Clinical Isolation and Molecular Diagnosis of Cutaneous Leishmaniasis using RFLIP for ITS1 Gene in some
Nada Noori Younis1
and Sara Murad Muhammed2
Ministry of Health Central Health Laboratory1 Uruk University2
Keywords:Leishmania tropica,Leishmania major,ITS1, RFLIP.
This study was aimed to find out the most effective clinical samples and diagnosis methods in chronic cutaneous leishmaniasis (CCL). Smear, aspiration fluid, and filter paper, samples were taken from 88 skin lesions of suspected cases with CCL in some Baghdad hospitals, and they were compared using microscopic examination, culture, and molecular methods.Samples from the clinical patients were examid which showed throug PCR 93%(82/88) ,Microscopic examination 70%(62/88), culture 25%(22/88).During the study ,products in RFLIP for ITS1, by restriction the amplified ITS1 gene product of Leishmania by the endonuclease Hae III, gave two fragments 60 and 200 bp were identified(57 patient) as L.tropica and two fragments of 140 and 220 bp (25 patient) as L. major ,and the filter paper most effective clinical samples from lesion.In conclusion the PCR –based assays tested on our increased the speed and sensitivity of the diagnosis DNA –PCR very important to the species identification ( L. tropica and L. major).
Leishmaniasis is a disease caused by Leishmania parasite and transmitted to mammals and human beings by Phlebotomine sand flies and it causes skin infections[ 6,10]
)Twenty-one species of Leishmania have been reported to cause human infection[ 6 ].
Each year, 1.5–2 million new cases are reported and 70,000 deaths occurred. The number of disease and death cases showed about 2.4 million people affected throughout the world . Leishmaniasis can produce various symptoms in mammalian host depending on the host genetic makeup and species of the Leishmania parasite( 15 ). Approximately, 90% of the cases of the cutaneous leishmaniasis were observed in Iran, Afghanistan, Pakistan, Saudi Arabia, Brazil, Peru,Iraq and Syria .
The lesions are mostly found on the exposed areas of the skin [3, 16] The lesion or ulcer leaves a scar on infected area . Secondary bacterial or fungalinfection of the ulcers causes increased tissue destruction and disfiguring of the skin  .Several techniques have been described for the identification of Leishmania at the molecular level. These techniques include sequence analysis of multicopy genes, restriction fragment length polymorphism, inter genic spacer regions, DNA fingerprinting, polymerase chain reaction (PCR), and randomly amplified polymorphic DNA[8,1].
The accurate identification and diagnosis which are concerned with epidemiology, clinical finding, and management and treatment of the patient must be based on molecular diagnosis.
http://annalsofrscb.ro 775 Materials and methods
Samples were collected from the lesions of patients with clinical suspected cutaneous leishmaniasis from some Baghdad Hosptails(Al-Yarmook, AL-Karama , Al- Kadhimiya, Al-Kindi) The participants suspected of having CL were informed about the study and given a questionnaire. According to the results obtained by the questionnaire, aclassification of existing lesions was done for the following categories: acute CL (ACL) less than 1 year in duration, CCL more than 12 months.
Samples collected by Aspiration fluid, Smear , Filter paper.
All of the smears were fixed by dipping in absolute methanol and stained with Giemsa 10% stain, and then, they were examined under a light microscope with magnification at 1,000×. Some of aspiration fluid was also smeared onto a glass slide, fixed with methanol, stained with Giemsa, and examined under a microscope. All of the preparations where amastigote was observed were accepted to be positive, and those preparations where amastigote was not observed were negative.
Approximately 0.2 mL aspiration fluids were inoculated into a 2-mL sterile tube containing 0.1 mL Novy-MacNeal-Nicolle (NNN) medium supplemented with 10%
fetal calf serum (FCS; Sigma Aldrich Chemical, France), antibiotics (penicillin and streptomycin at 50 U/mL), and an antifungal agent (fucytosine). The cultures were incubated at 26°C and observed every week for 1 month. Promastigote-observed cultures were accepted to be positive, and cultures where promastigote was not observed were negative.
Filter paper The skin lesion area was cleaned with 70% alcohol andsterile Whatman 5-mmfilter
papers (Whatman House, Maidstone, United Kingdom) was gentlypattedonto the lesion around the edges of the cuts and allowed to air drythoroughly and then cut and
but in the appendrof conten 400ml BPS to use for DNA extraction.
