Detection of cutaneous leishmaniasis based on ITS1 gene by PCR-RFLP technique Hekmat Ahmed Al-Fahdawi 1

Detection of cutaneous leishmaniasis based on ITS1 gene by PCR-RFLP technique

Hekmat Ahmed Al-Fahdawi 1, Sarab Fawzi Al-Ani 2
Thamir abdalmajed Al-Kubaisi 3
1,2- Department of Microbiology, College of Medicine, University of Anbar,
3- Department of Medicine, College of Medicine, University of Anbar, Iraq.

ABSTRACT
Background: Cutaneous leishmaniasis is a vector-borne disease transmitted by biting of the sandfly, it is a severe health problem in many countries and endemic in most regions of Iraq.
Objectives: This study was conducted to find the best method for diagnosis of cutaneous leishmaniasis, detect the genotypes of Leishmania tropica and Leishmania major in Ramadi (Iraq) by PCR-RFLP technique.
Materials &methods: One hundred twenty-two patients 68 were males while the females gender were 54 with age ranged 1-68 years, CL who attended to Department of Dermatology in Al-Ramadi Teaching Hospital and dermatology Private clinics, during the period between November 2017 to April 2018. The Molecular study was carried out to detect the ITS1 gene by (PCR). The restriction fragment length polymorphism (RFLP) was adopted on ITS1-PCR product and after HaeIII digestion at 37Cº for 2 hours.
Results: Laboratory examination of 122 cases showed 62 infection cases in Cutaneous leishmaniasis by using PCR technique and in infection proportion reaches at 51% out of the total number of the cutaneous cases which are similar to leishmaniasis during the months of the study. The demographic study dealt with age, gender, number of lesions and body site of infection, demonstrated that the majority of patients at the age of 1-10 years with percent reachedo 28.7%. Also Males (55.7%) had higher infection than females (44.3%), upper limbs had the highest percentage (48%) when compared with other sites of infection, single lesion was documented in 55% of patients, while two lesions were observed in 25% and multiple (3-10) lesions were observed in 20%. Different techniques were used for diagnosis of CL including routine method performed by direct microscopic smear from lesion which showed amastigotes in the macrophage in 50 (41%) positive case. The Molecular study was carried out to detect the ITS1 gene (internal transcribed spacer1) by (PCR). DNA extracted from 122 samples showed 62 (51%) were positive for (ITS1)gene, The restriction fragment length polymorphism (RFLP) was adopted on ITS1-PCR product and after HaeIII digestion at 37Cº for 2 hours obtained two fragments of 60 and 200 bp 42 as L.tropica, and two fragments of 140 and 210 bp were identified 20 as L.major, genotype techniques were performed for all positive samples.
Conclusion: CL is highly spread with single lesions more than multiple lesions and molecular detection showed that L.tropica more common than L.major.

Keywords: Cutaneous leishmaniasis (CL), ITS1, gene, PCR-RFLP.
INTRODUCTION :
Leishmaniasis is a parasitic disease caused by haemoflagellate Leishmania. The disease is widespread and may cause serious health problems in communities throughout the Mediterranean regions and the Middle East, including Iraq (Ashford et al.,1992;CDC,2004). There are an estimated 12 million cases worldwide, and there are about 1.5 million new cases of cutaneous leishmaniasis each year, of which over 90% occur in Afghanistan, Algeria, Iran, Iraq, Saudi Arabia, Syria, Brazil and Peru (Markle et al.,2004). Old World disease primarily is caused by Leishmania tropica in urban areas and Leishmania major in dry desert areas (CDC ,2004)
There are three main types of leishmaniasis: cutaneous leishmaniasis (CL) caused by Leishmania tropica, L. mexicana and L. major. Visceral leishmaniasis (VL) caused by L. donovani and L. infantum and mucocutaneous leishmaniasis (MCL) caused by L. braziliensis (Igbineweka et al.,2012).Cutaneous Leishmaniasis is caused by different species of Leishmania with a specific inclination of each species to a particular geographical region. (WHO,2014). It spread by sand fly bites, afflicts hundreds of thousands of the world’s poorest people in tropical countries. Leishmania spp are intracellular protozoa have a complex digenetic life cycle, requiring a susceptible vertebrate host and a permissive insect vector, which allow their transmission, emphasized the importance of animal reservoirs in facilitating transmission of CL (WHO,2015).
In Iraq, two species are present: L tropica, the agent of anthroponotic cutaneous leishmaniasis (ACL), and L. major, the agent of zoonotic cutaneous leishmaniasis (ZCL). Both ACL and ZCL were reported as causative agents of leishmaniasis in Iraq, but ACL is found mainly in suburban areas (WHO,2003).
Only a minority of infected humans develops the disease: most are infected at a sub-clinical level, These asymptomatic hosts help sustain VL transmission in endemic areas and represent a major challenge for infection control (Singh,2006).
The Cutaneous leishmaniasis lesions appeared as the sores which can change in size and appearance over time. It may start out as papules (bumps) or nodules (lumps) and may end up as ulcers (like a volcano, with a raised edge and central crater); skin ulcers may be covered by scab or crust. The sores usually are painless but can be painful. Some people have swollen glands near the sores (for example, under the arm, if the sores are on the arm or hand). The lesions of CL in normal infection appeared in the arms, legs, faces and ears, showed solid, dry like volcano area in shape and characterized by erythematous papule, with ulcerative border (CDC,2012). In such cases, the diagnosis should be confirmed by examination of smears from lesions, culture, and histopathological examination (Singh et al.,2003). In developing countries such as Iraq, laboratory equipment and materials such as ELISA test kits or PCR technique materials are not available and dermatologists mostly have to rely on the clinical characteristics of the lesion. Giemsa or Leishman-stained smears obtained from the lesions are a rapid means of diagnosis (Ramírez et al.,2000).
The diagnosis of CL is based on clinical features and laboratory tests, including a direct parasitological examination and/or indirect testing with serology and molecular diagnostics (Singh et al.,2003). A universal PCR method targeting the internal transcribed spacer 1 (ITS1) region, which occurs between the genes encoding 18S rRNA and 5.8S rRNA, has proved useful in the direct diagnosis and identification of the Leishmania parasite because this region is highly conserved among species. On the other hand, there are other targeted genes using in the molecular description. The species identification requires additional processing by restriction fragment length polymorphism (RFLP) and DNA sequencing, these last tests are more sensitive but may be applied only in particular centres (Al-Nahhas et al.,2013).
The evidence confirmed increasing in percentage of infection due to a bad situation for hundreds of thousands of people who exposed to the displacing and dived in camps, in addition to the presence of the war and bad conditions, and presence of swamps near their camps that important for reproduction sand fly (Younis, 2018).

