Abstract
ObjectiveLeishmaniasis 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.ResultsITS1-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.
Highlights
Different species of Leishmania cause a wide range of clinical manifestation, including a simple self-healing skin lesion called cutaneous leishmaniasis (CL), a systemic fatal form called visceral leishmaniasis (VL), and mucocutaneous leishmaniasis (MCL)
The results showed no LRV1 in any of the samples but 7 treatment response (TR) isolates and 2 treatment failure (TF) isolates showed positive for LRV2
All 30 isolates were checked for LRV1 and LRV2, 7 TR isolates and 2 TF isolates showed positive amplification for LRV2
Summary
Patients Upon the results pf PCR, only the L. major isolates were included in this study. There was no significant difference between the age of the patients with TR isolates (25 years) and TR isolates (26 years). The expected size of PCR amplicons was around350 bp (Additional file 1: Figure S1). After extraction of RNA and synthesis of cDNA, in order to control the system for amplifiability, PCR was done using the specific primer pair of kmp. The amplification results showed LRV2 in 7 TR isolates and 2 TR isolates collected from patients. Melting curve analysis For verification of the specific amplification for kmp and LRV2, the melting curve analysis was done. The results showed that the amplifications were done specific with the melting temperature of 82.6 °C for kmp and 79.8 °C for LVR2
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