (1999) Leishmaniasis. Lancet 354:1191–1199PubMedCrossRefGoogle Scholar
  4. 4.
    Arévalo J, Ramírez L, Adaui V, Zimic M, Tulliano G, Miranda-Verástegui C, Lazo M, Loayza-Muro R, De Doncker S, Maurer A, Chappuis F, Dujardin JC, Llanos-Cuentas A (2007) Influence of Leishmania (Viannia) species on the response to antimonial treatment in patients with American tegumentary leishmaniasis. J Inf Dis 195:1846–1851CrossRefGoogle Scholar
  5. 5.
    Llanos-Cuentas A, Tulliano G, Araujo Castillo R, Miranda-Verástegui C, Santamaría-Castrellon G, Ramírez L, Lazo M, De Doncker S, Boelaert M, Robays J, Dujardin JC, Arévalo J, Chappuis F (2008) Clinical and parasite species risk factors for pentavalent antimonial treatment failure in cutaneous leishmaniasis in Peru. Clin Infect Dis 46:223–231PubMedCrossRefGoogle Scholar
  6. 6.
    García AL, Parrado R, De Doncker S, Bermúdez H, Dujardin JC (2007) American tegumentary leishmaniasis: direct species identification of Leishmania in non-invasive clinical samples. Trans R Soc Trop Med Hyg 101:368–371PubMedCrossRefGoogle Scholar
  7. 7.
    Pérez JE, Veland N, Espinosa D, Torres K, Ogusuku E, Llanos-Cuentas A, Gamboa D, Arévalo J (2007) Isolation and molecular identification of Leishmania (Viannia) peruviana from naturally infected Lutzomyia peruensis (Diptera: Psychodidae) in the Peruvian Andes. Mem Inst Oswaldo Cruz 102:655–658PubMedCrossRefGoogle Scholar
  8. 8.
    Ampuero J, Pereira Rios A, Carranza-Tamayo CO, Romero GAS (2009) Genus-specific kinetoplast-DNA PCR and parasite culture for the diagnosis of localized cutaneous leishmaniasis: applications for clinical trials under field conditions in Brazil. Mem Inst Oswaldo Cruz 104:992–997PubMedCrossRefGoogle Scholar
  9. 9.
    Deborggraeve S, Laurent T, Espinosa D, Van der Auwera G, Mbuchi M, Wasunna M, El-Safi S, Al-Basheer AA, Arévalo J, Miranda-Verástegui C, Leclipeteux T, Mertensa P, Dujardin JC, Herdewijn P, Büscher P (2008) A simplified and standardized polymerase chain reaction format for the diagnosis of leishmaniasis. J Infect Dis 198:1565–1572PubMedCrossRefGoogle Scholar
  10. 10.
    Gadisa E, Kuru T, Genet A, Engers H, Aseffa A, Gedamu L (2010) Leishmania donovani complex (Kinetoplastida, Trypanosomatidae). Comparison of deoxyribonucleic acid based techniques for typing isolates from Ethiopia. Exp Parasitol 126:203–208PubMedCrossRefGoogle Scholar
  11. 11.
    Montalvo AM, Fraga J, Montano I, Monzote L, Marín M, Van der Auwera G, Dujardin JC, Vélez ID, Muskus C (2010) Differentiation of Leishmania (Viannia) panamensis and Leishmania (V.) guyanensis using BccI for hsp70 PCR-RFLP. Trans R Soc Trop Med Hyg 104:364–367CrossRefGoogle Scholar
  12. 12.
    Fraga J, Montalvo AM, De Doncker S, Dujardin JC, Van der Auwera G (2010) Phylogeny of Leishmania species based on the heat-shock protein 70 gene. Infect Genet Evol 10:238–245PubMedCrossRefGoogle Scholar
  13. 13.
    Montalvo AM, Fraga J, Monzote I, Montano I, De Doncker S, Dujardin JC, Van der Auwera G (2010) Heat-shock protein PCR-RFLP: a universal simple tool for Leishmania species discrimination in the New and Old World. Parasitology 137:1159–1168PubMedCrossRefGoogle Scholar
  14. 14.
    García AL, Kindt A, Bermúdez H, Llanos-Cuentas A, De Doncker S, Arévalo J (2004) Culture-independent species typing of neotropical Leishmania for clinical validation of a PCR-based assay targeting heat shock protein 70 genes. J Clin Microbiol 42:2294–2297PubMedCrossRefGoogle Scholar
  15. 15.
    Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA 4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  16. 16.
    Rodríguez N, Guzman B, Rodas A, Takiff H, Bloom BR, Convit J (1994) Diagnosis of cutaneous leishmaniasis and species discrimination of parasites by PCR and hybridization. J Clin Microbiol 32:2246–2252PubMedGoogle Scholar
  17. 17.
    Ramírez JR, Agudelo S, Muskus C, Alzate JF, Berberich C, Barker D, Vélez ID (2000) Diagnosis of cutaneous leishmaniasis in Colombia: the sampling site within lesions influences the sensitivity of parasitological diagnosis. J Clin Microbiol 38:3768–3773PubMedGoogle Scholar
  18. 18.
    Bastrenta B, Mita N, Buitrago R, Vargas F, Flores M, Machane M, Yacsik N, Torrez N, Le Pont F, Brenière F (2003) Human mixed infections of Leishmania spp. and Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme. Mem Inst Oswaldo Cruz 98:255–264PubMedCrossRefGoogle Scholar
  19. 19.
    Mendes DG, Lauria-Pires L, Nitz N, Lozzi SP, Nascimento RJ, Monteiro PS, Rebelo MM, Rosa Ade C, Santana JM, Teixeira AR (2007) Exposure to mixed infections with Trypanosoma cruzi, Leishmania braziliensis and Leishmania chagasi in the human population of the greater Amazon. Trop Med Int Health 12:629–636PubMedCrossRefGoogle Scholar
  20. 20.
