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Table of Contents
Year : 2020  |  Volume : 57  |  Issue : 1  |  Page : 52-57

DNA-based detection of Leishmania and Crithidia species isolated from humans in cutaneous and post-kala-azar dermal leishmaniasis from Shiraz and Kharameh, southern Iran

1 Research Center for Health Sciences, Institute of Health, Department of Vector Biology and Control of Diseases, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran
2 Basic Sciences in Infectious Diseases Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
3 Research Research Center for Health Sciences, Institute of Health, Department of Vector Biology and Control of Diseases, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran
4 Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

Date of Submission26-Apr-2018
Date of Acceptance15-Nov-2018
Date of Web Publication05-Feb-2021

Correspondence Address:
Dr Kourosh Azizi
Research Center for Health Sciences, Institute of Health, Department of Vector Biology and Control of Diseases, School of Health, Shiraz University of Medical Sciences, Shiraz
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.309518

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Background & objectives: Leishmania major and L. tropica are the main pathogens of cutaneous leishmaniasis (CL) in several rural and some urban regions of Iran, respectively. The aim of this study was to detect Leishmania species, and update the distribution data of these species in humans suspected to CL in two endemic foci in southern Iran.
Methods: From March 2016 to March 2017, 276 positive samples from of 350 suspected cases were diagnosed and compared by different diagnostic methods, viz. microscopy, culture, and PCR. In PCR assay, four different gene identifications were performed including minicircle kDNA, and cysteine protease B genes for Leishmania detection, and glyceraldehyde-3-phosphate dehydrogenase, and internal transcribed spacer 1 genes for Crithidia detection.
Results: In total, 68% (235/350) and 65.3% (177/271) of patients suspected of leishmaniasis were positive by microscopy and cultivation methods. In PCR assay, L. major, and L. tropica were detected in 86.2% (238/276), and 13.1% (36/276) of CL cases, respectively. Also, dermal L. infantum strain was isolated from 0.7% (2/276) of post-kala-azar dermal leishmaniasis patients. In addition, Crithidia fasciculata was detected in two CL patients chronically infected with L. major.
Interpretation & conclusion: It appears that the epidemiology of CL has changed during the last decades and can complicate the control strategy aspects of CL in southern Iran. Therefore, more epidemiological, ecological, and gene polymorphism studies are needed to understand the pathogenic role of these species in human, as a main host of leishmaniasis in Iran.

Keywords: Cutaneous leishmaniasis; human; Iran; Leishmania; PCR; post-kala-azar dermal leishmaniasis

How to cite this article:
Kalantari M, Motazedian MH, Asgari Q, Soltani A, Mohammadpour I, Azizi K. DNA-based detection of Leishmania and Crithidia species isolated from humans in cutaneous and post-kala-azar dermal leishmaniasis from Shiraz and Kharameh, southern Iran. J Vector Borne Dis 2020;57:52-7

How to cite this URL:
Kalantari M, Motazedian MH, Asgari Q, Soltani A, Mohammadpour I, Azizi K. DNA-based detection of Leishmania and Crithidia species isolated from humans in cutaneous and post-kala-azar dermal leishmaniasis from Shiraz and Kharameh, southern Iran. J Vector Borne Dis [serial online] 2020 [cited 2022 May 21];57:52-7. Available from: https://www.jvbd.org/text.asp?2020/57/1/52/309518

  Introduction Top

About 20 Leishmania species cause different forms of Leishmaniasis and over 90 infected sandfly species transmit the parasites between human and other mammalians in the world[1]. The disease is also associated with environmental changes and hygiene poverty (such as urbanization, malnutrition, poor housing, etc.), and annually up to 1 million new cases and 30,000 deaths are reported globally[1].

Cutaneous leishmaniasis (CL) caused by L. major and L. tropica is more common clinical form of the disease and prevalent in different regions of Iran, with varying incidence rate[2],[3]. Zoonotic and anthroponotic CL are endemic in rural areas and in some important cities of Iran, respectively, and mammals play an important role in the maintenance and transmission of the parasite[4],[5].

