Development of a species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis

Abstract

We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of Leishmania donovani in a species-specific manner among Old World leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz., Mycobacterium tuberculosis, Mycobacterium leprae, and Plasmodium spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples.

FIG. 1

Sensitivity of the PCR assay. Shown are the results of PCR amplification of the serially diluted L. donovani (DD8) DNA analyzed on agarose gels. DNA was extracted from parasite cultures and amplified as described in Materials and Methods. Lane M, 1 kb Ladder (Gibco BRL); lane 1, 10 ng of DNA; lane 2, 1 ng of DNA; lane 3, 10 pg of DNA; lane 4, 1 pg of DNA; lane 5, 10 fg of DNA; lane 6, 1 fg of DNA.

FIG. 2

Sensitivity of PCR amplification of Leishmania kDNA followed by Southern blot analysis. The PCR contained 100 ng of human genomic DNA and the indicated amount of total DNA from L. donovani DD8. The PCR product was probed with parasite kDNA and exposed for about 1 h. Lane 4 represents a PCR containing only human DNA as a control.

FIG. 3

Amplification of parasite DNA from various strains and isolates of Leishmania. DNA (1 ng) isolated from parasite cultures was subjected to PCR and analyzed. Lane 1, L. donovani AG83; lane 2, L. donovani DD8; lane 3, L. donovani IICB8; lane 4, L. donovani IICB6; lane 5, L. donovani IICB 7 (PKDL origin); lane 6, L. donovani 1S; lane 7, L. donovani WR684; lane 8 L. donovani infantum; lane 9, L. tropica WR683; lane 10, L. major LV 39, lane M, 1-kb ladder, lane 11, Plasmodium; lane 12, M. leprae; lane 13, M. tuberculosis.

FIG. 4

DNA amplification from recent field isolates of KA and PKDL. DNA (1 ng) extracted from cultures of parasite isolates was used for PCR amplification. Lanes: M, 1-kb ladder; 1, KA-1; 2, KA-2; 3, KA-3; 4, KA-4; 5, KA-5; 6, PK-1; 7, PK-2; 8, PK-3; 9, PK-4; 10, PK-5; 11, isolate from a patient with cutaneous leishmaniasis.

FIG. 5

PCR assay with clinical samples of KA and PKDL. DNA (100 ng) isolated from clinical samples was used for PCR amplification. Lane M, 1-kb ladder, lane 1, KA (bone marrow); lane 2, KA (blood); lane 3, malaria (blood); lane 4, tuberculosis (blood); lane 5, control from the area of endemicity (blood); lane 6, PKDL (skin lesion); lane 7, leprosy (lesion).

FIG. 6

Sequence of PCR product with DNA isolated from L. donovani DD8 strain and isolates and clinical samples from patients with KA and PKDL. PCR products obtained with DNA isolated from L. donovani DD8 strain, parasite isolates from patients with KA and PKDL (two each) and clinical samples (two each of KA blood and PKDL tissue) were subjected to sequence analysis. Identical sequences were obtained for the PCR products in each case, which matched exactly with the published sequence of a 792-bp kDNA mini-circle segment of the DD8 strain of L. donovani (GenBank accession no. Y11401). The positions of primers are indicated in boldface type.