Genotyping the causative agents of cutaneous leishmaniasis in Ahvaz southwest of Iran

Abstract

With sand flies as the main vectors, Leishmania species cause ancient zoonotic diseases called leishmaniasis. Iran is an endemic country regarding cutaneous leishmaniasis. A number of 100 smear slides were collected from cutaneous lesions referred to Ahvaz health centers. The DNA was extracted and ITS1–PCR using LITSR and L5.8S primer pair was performed to detect the genus Leishmania. Then, enzymatic digestion of PCR products was done by HaeIII (species detection), TaqI (strain detection), DpnI and HpaII (mutation assessment). Furthermore, 50 samples were sent for sequencing. Microscopic examination showed amastigote form in all 100 slides. Also, molecular identification confirmed the infection of all cases to Leishmania genus, representing a 350 bp band. HaeIII digestion yielded 150 and 200 bp bands, indicating Leishmania major, while 130 and 200 bp fragments following TaqI digestion suggested A1 strain of the parasite. Moreover, no likely mutations was detected in ITS1 fragment of obtained parasites using DpnI (140 and 200 bp digestion) and HpaII (without digestion). The sequencing result also was consistent with our findings, having 100% homology to A1 strain sequence (AY550178). Leishmania major A1 strain was the predominant species in clinical samples of Ahvaz. Nevertheless, future researches should address the parasite strains in other foci and hosts of epidemiological significance.

Introduction

Leishmaniasis are a group of ancient parasitic diseases which human being has encountered since antiquity. With over 21 species, Leishmania parasites cause diverse spectrum of clinical manifestations, being categorized into cutaneous, diffuse cutaneous, muco-cutaneous and visceral forms (Srivastava et al. 2016). Over 100 countries and 350 million individuals around the world are in endemic regions and at-risk of infection (de Vries et al. 2015).Ten nations including Colombia, Peru Brazil, Costa Rica, Syria, Iran, Afghanistan, Algeria, North Sudan and Ethiopia are the most endemic regions regarding cutaneous leishmaniasis (CL). Several risk factors such as migration, urbanization as well as man-related proceedings has provoked environmental changes, which impacts the diffusion of CL (Norouzinezhad et al. 2016).

Iran, located at Eastern Mediterranean region, is considered as endemic territory for CL with approximately 20,000 cases per annum, predominantly in Isfahan and Fars provinces. However, the actual incidence rate is four- to fivefold more than currently-documented reports (Karamian et al. 2016; Khademvatan et al. 2012). CL is the second most prevalent arthropod-borne disease following malaria in Iran (Moemenbellah-Fard et al. 2012), which manifests as anthroponotic (dry, urban) and zoonotic (wet, rural) forms (de Vries et al. 2015; Karamian et al. 2016). Leishmania tropica (L. tropica) is the causative agent of urban CL, which mostly emerges in highly crowded cities with considerable daily immigrants (Moemenbellah-Fard et al. 2015). On the other hand, rural disease, caused by Leishmania major (L. major), frequently occurs in rustic areas in the west, southwest, northeast and central Iran, where rodent reservoir hosts inhabit (Farahmand et al. 2011; Reithinger et al. 2007).

Molecular identification of Leishmania species is of utmost clinical importance to design for better treatment strategies and combating resistant isolates. Internal transcribed spacer-1 (ITS1), a potential genetic hallmark of ribosomal RNA (rRNA), is a strictly conserved fragment for phylogenetic classification of closely-related organisms. PCR–RFLP technique by specific endonucleases using ITS1 region is a promising method to detect parasite species and strains as well as mutation assessment. In current investigation, we employed such technique to provide molecular evidence on the presence of the causative agents of cutaneous leishmaniasis in clinical samples from Ahvaz, southwestern Iran (Doudi et al. 2010; Monroy-Ostria et al. 2014).

Materials and methods

Study area and sample preparation

Ahvaz, the capital of Khuzestan province, is located in southwestern Iran with about 1,300,000 population. The only navigable river of Iran, i.e. Karun passes through the city, providing a favorable ecological habitat for probable localization of different species. Ahvaz possesses subtropical hot desert climate, representing short winters and long, very hot summers. The regular temperature during summer reaches 45 °C with many sandstorms and dust storms. The minimum temperature in winter falls to about 5 °C.

In this study, a number of 100 smear with diagnosis in cutaneous leishmaniasis and laboratory approval, were obtained from cutaneous lesions of cases referring to the health centers of the Ahvaz city (North, South, West, East, and Center) during 2016. The whole specimen was microscopically examined to observe amastigote form of Leishmania and confirm CL cases.

