A real-time polymerase chain reaction assay for the identification and quantification of American Leishmania species on the basis of glucose-6-phosphate dehydrogenase.
A real-time polymerase chain reaction (PCR) test was developed on the basis of the Leishmania glucose-6-phosphate dehydrogenase locus that enables identification and quantification of parasites. Using two independent pairs of primers in SYBR-Green assays, the test identified etiologic agents of cutaneous leishmaniasis belonging to both subgenera, Leishmania (Viannia) and Leishmania (Leishmania) in the Americas. Furthermore, use of TaqMan probes enables distinction between L. (V.) braziliensis or L. (V.) peruviania from the other L. (Viannia) species. All assays were negative with DNA of related trypanosomatids, humans, and mice. The parasite burden was estimated by normalizing the number of organisms per total amount of DNA in the sample or per host glyceraldehyde-3-phosphate dehydrogenase copies. The real-time PCR assay for L. (Leishmania) subgenus showed a good linear correlation with quantification on the basis of a limiting dilution assay in experimentally infected mice. The test successfully identifies and quantifies Leishmania in human biopsy specimens and represents a new tool to study leishmaniasis.
Castilho
Tiago Moreno
TM
Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06510, USA. tiago.castilho@yale.edu
Camargo
Luís Marcelo Aranha
LM
McMahon-Pratt
Diane
D
Shaw
Jeffrey Jon
JJ
Floeter-Winter
Lucile Maria
LM
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D016054:DNA, Protozoan / Q000032:analysis; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007891:Leishmania / Q000145:classification / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis* / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011237:Predictive Value of Tests; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
D017931:DNA Primers; D016054:DNA, Protozoan; D005954:Glucosephosphate Dehydrogenase