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. CONCLUSION: Antigen genes constitute valid targets for PCR-based Leishmania typing without the need for isolation of parasites.
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. CONCLUSION: Antigen genes constitute valid targets for PCR-based Leishmania typing without the need for isolation of parasites.
Authors: Diego Peres Alonso; Dorcas Lamounier Costa; Ivete Lopes de Mendonça; Carlos Henrique Nery Costa; Paulo Eduardo Martins Ribolla Journal: Am J Trop Med Hyg Date: 2010-05 Impact factor: 2.345
Authors: S Hendrickx; G Boulet; A Mondelaers; J C Dujardin; S Rijal; L Lachaud; P Cos; P Delputte; L Maes Journal: Parasitol Res Date: 2014-03-11 Impact factor: 2.289
Authors: Tim Downing; Hideo Imamura; Saskia Decuypere; Taane G Clark; Graham H Coombs; James A Cotton; James D Hilley; Simonne de Doncker; Ilse Maes; Jeremy C Mottram; Mike A Quail; Suman Rijal; Mandy Sanders; Gabriele Schönian; Olivia Stark; Shyam Sundar; Manu Vanaerschot; Christiane Hertz-Fowler; Jean-Claude Dujardin; Matthew Berriman Journal: Genome Res Date: 2011-10-28 Impact factor: 9.043
Authors: Ruben t'Kindt; Richard A Scheltema; Andris Jankevics; Kirstyn Brunker; Suman Rijal; Jean-Claude Dujardin; Rainer Breitling; David G Watson; Graham H Coombs; Saskia Decuypere Journal: PLoS Negl Trop Dis Date: 2010-11-30