Visceral leishmaniasis (VL) is endemic in southern and northwestern parts, and sporadic in all geographical regions of Iran[6],[7]. In addition, post-kala-azar dermal leishmaniasis (PKDL) cases are reported from northwest and southern regions of the country[8].

In Trypanosomatidae family, genus of Leishmania (about 20 species) and Trypanosoma are pathogenic for humans. Other genus such as Phytomonas and Crithidia are recognized as pathogenic for plants and arthropods, respectively[9].

To predict and develop new control strategies of CL, the current study was designed to identify Leishmania species involved in human infections in Shiraz and Kharameh foci of Fars province, southern Iran.

  Material & Methods Top

Sample collections and parasite culture

During March 2016 to March 2017, cutaneous slit biopsies were prepared from 350 human cases suspected of CL in Shiraz (the capital city of Fars province) and Kharameh (80 km northeast of Shiraz), which are situated at 29° 59′18 ″ North, 52° 58′37 ″ East, and 29°50′ ″20 N, 53°31′24 ″ E, respectively [Figure 1]. Patient data, such as age, gender, number and location of each lesion were recorded in a distinct form. At least two biopsies were obtained for microscopy, culture, and molecular assays[6].
Figure 1: Map of Iran showing the locations of 6 study areas within or around Shiraz and Kharameh cities in Fars province, southern Iran, 2016. The numbers 1, 2 and 3 correspond to Sange-siah regions in downtown, Soltan-abad, and Zafar-abad villages in suburb of Shiraz city, respectively; and the numbers 4, 5 and 6 refer to Kheir-abad and Kouh-gari, Kharameh city and Moez-abad in suburb of Kharameh, respectively.

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For culturing, the biopsies were transferred to liquid phase of modified Novy-MacNeal-Nicole (NNN) medium culture (contained horse blood agar base and an overlay Locke’s solution) in sterile condition and placed in incubator (temperature of 24 ± 2°C). The inoculated samples were checked under invert microscope to observe motile promastigotes of Leishmania, every 2 to 3 days. For mass cultivation, positive cultures were transferred to RPMI-1640 medium (Gibco, Frankfurt, Germany) enriched with 10% inactivated fetal calf serum (Sigma, Cat No. 308056), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco, Frankfurt, Germany). Approximately, 5 × 106 promastigotes/ml were used for supplementary assays[10]. Some parasites were carried to semi-solid Locke-blood agar medium and transported to 4°C for more adaptation and investigation in prolonged time. Furthermore, some of the promastigotes were transferred to cryo tubes containing 5% dimethyl sulfoxide (DMSO) and stored in –70°C and liquid nitrogen, respectively. Additionally, some promastigotes were collected by centrifugation (10,000 g for 10 min) and washed thrice in cold sterile PBS (pH 7.2). Parasite pellets were stored at –20°C until use[10].

DNA extractions

The glass slides and culture sediments of human samples were scraped and extracted by kit extraction (YTA genomic DNA extraction mini kit, Cat No. YT9030) method. Briefly, samples were transferred to micro-tubes, and 20 μl proteinase K and 200 μl lysis buffer were added to the samples, then mixed and incubated at 60°C for 15 min for complete lysation. Afterwards, 200 μl of absolute ethanol was added to the samples and mixed by pulse vortexing for 30 sec. The sample mixtures containing any precipitate were carefully transferred to column micro-tubes and centrifuged for 1 min at 8000 rpm and washed by washing buffers several times to remove impurities from column micro-tubes. Finally, 100–200 μl of elution buffer or ddH2O was added to the membrane centre of the column tubes and after 3 min, they were centrifuged for 2 min at 14000 rpm to elute the DNA and stored at –20°C for PCR assays[11],[12].

