Dataset Information

Relationship of Leishmania RNA Virus (LRV) and treatment failure in clinical isolates of Leishmania major.

ABSTRACT:

Objective

Leishmaniasis is caused by different Leishmania spp. Treatment failure (TF) of cutaneous leishmaniasis (CL) is a serious issue that may be due to various reasons, previous studies suggested Leishmania RNA virus (LRV) as a potential cause of TF. Two variant groups of LRV1 and LRV2 are reported. In this study, the presence of LRV1/LRV2 was compared in TF with treatment response (TR) isolates of L. major. Clinical isolates of 15 TF and 15 TR were collected from CL patients referred to the Health Centers of Isfahan. Genomic DNA was extracted to identify Leishmania spp. using ITS1-PCR-RFLP. Identification of LRV1/LRV2 was performed using SYBR Green Real-Time PCR. The statistical analysis to test relationship between the treatment response with Glucantime and the presence of LRV were performed using SPSS 16.0 with Fisher's Exact test. P value of less than 0.05 was considered significant.

Results

ITS1-PCR-RFLP results showed that every isolate was identified as L. major. The results showed no LRV1 in any of the samples but 7 TR isolates and 2 TF isolates showed positive for LRV2. Statistical analysis showed no significant difference between the presence of LRV2 and response to Glucantime (p-value = 0.1086). Therefore, other mechanisms might be responsible for TF.

SUBMITTER: Abtahi M

PROVIDER: S-EPMC7074996 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundLeishmaniasis is a prevalent tropical disease caused by more than 20 Leishmania species (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. SbV must 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 gene COXII and kDNA minicircle and expression analysis of AQP1 were 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 as L. major. No amplification failure was observed in the cases of barcode gene COXII 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 using COXII. 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 gene COXII and the minicircle kDNA were suitable for identification, differentiation and phylogenetic analysis in treatment failure clinical isolates of Leishmania major. Also, AQP1 gene expression analyses showed that treatment failure isolates had less expression than TR isolates. The isolate with TF and overexpression of the AQP1 gene 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.

Project description:IntroductionLeishmania RNA virus type 1 (LRV1) is an endosymbiont of some Leishmania (Vianna) species in South America. Presence of LRV1 in parasites exacerbates disease severity in animal models and humans, related to a disproportioned innate immune response, and is correlated with drug treatment failures in humans. Although the virus was identified decades ago, its genomic diversity has been overlooked until now.Methodology/principles findingsWe subjected LRV1 strains from 19 L. (V.) guyanensis and one L. (V.) braziliensis isolates obtained from cutaneous leishmaniasis samples identified throughout French Guiana with next-generation sequencing and de novo sequence assembly. We generated and analyzed 24 unique LRV1 sequences over their full-length coding regions. Multiple alignment of these new sequences revealed variability (0.5%-23.5%) across the entire sequence except for highly conserved motifs within the 5' untranslated region. Phylogenetic analyses showed that viral genomes of L. (V.) guyanensis grouped into five distinct clusters. They further showed a species-dependent clustering between viral genomes of L. (V.) guyanensis and L. (V.) braziliensis, confirming a long-term co-evolutionary history. Noteworthy, we identified cases of multiple LRV1 infections in three of the 20 Leishmania isolates.Conclusions/significanceHere, we present the first-ever estimate of LRV1 genomic diversity that exists in Leishmania (V.) guyanensis parasites. Genetic characterization and phylogenetic analyses of these viruses has shed light on their evolutionary relationships. To our knowledge, this study is also the first to report cases of multiple LRV1 infections in some parasites. Finally, this work has made it possible to develop molecular tools for adequate identification and genotyping of LRV1 strains for diagnostic purposes. Given the suspected worsening role of LRV1 infection in the pathogenesis of human leishmaniasis, these data have a major impact from a clinical viewpoint and for the management of Leishmania-infected patients.

Dataset Information

Relationship of Leishmania RNA Virus (LRV) and treatment failure in clinical isolates of Leishmania major.

Objective

Results

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