Detection and Identification of Leishmania major, Leishmania tropica, and Leishmania infantum in Clinical Samples Based on Size Polymorphism of Partially Amplified Ribosomal DNA.

Accurate Leishmania species identification is crucial for epidemiological aspects and disease management. This study aimed to detect and identify the main causative species of human cutaneous and visceral leishmaniasis in Iran using nested polymerase chain reaction (PCR) based on size polymorphism of the ribosomal DNA (rDNA) intergenic region. In total, 180 clinical samples, including 156 confirmed positive cases, 24 confirmed negative cases, and 33 suspected cases that tested negative by microscopy, were collected from skin lesions, bone marrow, and lymph nodes across five provinces in Iran. DNA was extracted from the samples, and the nested PCR-amplified fragments were differentiated with agarose gel electrophoresis. The nested PCR assay successfully identified Leishmania major, Leishmania tropica, and Leishmania infantum with high accuracy. Among 156 microscopy-positive samples, L. major was detected in 84 cases, L. tropica was detected in 64 cases, and L. infantum was detected in 8 cases. The results obtained were fully consistent with those from a commercial real-time PCR diagnostic kit. Phylogenetic analysis supported the differentiation of Leishmania species. However, among 33 suspected cases, 14 samples were negative by all three methods (microscopy, commercial PCR kit, and nested PCR), 7 samples tested positive by both molecular methods, 8 samples were positive only with the commercial PCR kit, and 4 samples were positive exclusively by nested PCR. The findings indicate that the nested PCR assay targeting size polymorphisms in the rDNA region is a cost-effective method for detection and identification of Leishmania species in clinical specimens, particularly when microscopy results are inconclusive.