Nucleic and kinetoplastic DNA-based detections of Leishmania

DNA extracted of smears and culture sediments of examined human biopsies were selected for Leishmania detections. The samples were examined for identification of the Leishmania species by a modified PCR assay to amplify the variable area of any Leishmania minicircle kDNA gene in the human DNA samples. Each 25 μl reaction mixture (total volume) contained 12 μl master mix buffer (Ampliqon taq DNA polymerase master mix red, Cat No. A180301 (containing 1.5 mM MgCl2 and 2 × concentration of taq DNA polymerase), 1 μl of each primer; forward LINR4 (5′-GGG GTT GGT GTA AAA TAG GG-3′) and reverse LIN17 (5′- TTT GAA CGG GAT TTC TG-3′) (with 10 pmol concentrations), 3 μl DNA sample and 8 μl double distilled water. A programmable thermocycler (Eppendorf AG Master-cycler Gradient, Germany) was used for amplifying the mixtures for 5 min at 94°C (one cycle) followed by 30 cycles at 94°C, 52°C, and 72°C for 30 sec, 30 sec, and 60 sec, respectively; followed by 72°C for 5 min as the final elongation. Finally, 5 μl of each PCR result was electrophoresed in 1.2% agarose gel. Reference strain of L. major (MHOM/IR/54/LV39) was used as standard kDNA. A band of 650 bp was considered as indication of the presence of L. major kDNA in the tested smears[11].

To study the phylogenetic relations of leishmaniasis, an advanced PCR method based on cysteine protease B (CPB) gene detection was used to differentiate L. donovani from L. infantum[13]. In this method, briefly, each 30 μl reaction mixture contained 12 μl master mix buffer (Ampliqon taq DNA polymerase master mix red, Cat No. A180301), 1 μl of each primer; forward cpbF (5′-CGT GAC GCC GGT GAA GAA T -3′) and reverse cpbE (5′- CGT GCA CTC GGC CGT CTT-3′) (with 6 pmol concentrations), 3 μl DNA sample and 8 μl double distilled water. The thermocycler was programmed for 5 min at 94°C (one cycle) followed by 30 cycles at 94°C, 62°C, and 72°C for 30 sec, 60 sec, and 60 sec, respectively, and finally followed by 72°C for 10 min. Reference strain of L. infantum (MHOM/DZ/82/LIPA59) was used, and the bands of 702 bp and 741 bp indicated the presence of L. infantum strain LIPA59 in the patient’s smears[13].

Crithidia detections

All the DNA extracted from human biopsies were selected for Crithidia genus detection by using a sensitive PCR method which was applied to amplify any Crithidia glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene[14]. By using the gene bank information, the specific primers of 5′-TCCATGTGCGAGGACAACGTGCT3′ and 3′-CGCGTCGTTGATGAAGTCGCT-5′ were designed through Gen-Script online PCR primer tool design[14]. Each 25 μl reaction mixture contained 12 μl master mix buffer, 1 μl of each primer (with 10 pmol concentrations), 6 μl double distilled water, and 5 μl DNA sample. Thermocycler was programmed for an initial temperature of 94°C for 5 min (one cycle), followed by temperatures of 94°C for 30 sec, 55°C for 1 min, and 72°C for 1.5 min (30 cycles), and continued for one cycle by a final temperature of 72°C (for 5 min)[14].