MATERIALS AND METHODS :
This study is carried out on Iraqi patients included 122 patients suspected of cutaneous leishmaniasis admitted to Al-Ramadi teaching Hospital and some private clinics during the period from 1st November 2017 to 1st April 2018. Approval for this study was obtained from the Ethical Committee of the University of Anbar.
According to (Eksi et al.,2017) with some modification in the method of collecting the aspiration fluid, the lesion sites were cleaned with 70% alcohol. there my a syringe (1ml,30G*1/2 )when intradermal pentostam injection at the periphery was done, we were aspirated the fluids and the blood that oozing from the sites by capillary tubes with anti-coagulants and prepared slide to examine directly by microscopy after staining and the other was saved in screw cap container with the Sterile NaCl solution as dilution fluid and incubated in refrigerator (4 c) until DNA extraction.

Microscopic examination conducted on each sample of aspiration prepared the smear by transferring a portion of the sample onto a clean slide. Staining with Giemsa or Leishman’s stain solution and examined under a light microscope with a 100_objective lens. Preparation showing amastigotes is considered to be positive (+ ve ?) for Leishmania spp. and preparation with no amastigotes is considered negative (–ve) for Leishmania spp. All results were recorded (Rahi, 2015; Eksi et al.,2017).
The isolation of Leishmania spp. DNA was extracted from the aspiration fluids by using the (DNA extraction for the intracellular organism) Genomic DNA Mini Kit (Geneaid, Taiwan) according to the manufacturer’s protocol and stored at -20oC. DNA samples prepared from aspiration blood were quantified by Ultraviolet spectrophotometer (Unico, USA) reading at 260 and 280 nm (Sambrook et al.,1989). All samples were stored at -20 oC until use.
All suspension samples examined for DNA extraction which was assayed by PCR amplification process. The specific primers were synthesized from IDT (USA), The forward primer (LITSR) 5-CTGGATCATTTTCCGATG-3 and reverse primer (L5.8S) 5-TGATACCACTTATCGCACTT-3, specific to the ribosomal ITS1 region of Cutaneous leishmaniasis the PCR program. of ITS1 region According to (El Tai et al.,2000; Rio de Janeiro, 2009). as the following: Initial Denaturation 95 oC for 5 min. Denaturation95 oC30 sec. Annealing48 oC30 sec. Extension72 oC1 min. Final Extension72 oC6 min. for 35 cycles. The PCR products of ITS1 gene were detected and separated by 2% of agarose gel with (2 ?l of 10mg/ml) ethidium bromide and carried out for one hour with 5volt\cm electrophoresis, followed by detection of the specific bands (350 bp for ITS1) under Ultra Violet Light (Sambrook et al.,1989).
Sample Positive for PCR-products gene was performing of RFLP procedure with endonuclease restriction enzyme HaeIII (Takara Bio Inc, Japan) according to (Eroglu et al.,2011; Eksi et al.,2017) for restricted of specific sequences for each genotype of cutaneous leishmaniasis For the purpose of knowledge subgenotypes that infected human and enhance the accuracy of diagnosis. As the following steps 10µ? of PCR product was placed into Eppendorf tube (1.5 ml),3 µ? from 10X M buffer enzyme,0.5µ? of restriction enzyme,1.5 µ? of deionized water were added to all of these components which final volume of reaction mixture was 15µ? and the Reaction mixture was incubated at 37oC for three hours. When Incubation period was completed, add 3 µ? of 10X Loading Buffer to stop enzyme reaction and apply on agarose gel electrophoresis ( 2.5% agarose) and visualized with ethidium bromide staining and carried out for observation of restricted bands by restriction enzymes.

According to (Simon 2006) all result was statistically analyzed. Inferential statistics such as Chi-square test by using the SPSS statistical program was used to test whether or not significant differences between proportion and means exist.
RESULTS :
The patients were of different sex, out of 122 specimens, 68 (55.7%) were male while the female gender was 54 (44.3%), the ages were distributed between a year to 68 years where distribution into 7 age groups. The total number of specimens that gave positive results of cutaneous leishmaniasis by microscopic examination was 50 (41 %) patients. while the specimens that gave positive results by PCR technique for ITS 1 gene was 62 (51%). In addition to that the samples that gave positive result by PCR assay were inserted to nucleic acid restriction enzymes (Hae III) for restricted the ITS1region of the cutaneous leishmaniasis where Hae III was used for restricted the gene ITS1(350 bp) into (210 bp, 140 bp )for Leishmania major and (200 bp, 60 bp) for Leishmania tropica. There is a statistically significant difference between the rate of infection and the age groups, P