التشخيص الجزيئي والتحليل الوراثي لـ5.8s rDNA جين لداء الليشمانيات الجلدي المعزول من مرضى في كربلاء المقدسة

Molecular diagnosis and phylogenetic analysis of 5.8s rDNA of gene of cutaneous leishmaniasis isolated from patients in holy Karbala

Abstract

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. There are about 12 million cases worldwide, and there are about 1.5 million new cases of cutaneous leishmaniasis each year.
Cutaneous leishmaniasis (CL) is a major public health problem and an endemic disease in Iraqi population, so this study was conducted to find the best method for the diagnosis, by “Molecular diagnosis and phylogenetic tree analysis to detection of the cutaneous leishmaniasis species in Karbala city by Nested PCR.
Results of laboratory with microscope examination of one hundred patients cases showed (92) cases were infected while (8) is not in Cutaneous leishmaniasis that 72 males that percentage (78.3)% and 20 females that percentage (21.7)% with age ranged 10-59 years, attended to Department of Dermatology Al-Husseini Hospital in Karbala during the period between November 2017 to end January 2018.
The most infected rate of patients was between the ages of 40-49 years (40.2%), while the lowest infection rate was of patient’s ages 10-19 years (6.5%).
The most infected rate of occupation patients in solder was 34 (36.9%), while the lowest infection rate in kids was 3 (3.2%), and the Most infected rate of patients was found in hands 44 (47.8) while the lowest infection rate was in Legs 11(12%).
The geographic distribution of the patients shows that 62% of the patients were from the cities of Telaefar and Dialla, while only 30% were from Karbala city. The number of lesions differs between patients. Most (65%) of the patients had a multiple lesions in comparison to the single lesions which was found in (35%) of patients.
The direct microscope examination was performed in two methods aspiration and direct microscope examination then stained with Leishman stain. Then, the positive samples were cultured on the modified Novy-MacNeil-Nicolle medium from (15 – 30) day and then active in Roswell Park Memorial Institute medium). Only 20) sample growth.
A more specific diagnosis of the causative agent of cutaneous leishmaniasis was performed using molecular tests. Choosing 20 sample of the positively cultured samples, of the total number of pathogens that
Showed positive results were studied by Nested-PCR using two types of primers to determine the species. Because large 5.8s.r DNA gene through Nested PCR showed that all the samples were L.major.
The Nested PCR products of twelve isolates send to sequenced in the South Korea then compared the local species which isolated from current study with the global strains in the NCBI gene bank then recorded choose rendomly isolate K1.
It was concluded that CL is highly spread with multiple lesions more than single lesions of Cutaneous leishmaniasis diverse clinical manifestation “is prevalent in karbala city, and the molecular detection showed L.major was the main species causing cutaneous leishmaniasis in comparing with L.tropica in the present study, Nasted-PCR is a reliable method for the diagnosis and identification of Leishmania species because high specific and sensitive can applied in epidemiologic investigations, All L.major isolates in Karbala city is one isolate for sequencing 5.8s.rDNA gene and result of the (12) sequence where all similar genetically with very little change is not significant and then compare with global strain in the NCBI gene bank were similar genetically (one isolated) with Iranian isolated , finally Iraqi L.major isolate was genetically closest with Iranian L.major isolates because Iraq and iran are on the same geographic line and genetically distant for Chinese isola