For species detection, primers of internal transcribed spacer 1 (ITS1) gene were used[15]. Each 25 μl reaction mixture (total volume) contained 12 μl ampliqon master mix buffer, 1 μl of each primer of LITSr (5′-CTGGATCATTTTCCGATG-3′) and L5.8s (5′-TGATACCACTTATCGCACTT-3′) (with 10 pmol concentrations), 5 μL DNA sample and 6 μL double distilled water. Eppendorf Master-thermocycler was used for amplifying the mixtures for 5 min at 95°C (one cycle) followed by 30 cycles at 95°C for 20 sec, 53°C for 30 sec, and 72°C for 1 min, followed by a final elongation at 72°C for 6 min. The PCR product was finally visualized by ultra violet transillumination. To detect the Crithidia species in RFLP analysis the products of PCR were digested under the effect of restriction enzyme HaeIII (Fermentas) and the related buffer in 37°C for 2 h. Analysis of digestion was done using 2% agarose gel electrophoresis in Tris-acetate-EDTA (TAE) buffer. Afterwards, the observed bands of 400 bp, and 450 bp were compared with reference strains of Crithidia (as the standard kDNA) for the presence of C. luciliae, and C. fasciculate in the tested smears, respectively[15].

Ethical statement

The study was designed and confirmed by the Ethics Committee of the Research Vice-chancellor of Shiraz University of Medical Sciences, Iran (No. IR.SUMS.REC.1395.S475). Informed and free consent was obtained from the volunteer patients.

  Results Top

Patient’s data

Among 350 suspected cases of CL, 276 patients were confirmed at least by one of the performed diagnostic techniques of direct microscopy, culture, and PCR. Majority of the CL patients (157/276) were aged 11–30 yr; however, the numbers of male and female cases were similar [Table 1].
Table 1: The number of male and female cases of cutaneous leishmaniasis by age-group in Shiraz and Kharameh, Fars province, southern Iran

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Parasite detection

Detection of Leishmania: Out of 350 samples of suspected CL cases checked through microscopy, culture, and/ or PCR methods, 276 were confirmed by minicircle kDNA gene as Leishmania species infection; of which 238 (86.2%), 36 (13.1%), and 2 (0.7%) samples produced 650 bp, 760 bp, and 720 bp amplicon indicating L. major, L. tropica, and dermal L. infantum [Table 2], respectively. Two kala-azar cases (4 and 7 yr-old boys) admitted in Namazi hospital, Shiraz with dermal lesions, were confirmed by CPB gene. The fragments of 702 and 741 bp were amplified for strains of L. infantum LIPA59 [Figure 2]. No sample produced more than one amplicon indicating a mixed infection. Moreover, no amplicon and other sizes of amplicon were detected in the negative control samples.
Figure 2: Gel electrophoresis of samples prepared from the kala-azar patient of Kharameh based on nucleic DNA-detection. Lane 5: Molecular weight marker; Lane 1: Reference strains of Leishmania infantum LIPA59 (702 and 741 bp); Lane 2: Reference strains of L. infantum (702 bp); Lane 3: Reference strains of L. major as the negative sample; Lane 4: Sample prepared from a post kala-azar boy.

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Table 2: The frequency and prevalence of Leishmania/Crithidia positives in humans suspected leishmaniasis examined by parasitological, cultural, and molecular assays in Shiraz and Kharameh, Fars province, southern Iran

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Detection of Crithidia: Amplification of GAPDH gene was performed for Crithidia genus detection[14]. Thereafter, two cases were molecularly assessed for Crithidia species by ITS1 gene amplification. A 555 bp fragment was amplified for Crithidia species. The digestion with HaeIII restriction enzyme, showed presence of 450 bp and 240 bp fragments indicating C. fasciculata [Figure 3]. These two recent samples belonged to 15 and 19 yr-old girls who lived in Kharameh focus, and had the history of chronic L. major infections, before.
Figure 3: Gel electrophoresis of samples prepared from the CL patient of Kharameh based on RFLP Crithidia based detection. Lane 1: Molecular weight marker; Lane 2: Reference strains of C. fasciculata (555 bp), before HaeIII restriction enzyme effect; Lanes 3 and 4: Samples prepared from two patients, who had the history of chronic L. major, before enzyme effect; Lane 5: Reference strains of C. faciculata, after enzyme effect (450 bp, and 240 bp); Lanes 6 and 7: Samples prepared from two patients, who had the history of chronic L. major, after enzyme effect.

