Sequencing a specific kinetoplast DNA fragment of Leishmania donovani for polymerase chain reaction amplification in diagnosis of leishmaniasis in bone marrow and blood samples

A set of oligonucleotide primers I and II was developed by analyzing the specificity of a cloned kinetoplast DNA (kDNA) fragment of Leishmania donovani and sequencing the fragment. Polymerase chain reaction (PCR) was conducted with the primers to amplify a minicircle kDNA fragment (297 bp) to detect L. donovani in the bone marrow (22 samples), whole blood (16 samples), and serum (17 samples) of 22 patients with visceral leishmaniasis. All of 22 patients were diagnosed by microscopic identification. Control samples of bone marrow, whole blood, and serum were obtained from patients with leukemia and from healthy volunteers. In addition, 12 dogs were infected with L. donovani promastigotes for the PCR test. The total number of patients positive by PCR testing was 95.5% (21/22), with 91.0% (20/22) from the bone marrow, 68.8% (11/16) from the blood, and 29.4% (5/17) from the sera. Similar results were obtained in infected dogs. No amplification products were seen in control samples from humans or dogs. Our results suggest that PCR may be useful in detecting kDNA in the bone marrow and blood of patients with visceral leishmaniasis.