DNA extraction

At first, slides were washed by phosphate buffer saline (PBS) solution, then smears were scraped by surgical scalpel and contents were transferred to a 1.5 mL microtube. Following repeated washing with PBS, GeNet Bio PrimePrep DNA extraction kit from tissue (GeNet Bio, South Korea) was applied, according to the manufacturer’s protocol. The finally extracted DNA was maintained at − 20 °C for further use.

PCR–RFLP technique

Molecular identification of Leishmania clinical isolates was done using ITS1–PCR with LITSR (5-CTGGATCATTTTCCGATG-3) and L5.8S (5-TGATACCACTTATCGCACTT-3) primer pair and the subsequent RFLP method (El Tai et al. 2000; Kazemi-Rad et al. 2008; Schönian et al. 2003). DNA amplification was performed in a total volume of 25 μL, including 1.5 mM MgCl₂, 250 micro Molar dNTPs, 1 U Taq DNA polymerase and 20 pmol of each primer. Thermal cycler (Analytik Jena, Germany) conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 53 °C for 5 min and extension at 72 °C for 1 min. At last, an additional extension step was done at 72 °C for 5 min.

After accomplishment of DNA amplification process, the PCR products were digested using specific endonucleases. For this aim, HaeIII (Jena Bioscience, Germany) was first used for species determination, with 5 μL PCR product, 2.5 μL universal buffer, 15 U enzyme and sterile water. All contents were incubated at 37 °C for three h (Schönian et al. 2003). Strains were also delineated by using TaqI (Jena Bioscience, Germany) restriction enzyme, similar to HaeIII condition but incubation was at 65 °C for two h. Furthermore, mutation analysis was accomplished by DpnI and HpaII enzymes (Thermo Fisher Scientific, USA) in a total volume of 16 μL, with 6 μL PCR product, 2 μL universal buffer, 10 U enzyme and sterile water, being incubated at 37 °C for three h (Doudi et al. 2012).

Sequencing

A number of 50 PCR products were sent for sequencing (Microsynth, Switzerland) along with LITSR and L5.8S primer pair. Sequencing results were interpreted using MEGA X and BLAST Tool (http://www.ncbi.nlm.nih.gov/BLAST) and compared to other GenBank registered records. After A, G nucleotide evaluation in 113 position and comparing to standard A1 (AY550178) and B1 (AY573178) strains, our detected strains were confirmed.

Results

In current study, 100 smear slides obtained from cutaneous lesions of suspected cases referring to Ahvaz health centers during 2016, were microscopically confirmed to be positive with Leishmania spp. (Fig. 1).

Fig. 1.

L. magor amastigotes form observed under optical microscope (×1000)

Also, PCR method confirmed such infection in whole specimen, representing a 350 bp band specific to ITS1 region of genus Leishmania (Fig. 2).

Fig. 2.

Electrophoresis results of ITS1 PCR from smears. M: molecular marker (100 bp); P: standard L. major (MRHO/IR/75/ER); N: negative control; lane 1–12: PCR products sample

Following enzymatic digestion by HaeIII, all samples showed 150 and 200 bp bands, indicating of L. major (MRHO/IR/75/ER) (Fig. 3).

Fig. 3.

Restriction enzyme digestion profile of amplified ITS1 region with the restriction enzyme of HaeIII. M: molecular marker (50 bp); P: standard L. major; N: negative control; lane 1–12 Leishmania major

Additionally, 130 and 200 bp bands were formed following TaqI digestion, entitling A1 strain for detected Leishmania parasites (L. major A1) (Fig. 4).

Fig. 4.

Restriction enzyme digestion profile of amplified ITS1 region from L. major with the enzyme TaqI. M: Molecular marker (50 bp); P: standard L. major with TaqI; N: negative control; lane 1–12: L. major A1 (130 and 200 bp) L. major

PCR products undergoing DpnI digestion showed 140 and 200 bp band patterns, whereas no cutting site was identified for HpaII, which both suggest no mutation in ITS1 fragment of detected Leishmania parasites (Fig. 5).

Fig. 5.

Restriction enzyme digestion profile of amplified ITS1 region with the restriction enzyme of DpnI. M: molecular marker (50 bp); P: standard L. major; N: negative control

Sequencing results comparison to standard A1 (AY550178) and B1 (AY573178) strains registered in GenBank demonstrated 100% homology to A1 (AY550178) strain. Thereby, all clinical isolates were related to A1 strain (L. major A1), and no other strain/genotypes were detected in Ahvaz region.

