Development of a genetic assay to distinguish between Leishmania viannia species on the basis of isoenzyme differences.
BACKGROUND
Tegumentary leishmaniasis in Latin America is caused mainly by Leishmania viannia braziliensis complex parasites. L. braziliensis and Leishmania viannia peruviana are the 2 predominant Leishmania species in Peru. L. braziliensis is more virulent, because it can cause mucocutaneous leishmaniasis, known as espundia, that results in severe facial destruction. Early identification of the species that causes the initial cutaneous infection would greatly help to prevent mucocutaneous leishmaniasis, because it would allow more aggressive treatment and follow-up. However, because of the close genetic similarity of L. braziliensis and L. peruviana, there currently exists no simple assay to distinguish between these species.


METHODS
We cloned the mannose phosphate isomerase gene from both L. braziliensis and L. peruviana. It is the only known isoenzyme capable of differentiating between L. braziliensis and L. peruviana in multilocus enzyme electrophoresis. Interestingly, only a single nucleotide polymorphism was found between the mannose phosphate isomerase genes from L. braziliensis and L. peruviana, resulting in an amino acid change from threonine to arginine at amino acid 361. A polymerase chain reaction assay was developed to distinguish the single nucleotide polymorphism of the mannose phosphate isomerase gene to allow for the specific identification of L. braziliensis or L. peruviana.


RESULTS
This assay was validated with 31 reference strains that were previously typed by multilocus enzyme electrophoresis, successfully applied to patient biopsy samples, and adapted to a real-time polymerase chain reaction assay.


CONCLUSIONS
This innovative approach combines new genetic knowledge with traditional biochemical fundamentals of multilocus enzyme electrophoresis to better manage leishmaniasis in Latin America.
Department of Microbiology and Immunology, McGill University, Montreal, Canada.
Wen-Wei
WW
Zhang
Cesar
C
Miranda-Verastegui
Jorge
J
Arevalo
Momar
M
Ndao
Brian
B
Ward
Alejandro
A
Llanos-Cuentas
Greg
G
Matlashewski
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D017125:Genes, Protozoan; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000145:classification / Q000235:genetics; D007843:Latin America; D007891:Leishmania / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D007897:Leishmaniasis, Mucocutaneous / Q000175:diagnosis*; D008359:Mannose-6-Phosphate Isomerase / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D007527:Isoenzymes; D008359:Mannose-6-Phosphate Isomerase