The samples were subjected to DNA extraction by using Isolation Kit (Qiagen, Hilden,Germany) manufacturer protocol and extracted DNA were stored at −20°C for further process
Agarose Gel Electrophoresis After genomic DNA extraction for skin samples, agarose gelelectrophoresis was
adopted to confirm the presence and integrity of the extracted DNA .
-ITS1 PCR- RFLP The ITS1 PCR was performed on all 88 extracted DNA samples. Amplification reaction was performed in volum 20 ml containing PCR Master mix (Bioneer,Koera) with primers Forward LITSR(5\- CTGGATCATTTTCCGATG-3\) and ReverseITS1R-TR1 (5\-GAAGCCAAGTCATCCATCGC-3\) 10p mol as mentioned by Davila and Momen(2000) and DNA 5mlnucleus free water as negative control . Rection were amplified in thermocycler (Appendorf USA) as follows ;initial denaturation at 95oC for 5min by 35 cycles of 94oCfor 30 sec ,48oC for 30 sec ,72oC for 1min and final extension 72oCfor 10min.At the end PCR products were analyzed using 1.5% gel electrophoresis .The diagnostic criteria of cutaneous Leishmaniasis in ITS1 PCR was based on the observation of expected band 350 bpinPCR product and after enzymatic digestion by the observation of its pattern, species of Leishmaniawere identified.
.RFLIP analysis of amplified ITS1 All positive samples of ITS1 PCR product were digested with restriction enzyme HaeIII and the Leishmaniaspecies were identified based on pattern of enzymatic digestion the PCR products , (10ml) of PCR product weredigested with 1ml of HaeIII (fast digest fermentas)at 37oC for 30 min using conditions recommended by the supplier, the restriction fragments were subjected to electrophoresis in 4% agarose gel.
Chi-square test was used to significant compare betweenpercentage in this study(SAS, 2012).
Results and Discussion
Leishmania spp the productivity and the potential isolation of (amastigotes) in NNN media .were assessed during the present study for evaluating the efficiency in the diagnosis of different life stages of the parasite. The results of microscopic examination, cultivation , extracted DNA for ITS1 genRFLIP PCR of the specimens from the patients having suspected CL lesions.
The results revealed that 70%( 62/88) of cases were positive by the Microscopic examination)(fig- 1) , where as only 25%( 22/88) by culture(fig- 2). In molecular method by RFLIP PCR it was found 93%(82/88) for ITS1 gene(fig-3) ; 69%(57/82) were typed as L.tropica and 31%(25/82) were typed as L.major (fig-4) while PCR positive for ITS1 gene .Highly Significant (P<0.01) correlation was observed
between diagnosis methods (Table 1). .
(Fig -2) Promastigote in culture NNN ( fig- 1) Amastigote in WBC(monocyte)
Fig- 3] Gel electrophoresis of ITS1PCR products of Leishmania on 4% Agarose gel ,Wells 1-14 ,L 100bp DNA ladder marker
Table 1: Comparative detection of Leishmania in direct microscopy, culture and RFLIP for (ITS1) genes
χ2: P- value Total
5.032 * (0.039) 88
** (P<0.01)-Highly significant
This result confirmed by previous study conducted by [9 ] obtained same result when used conventional PCR for amplification ITS1 gene.
The results of restriction the amplified ITS1 gene product of Leishmania by the endonucleaseHae III,gave two fragments 60 and 200 bp were identified as L.tropica and two fragments of 140 and 220 bp as L. major(Figure, 2).
Fig (4)Gel electrophoresis of PCR- RFLP products of Leishmania isolates using HaeIII enzyme on 4% gel agarose 1-7 and 9-11:Leishmania tropica isolates,8:50 bp DNA ladder marker 12 ,13,14: L. major
In the present study, 57 L.tropica ( dry skin lesion)and 25 L.major (wet skin lesion)were diagnosed with different diagnostic methods of CL. These results were in agreement with many studies, in Iraq [16, 3] and Afghnistan [10 ,12, 13] in Colombia  who found the dry more than wet lesions, but in contrast with other study done in Indiaby [ 15] and in America  reported that dry skin lesion less than wet skin lesion.
The high frequency of L.tropica in this and other studies may be due to the presence of reservoir animals in large number in some areas in Iraq especially rodents and dogs as reported by CDC  .
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