Antigen genes for molecular epidemiology of leishmaniasis: polymorphism of cysteine proteinase B and surface metalloprotease glycoprotein 63 in the Leishmania donovani complex.
BACKGROUND
Efficient monitoring of endemic and resurgent visceral leishmaniasis (VL) requires discriminatory molecular tools that allow direct characterization of etiological agents (i.e., the Leishmania donovani complex) in host tissues. This characterization is possible through restriction fragment-length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified sequences (PCR-RFLP).


METHODS
We present 2 new PCR-RFLP assays that target the gene locus of cysteine proteinase B (cpb), an important Leishmania antigen. The assays were applied to the characterization of 15 reference strains of the L. donovani complex, and their discriminatory power was compared with that of PCR-RFLP analysis of the gp63 gene, another Leishmania antigen, and with that of multilocus enzyme electrophoresis (MLEE), which is the reference standard for parasite typing.


RESULTS
Restriction patterns of the cpb locus were polymorphic, but less so than gp63 patterns. When data for both loci were combined, differences between PCR-RFLP and MLEE results were encountered. Antigen gene analysis was more discriminatory and supported a different classification of parasites, one that fitted with their geographic origin. PCR-RFLP analysis of cpb also allowed direct genotyping of parasites in bone marrow aspirate and venous blood samples obtained from patients with VL.


CONCLUSIONS
Antigen genes constitute valid targets for PCR-based Leishmania typing without the need for isolation of parasites.
Laboratory of Molecular Parasitology, Prins Leopold Instituut voor Tropische Geneeskunde, Antwerp, Belgium.
Kelly Wilber
KW
Quispe Tintaya
Xu
X
Ying
Jean-Pierre
JP
Dedet
Suman
S
Rijal
Xavier
X
De Bolle
Jean-Claude
JC
Dujardin
D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics*; D003546:Cysteine Endopeptidases / Q000235:genetics*; D006801:Humans; D007893:Leishmania donovani / Q000201:enzymology*; D008666:Metalloendopeptidases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length
D000953:Antigens, Protozoan; D003546:Cysteine Endopeptidases; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania