Species Identification and Genotyping of Cutaneous Leishmaniasis in Clinical Samples Based on ITS1-PCR-Sequencing in Southeast Iran

Abstract

Background: Cutaneous leishmaniasis (CL) is one of the most common parasitic diseases in many regions of Iran. It has a major role in deprived societies. We aimed to identify Leishmania species based on molecular as ITS1-rDNA-PCR internal transcribed spacer 1 (ITS1) region, microscopy, and culture techniques in diagnosing cutaneous leishmaniasis.

Methods: From April 2018 to May 2020, we conducted a cross-sectional study involving 32 patients with suspected CL lesions in Sistan and Baluchistan Province, located in southeast Iran. Samples were subjected to microscopic examination, culture, and PCR amplification targeting the internal transcribed spacer 1 (ITS1) region. DNA sequencing was performed on PCR-positive samples for species identification and phylogenetic analysis.

Results: PCR demonstrated superior sensitivity (93.75%, 30/32) compared to culture (56.25%, 18/32) and microscopic examination (53.1%, 17/32). Molecular analysis revealed that L. major was the predominant causative agent of CL in the study area, with L. tropica occurring less frequently. Sequencing and phylogenetic analysis of the ITS1 region showed high intraspecies similarity among L. tropica isolates, while L. major isolates exhibited greater genetic diversity.

Conclusion: This study shows the co-existence of L. major and L. tropica in Mirjaveh, southeast Iran, with L. major as the primary cause. While L. tropica isolates displayed high genetic similarity, L. major samples were more diverse, indicating different epidemiological patterns. These findings highlight the importance of molecular methods for accurately identifying Leishmania species and understanding CL epidemiology in the region.

Keywords: Cutaneous leishmaniasis; Iran; Polymerase chain reaction; Sequence.

Fig. 1:

Map of Sistan and Baluchistan province in Iran, Mirjaveh, and suburbs (Mellekan..), Southeast Iran(Investigation region) Mirjaveh City and suburbs on the map are seen in red), Aerial view of Mirjaveh City, Captured using Google Earth, (google Earth,2024). Retrieved from. https://www.google.com/about

Fig. 2:

The images depict ulcerative lesions with characteristic crusting and surrounding erythema, which are typical of infections caused by L. tropica and L. major, the predominant species identified in the region. The lesions show the progression of ulceration, a hallmark of the disease

Fig. 3:

Detection of cutaneous leishmaniasis in patient samples by PCR amplification. The image shows electrophoresis of the 303-bp PCR product on a 1.5% agarose gel. Lane SM contains the 100 bp molecular size marker (ladder). Lane C+ represents the positive control for cutaneous leishmaniasis, while Lane C- is the negative control. Lanes 2, 4, 5, 6, 8, 9, and 10 correspond to patient samples that tested positive for Leishmania DNA, indicating active infection. The clear amplification bands in these lanes confirm the presence of the target Leishmania sequence in the samples

Fig. 4:

Phylogenetic tree of various L. tropica isolates, illustrating the evolutionary relationships among different genetic sequences based on accession numbers. Sequences ON733094 and ON733095 are highlighted as they are directly related to this study

Fig. 5:

The phylogenic tree of L.major is shown here and shows some species derived from polyclad clusters, illustrating the evolutionary relationships among different genetic sequences based on accession numbers. Sequences ON733096 ‘ ON733098 ‘ON733097 and ON733099 are highlighted as they are directly related to this study

Fig. 6:

Sequence alignment of the ITS1- rDNA of Leishmania spp. isolated from patients, the region from Mirjaveh, Sistan, and Blugestan, Iran samples. Text size (for Image only): Medium bold, Text color (for Image only): black, Background color: white, High consensus color: red, Low consensus color: blue, Neutral color: black. This shows variable nucleotides and homology between samples isolated from Mirjaveh city and suburb, Southeast Iran