Each patient enrolled to this study completed by writing the informed consent. All methods were done based on principles of the declaration of Helsinki. The study was approved by the Ethics Committee of Shahid Sadoughi University of Medical Sciences, Yazd, Iran. (IR. SSU. MEDICINE. REC. 1399.194).
Varzaneh is a city in the southeast of Isfahan province (Fig. 1). The city is located in the western part of Gavkhooni swamp, in longitude of 39’, 52° and latitude of 25’, 52° north, and is a well-known CL endemic area. The current population of the city is approximately 17 000.
The clinical isolates of Leishmania were collected from patients of Health Center Laboratory of Varzaneh, Isfahan, Iran, from September 2020 to March 2021. The CL primary diagnosis was performed using direct smear and microscopic observation of Leishman body in Giemsa-stained slides. After diagnosis, the samples were directly collected from the lesion, transferred into RNAlater solution (Merck, Darmstadt, Germany), and stored at -20° C for further analysis. Each CL case was administered with standard regimen treatment of glucantime (20 mg/kg/day for 20 days). Response to drug was evaluated by re-epithelialization of lesion and decreased inflamed border of the lesions at day 20 of drug administration. In cases of no response to drug, the additional administration period was applied. In no response cases, the patient was considered as treatment failure (TF) (Martínez et al. 2020; Vanaerschot et al. 2014) and the related isolate was focused for next experiments. The cases with the response to glucantime treatment were grouped as treatment response (TR).
Verification and identification
The isolates were verified and identified using an Internal Transcribed Spacer (ITS)-1-PCR–RFLP (Eslami et al. 2016) using the specific primer pair of LITSR: 5′-CTTGGATCATTTTCCGATG-3′ and L5.8S 5′-TGATACCACTTATCGCATT-3′ in a final concentration of 0.5 µM for each. The reaction was conducted using thermo cycler (SimpliAmp, ABI, USA) in a 20 μl reaction solution with the amplification condition described by Eslami et al. (2016) Positive and negative controls were run in each reaction using L. major (MRHO/IR/75/ER) and ddH2O, respectively. The amplification analysis was carried out using 1.5% agarose gel electrophoresis using gel documentation. The gel was stained using DNA Green Viewer (Pars Tous, Iran, Mashhad). The amplified fragment with the length from 300 to 350 bp was considered as Leishmania. In order to species identification, RFLP was performed using digestion of the amplicon by Hae III restriction enzyme at 37 °C for 2 h. Digested fragments were assessed using 3% agarose gel electrophoresis stained with DNA Green Viewer alongside with 50 bp DNA ladder (CinnaGen, Iran, Tehran). The fragments with the length of 220 and 127 bp identified L. major; 220 and 50 bp recognized L. tropica; and 200, 100, and 50 bp known L. infantum.
RNA extraction and cDNA synthesis
Total RNA was extratced using the total RNA extraction kit (Vivantis, Malaysia) based on description by the manufacturer. DNase I (CinnaGen, Iran, Tehran) was used to treat the extracted RNA to avoid any genomic contamination. The RNA quantity was estimated using Nanodrop (Thermo Fisher Scientific, USA). Then, complementary DNA (cDNA) was synthesized from total RNA (1 µg) using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) based on the manufacturer’s instructions. The primers used in this step were random hexamer and oligodT primer.
Gene expression analysis
The expression profiling of ABCG2, ABCI4, ABCC7, ABCB4, and ABCC3 genes involving drug efflux were assessed using SYBR Green real-time PCR. The GAPDH gene (Eslami et al. 2016) was considered for normalization purposes, referred as internal or endogenous control. All primer pairs related to the ABCG2, ABCI4, ABCC7, ABCB4, and ABCC3 were designed in this study (Table 1). Amplifications were done in a total volume of 20 µl containing 2 µl cDNA, 10 µl SYBR Green Real Time master mix (2X; ABI, USA), and primer pairs (with the final concentration shown in Table 1) using a Step One thermocycler (Applied Biosystem, USA). The thermal conditions of the reaction were 95° C for 10 min in order to the first denaturation, followed by 40 cycles of 95° C for 10 s and 60 ° C for 10 s. The specificity of the reaction products, no nonspecific products, and no primer dimers were confirmed by melting curve, which consisted of temperatures between 60 and 95° C with a heating rate of 0.3 °C/s. All reactions were done in duplicate. In cases of necessary, the reactions were repeated in more duplicate set. The relative amount of amplification by each primer pair was determined based on threshold cycle (Ct) value of the interest gene, normalized to that of reference GAPDH gene. Gene expression analysis was done using 2-ΔΔCT method using follow formula:
ΔΔCT= (CTtarget gene in sample - CTGAPDH in sample) – (CTtarget gene in standard - CTGAPDH in standard)
Experiments were carried out in duplex and the data are presented as mean. Statistical comparisons between groups were performed using t-test. P value ≤ 0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 26.0 (Chicago, IL).