Molecular characteristic of treatment failure clinical isolates of Leishmania major

Ethical statement

The current study was approved by the Ethics Committee of Shahid Sadoughi University of Medical Sciences, Yazd, Iran (Approval ID IR.SSU.MEDICINE.REC.1396.323). All patients who participated in this study signed a written informed consent form before sampling.

Population study

In this study, the main target population was the patients referred to the Diagnosis Laboratory Center in Isfahan Province, Iran, from October 2017 to December 2019 and diagnosed with CL but categorized as TF. The inclusion criteria were CL patients failed to be treated with the standard course of glucantime regimen, without any interruption during the treatment, previous anti-Leishmania treatment except for glucantime, and anti-Leishmania co-therapy. Some treatment-responsive isolates were considered as the standard samples to compare the data.

The main objective of the research was to assess at molecular level the isolated from treatment failure prior to treatment of CL, to screen the selected marker of interest. The patients were treated with the standard regimen of glucantime. Response to the treatment was defined as re-epithelialization of lesions and decreased inflamed borders of the lesions. The patients with lesions and without a response at the end of the complete standard treatment course were considered treatment failures (TF, Martínez et al., 2020; Vanaerschot et al., 2014). The cases who responded to glucantime were considered as TR.

Sampling

Samples were collected by scrubbing the lesion edge after disinfecting with 70% alcohol. Two slides were prepared for either direct microscopic examination or molecular analysis for each patient. The biopsy was taken from the edges of lesion skin, transferred into RNAlater solution (Ambion, Inc., Austin, TX, USA), and then stored at −20 °C until the use.

Detection and identification of the isolates

A microscopic examination was carried out to find the Leishman body. The DNA was extracted from smears using the DNA extraction kit (GeneAll, South Korea) based on the manufacturer’s instructions. The quantity of isolated DNA was evaluated using nanodrop (ABI, Vernon, CA, USA). To detect the Leishmania genus, ITS1-PCR was performed using the primer pair of LITSr-F and L5.8s-R (Eslami et al., 2012). Each amplification reaction mixture was included 1 X PCR buffer, 0.2 mM dNTP, 1.5 mM MgCl₂, 1.5 U Taq DNA polymerase, 0.5 µM for each primer, and 100 ng DNA. The thermal profile of PCR amplification was comprised of the first denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 50 °C for 45 s, and elongation at 72 °C for 45 s. The final elongation was done at 72 °C for 5 min. Positive and negative controls were included in each run using DNA extracted from L. major (MRHO/IR/75/ER) and ddH₂O, respectively. The amplification product was analyzed using 1% agarose gel electrophoresis. The expected amplicons size was about 300–350 bp for the verification of Leishmania spp. Subsequently, the amplification products were digested with Hae III (Bsu RI) restriction endonuclease enzyme for three h at 37 °C; heat inactivation was done and then analyzed on 3% agarose gel. Digestion product produced two main bands of 220 and 140 bp was indicative of L. major species. L. major (MRHO/IR/75/ER) was included as a positive control. All tests were done in triplicate.

Molecular analysis by COXII

To amplify COXII, the primer pair of COXII-F 5′-GCATAAATCCATGTAAAACACCACA-3′ and COXII-R 5′-TGGCTTTTATWTTATCATTTTGAATG-3′ was used (Oliveira et al., 2011). Each amplification reaction mixture was included 1 X PCR buffer, 0.2 mM dNTP, 1.5 mM MgCl₂, 1.5 U Taq DNA polymerase, 0.5 µM for each primer, and 100 ng DNA. The thermal profile of PCR amplification was compromised of the first denaturation at 94 °C, followed by 35 cycles of denaturation at 94 °C for 60 s, annealing at 57 °C for 20 s, and elongation at 72 °C for 30 s. The final elongation was done at 72 °C for 5 min. The positive and negative controls were included in each run using DNA extracted from L. major (MRHO/IR/75/ER) and ddH₂O, respectively. The 1.5% agarose gel electrophoresis was used for the amplification product. The expected amplicon size was 602 bp.

Molecular analysis by kDNA minicircle

Molecular analysis of kDNA minicircle was performed as described previously (Aghai-Maybodi et al., 2018). Positive and negative controls were included in each run as L. major (MRHO/IR/75/ER) and ddH₂O, respectively. Amplicon size of 120 bp was considered as positive results.

Sequencing and molecular analysis

PCR amplifies fragments of COXII in all 5 TF were subjected to direct sequencing. Sixteen treatment responsive isolates were also sequenced for the sake of comparison between TF and TR isolates. The same has been done for kDNA minicircle amplicons. The repairing and then BLAST for all sequences were done. All the mentioned sequences were submitted in the GenBank, NCBI with the accession numbers from MK972457 to MK972460. Similar sequences were searched using BLASTn (Altschul et al., 1997). The included sequences are in Table 1. Multiple alignments using T-COFFE were done (Notredame, Higgins & Heringa, 2000). The software of MEGA 10.0.5 was used for phylogenetic analysis (Kumar, Stecher & Tamura, 2016). The evolutionary history was inferred using the maximum parsimony method with Bootstrap 500. This method showed the most Bootstrap.

