Heat-shock protein 70 PCR-RFLP: a universal simple tool for Leishmania species discrimination in the New and Old World.
BACKGROUND
Species typing in leishmaniasis gains importance in diagnostics, epidemiology, and clinical studies. A restriction fragment length polymorphism (RFLP) assay of PCR amplicons from a partial heat-shock protein 70 gene (hsp70) had been described for the New World, allowing identification of some species.


METHODS
Based on an initial in silico analysis of 51 hsp70 sequences, most of which we recently determined in the frame of a phylogenetic study, species-specific restriction sites were identified. These were tested by PCR-RFLP on 139 strains from 14 species, thereby documenting both inter- and intra-species variability.


RESULTS
Our assay could identify Leishmania infantum, L. donovani, L. tropica, L. aethiopica, L. major, L. lainsoni, L. naiffi, L. braziliensis, L. peruviana, L. guyanensis, and L. panamensis by applying 2 subsequent digests. L. mexicana, L. amazonensis, and L. garnhami did not generate species-specific restriction fragment patterns.


CONCLUSIONS
Currently no assay is available for global Leishmania species discrimination. We present a universal PCR-RFLP method allowing identification of most medically relevant Old and New World Leishmania species on the basis of a single PCR, obviating the need to perform separate PCRs. The technique is simple to perform and can be implemented in all settings where PCR is available.
Montalvo
A M
AM
Instituto de Medicina Tropical Pedro Kourí, Departamento de Parasitología, La Habana, Cuba.
Fraga
J
J
Monzote
L
L
Montano
I
I
De Doncker
S
S
Dujardin
J C
JC
Van der Auwera
G
G
D000818:Animals; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D007891:Leishmania / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D007896:Leishmaniasis / Q000175:diagnosis* / Q000469:parasitology*; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis / Q000469:parasitology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D013045:Species Specificity
D018840:HSP70 Heat-Shock Proteins