Diagnosis of Leishmania using the polymerase chain reaction: a simplified procedure for field work.
Oligonucleotide primers directed to the minicircle kinetoplast DNA of Leishmania strains supported enzymatic amplification of this DNA by the polymerase chain reaction (PCR). A single product of 70 basepairs was obtained from parasites belonging exclusively to the L. braziliensis complex. Direct sequencing of the amplified product confirmed its minicircle origin. Skin biopsy specimens from human patients were used directly for the PCR. A pulse incubation of such specimens with deoxyribonuclease I prior to the PCR increased the reliability of the assay. Nuclease disruption of the kinetoplast network was expected to make more copies of the minicircle accessible to amplification. Comparative results between the PCR and conventional parasitologic detection procedures indicate that the DNA detection approach presented is by far more sensitive for diagnostic purposes. Innovations in the PCR protocol are presented that adapt the diagnosis of leishmaniasis to settings with minimal equipment and that are distant from central laboratories.
Departamento de Ciencias Fisiologicas, Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima.
M
M
Lopez
R
R
Inga
M
M
Cangalaya
J
J
Echevarria
A
A
Llanos-Cuentas
C
C
Orrego
J
J
Arevalo
D000818:Animals; D001483:Base Sequence; D004270:DNA, Circular / Q000032:analysis*; D018105:DNA, Kinetoplast; D016054:DNA, Protozoan / Q000032:analysis; D005069:Evaluation Studies as Topic; D006801:Humans; D007892:Leishmania braziliensis / Q000235:genetics*; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis*; D007897:Leishmaniasis, Mucocutaneous / Q000175:diagnosis*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012867:Skin / Q000469:parasitology
D004270:DNA, Circular; D018105:DNA, Kinetoplast; D016054:DNA, Protozoan; D009838:Oligodeoxyribonucleotides