RNA extraction and cDNA synthesis

Total RNA was extracted from all TF samples using the RNeasy Plus Mini Kit (Qiagen, HildenGermany). Five TR samples were also analyzed as standard control isolates. The RNA quality and quantity were analyzed using 1% agarose gel electrophoresis and spectrophotometer (Eppendorf BioPhotometer Plus, Eppendorf, Germany), respectively. Then, cDNA was synthesized using high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) with oligo dT and random hexamer primers based on the manufacturer’s instruction.

AQP1 expression analysis

AQP1 gene expression analysis was performed as described previously (Eslami et al., 2016). Thermal cycling profile applies as follows; initial denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 10 s, and annealing and extension at 60 °C for 20 s. The melting curve analysis verified the specificity of the real-time PCR reaction. The relative expression (RQ) analysis was done using the following formula:

$$\begin{array}{ll}{2}^{\Delta \Delta \text{Ct}}=& \left({\text{Ct}}_{\text{target\hspace{0.17em}gene\hspace{0.17em}of\hspace{0.17em}TF}(\text{AQP1})}-{\text{Ct}}_{\text{reference\hspace{0.17em}gene\hspace{0.17em}of\hspace{0.17em}TF}(\text{GAPDH})}\right)-({\text{Ct}}_{\text{target\hspace{0.17em}gene\hspace{0.17em}of\hspace{0.17em}TR}(\text{AQP1})}\\ & -{\text{Ct}}_{\text{reference\hspace{0.17em}gene\hspace{0.17em}of\hspace{0.17em}TR}(\text{GAPDH})})\end{array}$$

Statistical analysis

The statistical analysis was done using the non-parametric Mann–Whitney U test for AQP1 gene expression between two groups of TF and TR isolates.

Detection and identification of the isolates

Molecular identification of the isolates using PCR-RFLP produced fragments of 300–350 bp, hence Leishmania spp. was confirmed. RFLP analysis was performed by Hae III restriction enzyme, which produced 220 and 140 bp fragments indicating L. major.

Molecular analysis and phylogenetic analysis by COXII

All TF isolates were successfully amplified using the primer pair of COXII (Supplemental File 1). The sequencings were successfully performed, and all of them were submitted at GenBank, NCBI, from MK972457 to MK972461.

Sequences of the COXII region from TF isolates were analyzed using nBLAST, T-COFFEE, multiple alignments, and MEGA (10.0.5) software. The multiple alignments of five TF isolates’ sequences were done with the Friedlin strain of L. major (Supplemental File 2). Accordingly, all five TF isolates had conserved areas in most sites compared to the European L. major (Friedlin), being significantly different from the standard European strain in several nucleotides except in terminal regions. The TF isolate with code 2 differed from the European strain with the dispersed point in two nucleotides, even though, all TF isolates were dissimilar with the European standard strain in four nucleotides (Supplemental File 2).

Sequences with complete homology were excluded, leaving only one representative to draw a phylogenetic tree using the barcode gene COXII. In Fig. 1, there are four major groups. The first group consists of only MH44388 Leishmania Varzaneh-63, while the second group consists of two clades. The first clade contains two sub-clades, and the first sub-clade consists of KY407541.1 Leishmania IMP/FARS3 and KY407539 Leishmania IMP/FARS1, and the second sub-clade contains KY407540 Leishmania IMP/FARS2. The second clade includes MH443402 Leishmania Varzaneh-114, when the third group includes only AF287688 Leishmania MRJX. The fourth group consists of 2 clades, and the first clade is divided into two sub-clades. The first sub clade contains Leishmania major and KU680818 Leishmania MHOM/SU/73/5ASKH. The second sub-clade is KU680818 Leishmania MRHO/IR/75/ER, and the second clade contains two sub-clades. The first sub-clade contains KF815210 Leishmania isolates 109 clone3, and the second contains MK972461 Leishmania Yazd 5 and KF815211 Leishmania isolates 9 (Fig. 1).

Molecular analysis and phylogenetic analysis by kDNA minicircle

All five TF isolates were assessed, and none of the sequences related to kDNA minicircle were deposited in GenBank, NCBI. In Fig. 2, four major groups are shown. The first group consisted of two clades, while the first clade contains two sub-clades. The first sub-clade contained isolates 4, 2 and second isolate 3. The second clade contained isolate 1. Besides, isolates 2 and 4 were very similar. The second group only consisted of isolate 5, and the third group included only isolate 331. The fourth group consisted of 2 clades. The first clade was isolate 63, and the second clade was divided into two sub-clades, isolates 115 and 116, and isolates 98 and 100. In this group, isolates 98 and 100 had high similarity with each other.

The gene expression of AQP1

The SYBR Green real-time PCR showed that one isolate had the overexpression among the TF isolates with a mean score of 2.75 ± 0.05. Except for the last isolate, the mean gene expression of AQP1 in 4 TF isolates was 0.7 ± 0.04 (Fig. 3). The difference was statistically significant (p = 0.016). The results of the AQP1 gene expression is detailed in Table 2.