Leishmania donovani persistence and circulation causing cutaneous leishmaniasis in unusual-foci of Nepal

Abstract

Cutaneous leishmaniasis cases have increased dramatically in recent years in Nepal. The study offers molecular identification of the Leishmania species using 40 patient's aspiration biopsy samples, targeting markers kinetoplast minicircle DNA (kDNA) and internal transcribed spacer-1 (ITS1). Among molecularly diagnosed 22 cutaneous leishmaniasis cases, L. donovani complex was identified in 13 instances and L. major in 9 cases. The ITS1 PCR was positive in 12 of the positive nested- kDNA PCR cases (12/22), confirming L. donovani complex in seven of the cases and L. major in five of the cases. In addition, the study conclude that concurrent occurrence of atypical cutaneous infections caused by L. donovani parasite in 59.1% of cases and typical cutaneous infections caused by L. major parasite in 40.9% of cases. A Phylogentic analaysis showed that the detected L. donovani species present null genetic distances from seven references of L. donovani, but slight differences between ITS1 sequences and not grouped into a significant monophyletic cluster.

Figure 1

Age & gender wise distribution of cutaneous leishmaniasis positive cases.

Figure 2

Map showing the distribution of positive cases of cutaneous leishmaniasis (CL) in the geographic regions, districts and provinces of Nepal. Map was prepared using QGIS 3.30 (http://qgis.osgeo.org).

Figure 3

Clinical features of cutaneous leishmaniasis, (1) Dry crusted lesion in upper chest (2) Dry erythematic plaque in arm, (3) Dry and raised lesion in hand, (4) Central invaginated and moist in hand, (5) Ulcerated lesion with Central invagination on left leg.

Figure 4

Agarose gel electrophoresis visualization of kDNA, ITS1 and ITS1-RFLP. (A1) Minicircle kDNA PCR-1 assay of clinical samples. (A2) Minicircle kDNA PCR-2 assay of clinical samples. PCR 1 amplicons of DNA isolated from the skin biopsies run for gel electrophoresis on 1.5% agarose gel and analysis. B1) ITS1 PCR assay of clinical samples. DNA isolated from the skin biopsies from the lesion was amplified and analyzed on 1.5% agarose gel. B2) ITS1 RFLP assay of ITS1 PCR positive samples. The grouping of the gels cropped were from different gels. The full length gels are included in supplementary information file. The size of the bands are defined as indicated by the arrows. Ladders-100 bp DNA ladder (Solis Biodyne), NC-Negative Control, L.D.- Control of L. donovani (LEM 138), CL- Cutaneous leishmaniasis sample.

Figure 5

Maximum Likelihood (ML) tree of ITS1 sequences from Leishmania species. ML tree of 29 ITS1 sequences from four Leishmania species, constructed by the software IQ-TREE v.2.2.0 from an alignment of 312 nucleotide positions. This tree showing the highest log likelihood (− 554.785) is based on the best model (K2P) determined by the program itself. The percentages of bootstrap values used as phylogenetic test and based on 50,000 replications are shown at the tree nodes. The sequences are labelled with their accession numbers and isolate names. The sequences CL17, CL23, CL29Culture and CL29Tissue, belonging to L. major and identified by black triangle symbols, and CL18, CL22, CL25 and CL-40 belonging to L. donovani and identified by black circle symbols are our isolates from Nepal of years 2018–2019.