    Mugasa CM, Deborggraeve S, Schoone GJ, Laurent T, Leeflang MM, Ekangu RA, El-Safi S, Saad AA, Basiye FL, De Doncker S, Lubega GW, Kager PA, Büscher P, Schallig HDFH (2010) Accordance and concordance of PCR and NASBA followed by oligochromatography for the molecular diagnosis of Trypanosoma brucei and Leishmania. Trop Med Intern Health 15:800–805CrossRefGoogle Scholar
  21. 21.
    Bensoussan E, Nasereddin A, Jonas F, Schnur LF, Jaffe CL (2006) Comparison of PCR assays for diagnosis of cutaneous leishmaniasis. J Clin Microbiol 44:1435–1439PubMedCrossRefGoogle Scholar
  22. 22.
    Romero GAS, Ferreira-Noronha E, Pirmez C, Silva-Pires FES, Fernandes O, Saad-Nehmec N, Cupolillo E, Firoozmand L, Cardoso da Graca G, Volpini A, Lopes-Santos S, Romanha AJ (2009) Sensitivity and reproducibility of a PCR assay for Leishmania detection using skin biopsy imprints on filter paper. Acta Trop 109:74–77PubMedCrossRefGoogle Scholar
  23. 23.
    Arévalo J, Ramírez L, Adaui V, Zimic M, Tulliano G, Miranda-Verástegui C, Lazo M, Loayza-Muro R, De Doncker S, Maurer A, Chappuis F, Dujardin JC, Llanos-Cuentas A (2007) Influence of Leishmania (Viannia) species on the response to antimonial treatment in patients with American tegumentary leishmaniasis. J Inf Dis 195:1846–1851CrossRefGoogle Scholar
  24. 24.
    da Silva LA, de Sousa CdS, da Graça GC, Porrozzi R, Cupolillo E (2010) Sequence analysis and PCR-RFLP profiling of the hsp70 gene as a valuable tool for identifying Leishmania species associated with human leishmaniasis in Brazil. Infect Genet Evol 10:77–83PubMedCrossRefGoogle Scholar
  25. 25.
    Dujardin JC, Bañuls AL, Llanos-Cuentas A, Alvarez E, De Doncker S, Jacquet D, Le Ray D, Arévalo J, Tibayrenc M (1995) Putative Leishmania hybrids in the Eastern Andean valley of Huanuco, Peru. Acta Trop 59:293–307PubMedCrossRefGoogle Scholar
  26. 26.
    Nolder D, Roncal N, Davies CR, Llanos-Cuentas A, Miles MA (2007) Multiple hybrid genotypes of Leishmania (Viannia) in a focus of mucocutaneous leishmaniasis. Am J Trop Med Hyg 76:573–578PubMedGoogle Scholar
  27. 27.
    Chappuis F, Sundar S, Hailu A, Ghalib H, Rijal S, Peeling RW, Alvar J, Boelaert M (2007) Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? Nat Rev Microbiol 5:873–882PubMedCrossRefGoogle Scholar
  28. 28.
    Cupolillo E, Grimaldi G, Momen H, Beverley SM (1995) Intergenic Region Typing (IRT) A rapid molecular approach to the characterization and evolution of Leishmania. Mol Biochem Parasitol 73:145–155PubMedCrossRefGoogle Scholar
  29. 29.
    Marfurt J, Nasereddin A, Niederwieser I, Jaffe CL, Beck HP, Felger I (2003) Identification and differentiation of Leishmania species in clinical samples by PCR amplification of the miniexon sequence and subsequent restriction fragment length polymorphism analysis. J Clinic Microbiol 41:3147–3153CrossRefGoogle Scholar
  30. 30.
    Marfurt J, Niederwiese I, Divine MN, Beck HP, Felger I (2003) Diagnostic genotyping of Old and New World Leishmania species by PCR-RFLP. Diag Microbiol Infect Dis 46:115–124CrossRefGoogle Scholar
  31. 31.
    Rotureau B, Ravel C, Couppie P, Pratlong F, Nacher M, Dedet JP, Carme B (2006) Use of PCR-restriction fragment length polymorphism analysis to identify the main new world Leishmania species and analyze their taxonomic properties and polymorphism by application of the assay to clinical samples. J Clin Microbiol 44:459–467PubMedCrossRefGoogle Scholar
  32. 32.
    Oshaghi MA, Ravasan NM, Hide M, Javadian EA, Rassi Y, Sedaghat MM, Mohebali M, Hajjaran H (2009) Development of species-specific PCR and PCR-restriction fragment length polymorphism assays for L. infantum / L. donovani discrimination. Exp Parasitol 122:61–65PubMedCrossRefGoogle Scholar
  33. 33.
    Simon S, Veron V, Carme B (2010) Leishmania spp. identification by polymerase chain reaction-restriction fragment length polymorphism analysis and its applications in French Guiana. Diagn Microbiol Infect Dis 66:175–180PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • A. M. Montalvo
    • 1
  • J. Fraga
    • 1
  • I. Maes
    • 2
  • J.-C. Dujardin
    • 2
    • 3
  • G. Van der Auwera
    • 2
  1. 1.Parasitology DepartmentInstitute of Tropical Medicine “Pedro Kourí” (IPK)HavanaCuba
  2. 2.Department of Biomedical SciencesInstitute of Tropical Medicine (ITM)AntwerpBelgium
  3. 3.Department of Biomedical SciencesUniversity of AntwerpAntwerpBelgium

Personalised recommendations