Discussion

Traditional identification of Leishmania species is a problematic issue due to diverse clinical pictures of CL in endemic areas as well as similar symptomatic diseases such as leprosy and fungal infections (Reithinger and Dujardin 2007). Fist attempts were based on imprecise biological and epidemiological classifications, and the following isoenzyme analysis typing. During last decades, however, nucleic acid-based techniques are more highlighted as golden standard methods for Leishmania identification (Mahnaz et al. 2006). Current investigation demonstrates evidence of Leishmania strain and possible mutations in clinical isolates from Ahvaz, southwestern Iran, using ITS1–PCR and the subsequent RFLP method. According to previous studies, the amplification of several standard Leishmania species using LITSR and L5.8S primer pair yields a 350 bp band, which was observed in all 100 clinical isolates. HaeIII digestion showed 150 and 200 bp fragments, suggestive of L. major species totally. Following TaqI activity, two cut sites yielded 130 and 200 bp bands, belonging to A1 strain of L. major (L. major A1), which was consistent with our sequencing results. As a consequence of rapid, convenient and less costly identification using RFLP, it is recommended as a more reliable method for parasite genotyping.

In Niknam et al. study, out of 341 Leishmania isolates, 283 L. major and 58 L. tropica were found in 11 geographical areas of Iran (Mahmoudzadeh-Niknam et al. 2012). Saki et al. demonstrated 14 Leishmania positive samples (12 L. major and 2 L. tropica) in Ahvaz, Dasht Azadegan, Shush, Hendijan and Ramhormoz regions (Saki and Khademvatan 2011). Furthermore, another investigation in Ahvaz revealed that among 100 positive samples for Leishmania, 97 isolates were L. major and 3 were L. tropica (Ghasemian et al. 2011). Maraghi and colleagues, also, detected L. major in 90% of isolates from Shush county, while the rest were dedicated to L. tropica species (Maraghi et al. 2007). According to the consistency of such investigations with our results, it is premised that L. major is the predominant Leishmania species in Khuzestan province, and in Ahvaz particularly. It seems that the presence of Phlebotomus papatasi and Tatera indica as vector and reservoir host for CL in this area, and several infection reports from various cities throughout Khuzestan province, entitle this region, Bushehr province as well as parts of Ilam province bordering Iraq as endemic territories for zoonotic cutaneous leishmaniasis (ZCL). Regarding a recent study on 20 clinical samples of CL in neighborhood country Iraq, 12 L. major samples belonged to L. major A and L. major B strains, while 8 L. tropica isolates were related to L. tropica A and L. tropica B strains (Ali et al. 2015).

Based on Tashakori and colleagues investigation on L. major isolates heterogeneity in ZCL endemic areas of Iran, including Kashan, Dezful, Dehloran and Damghan, five genotypes of L. major were identified (L. major A, L. major B, L. major C, L. major D and L. major E), with L. major A as the predominant genotype allover Iran, particularly in southwestern parts, while L. major C, L. major D and L. major E were only detected in a new focus in Damghan and L. major B were exclusively found in Kashan isolates (Mahnaz et al. 2006). Probably genetic persistency of an organism in reservoir host and avoiding various hostile conditions would postpone drug resistance development and formation of new strains. L. major is a zoonotic parasite with rodents as reservoir hosts; hence, L. major A, strains are naturally found in a region with L. major isolates. In addition, L. major A is possibly the most ancient genotype which is remarkably adaptive to the conditions of a new region, other strains are assumed to be derived from. Unlike L. major, L. tropica is anthroponotic and only human is involved, so such species are more exposed to various hostile pressures (treatments, environmental, seasonal, etc.) with enhanced chance of genome mutations; this issue would provoke new strain formation in L. tropica rather than L. major. In this case, Doudi et al. identified six genotypes of L. tropica (L. tropica A, L. tropica B, L. tropica C, L. tropica D, L. tropica E and L. tropica F) in three territories endemic for anthroponotic CL (Bam, Kermanshah and Mashhad) during 2010 (Doudi et al. 2012).

Conclusions

In total, findings of our investigation showed that L. major A was the prevailed genotype of CL in Ahvaz, and no other genotypes have been observed in the area so far. Consequently, given immigrants entering from Iraq to Khuzestan province on the one hand, and the key role of parasite genotype in clinical symptoms and pathogenesis on the other hand, more comprehensive researches are suggested to assess the genotypic picture of Leishmania isolates from different human, rodent and vector hosts throughout the province.

Funding

This study was based on a MSc. thesis with Grant No: OG-96131, who was supported by Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.