Molecular characteristic of treatment failure clinical isolates ofLeishmania major
Authors: Eslami Gilda , Hatefi Samira , Ramezani Vahid , Tohidfar Masoud , Churkina Tatyana V. , Orlov Yuriy L. , Hosseini Saeedeh Sadat , Boozhmehrani Mohammad Javad , Vakili Mahmood 
Published: March 2021

BackgroundLeishmaniasis is a prevalent tropical disease caused by more than 20Leishmaniaspecies (Protozoa, Kinetoplastida and Trypanosomatidae). Among different clinical forms of the disease, cutaneous leishmaniasis is the most common form, with an annual 0.6–1 million new cases reported worldwide. This disease’s standard treatment is pentavalent antimonial (SbV) that have been used successfully since the first half of the 20th century as a first-line drug. However, treatment failure is an increasing problem that is persistently reported from endemic areas. It is important to define and standardize tests for drug resistance in cutaneous leishmaniasis. SbVmust be reduced to its trivalent active form (SbIII). This reduction occurs within the host macrophage, and the resultant SbIIIenters amastigotes via the aquaglyceroporin1 (AQP1) membrane carrier. Overexpression of AQP1 results in hypersensitivity of the parasites to SbIII, but resistant phenotypes accompany reduced expression, inactivation mutations, or deletion of AQP1. Hence, in this study, a phylogenetic analysis using barcode geneCOXII and kDNA minicircle and expression analysis ofAQP1were performed in treatment failure isolates to assess the isolates’ molecular characteristics and to verify possible association with drug response.MethodsSamples in this study were collected from patients with cutaneous leishmaniasis referred to the Diagnosis Laboratory Center in Isfahan Province, Iran, from October 2017 to December 2019. Among them, five isolates (code numbers 1–5) were categorized as treatment failures. The PCR amplification of barcode gene COXII and kDNA minicircle were done and subsequently analyzed using MEGA (10.0.5) to perform phylogenetics analysis of Treatment failures (TF) and Treatment response (TR) samples. Relative quantification of the AQP1 gene expression of TF and TR samples was assessed by real-time PCR.ResultsAll samples were classified asL. major. No amplification failure was observed in the cases of barcode geneCOXII and kDNA minicircle amplification. Having excluded the sequences with complete homology using maximum parsimony with the Bootstrap 500 method, four major groups were detected to perform phylogenetic analysis usingCOXII. The phylogenetic analysis using the barcode target of minicircle showed that all five treatment failure isolates were grouped in a separate sub-clade.ConclusionsWe concluded that the barcode geneCOXII and the minicircle kDNA were suitable for identification, differentiation and phylogenetic analysis in treatment failure clinical isolates ofLeishmania major. Also,AQP1gene expression analyses showed that treatment failure isolates had less expression than TR isolates. The isolate with TF and overexpression of theAQP1gene of other molecular mechanisms such as overexpression of ATP-binding cassette may be involved in the TR, such as overexpression of ATP-binding cassette which requires further research.