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  Discussion Top

Fars province is an important endemic region of zoonotic CL and recent investigations have revealed increase in the prevalence and incidence rates of the disease[11],[16]. In the present study, two patients infected with L. infantum had a history of VL; and their skin lesions were limited to the face (typically open). But, in India, ulcerative lesions of PKDL are different; the lesions are closed, and shaped as nodular, popular, or macular[17]. By CPB gene amplification, two species of L. donovani and L. infantum were differentiable by their fragment length and characterized by 741 bp and a 702 bp products, respectively, except for one heterozygous strain with the two products that known as dermal L. infantum strain LIPA59. Moreover, in this method, other Leishmania species or kinetoplastids do not produce any amplification[13].

Leishmania donovani causes PKDL in Sudan and India and is reported in at least 10–15% of VL patients, however, few cases of PKDL are reported to be caused by L. infantum[18]. First report of PKDL due to L. infantum was observed in a patient infected with human immune-deficiency virus (HIV) type 1 in Australia[19]. The L. infantum has been identified from a PKDL patient in Sudan[20], besides, it has been detected from a VL patient, who was co-infected with HIV. In some reports, unknown causative agents of PKDL cases have been reported from northwest and south regions of Iran. However, a few CL cases caused by L. infantum, who had a history of VL, has been reported from east Azerbaijan and Ardabil provinces in northwest Iran, recently[8],[21],[22].

Cases of CL, associated with L. infantum, have been reported in the Middle East and Mediterranean basin, where L. infantum and L. donovani caused VL, and L. major, L. tropica, L. infantum/L. donovani hybrid, and dermal L. infantum strains caused CL[23]. Travel to an endemic area of this basin could be a risk factor for leishmaniasis and should be considered in strategy controls of the disease.

In two patients, who had the history of chronic L. major infections, C. fasciculata was detected concomitantly from the lesions. Recently, a novel TRYP6 gene from an Iranian L. major strain has been reported by Eslami et al[24] that exhibited homology with the related gene in Crithidia as a distinct genus of Kinetoplastida. In that study, 100 isolates of patients suspected to CL were analyzed for internal transcribed spacer 1 (ITS1) region, RNA polymerase II largest subunit (RPOIILS) and the mitochondrial DNA polymerase beta (DPOLB)[24]. The results of the ITS1 analysis (450, 240, and 310 bp fragments) were similar to the present study. Moreover, their molecular analysis by BLAST tool indicated that the selected sequence had a close similarity with C. fasciculata (97%), C. luciliae (90%) and L. infantum (40%)[25]. Furthermore, some molecular investigations have revealed that Crithidia species are co-detected from CL patients, and animal hosts in centre and south regions of Iran[14],[15], hence, it seems that this nonpathogenic parasite may improve its biological adaptations to survive in non-specific hosts. However, further enough experimental evidences and clinical findings are needed to prove the Crithidia spp. as a causative agent in human CL.

  Conclusion Top

The study indicates that L. major is the main causative agent of CL cases in many regions of Fars province, especially in rural areas, however, L. tropica is limited in the city centre areas of the province[2],[26],[27],[28]. Environmental aspects (increasing urbanization, population growth, movements toward focus of vectors, reservoirs, pathogens of leishmaniasis, etc.) have changed the epidemiology of CL during the last decades from a predominant zoonotic CL and can complicate the strategy control aspects of CL[29]. Therefore, more epidemiological, ecological, and gene polymorphism (in order to assess hybrid formation) studies are needed to understand the pathogenic role of these species in human, as a main host of leishmaniasis in Iran.

  Acknowledgements Top

This research was financially supported by the Research Vice-chancellor of Shiraz University of Medical Sciences, Iran and extracted from the results of an approved Ph.D. student thesis (No: 94-01-104-10873) conducted by the author, Mr. Mohsen Kalantari.

Conflict of interest: None

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2]

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