Leishmaniasis is a protozoan disease that, second only to malaria in numbers of fatalities, afflicts millions of people worldwide. It mainly distributed in East Africa, South Asia, South America, and Mediterranean Region, with an estimated 1.5 to 2 million cases in each year (Torres-Guerrero, E., et. al., 2017). Leishmaniasis is caused by species of parasites of the genus Leishmania. The common forms of leishmaniasis, such as cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (ML), and post-kala-azar dermal leishmaniasis (PKDL), visceral leishmania (VL). The parasite transmitted to humans by the bite of a sand fly, mainly Phlebotomus and Lutzomyia.
The infection may also transmitted by other means such as needle sharing, blood transfusions or from mother to child during pregnancy. (Fran§a, A.O., et. al.,2018) The diagnosis of leishmanial infection is being done through molecular biology based techniques like PCR amplification, Serological tests based on Immunoflourescent antibody test (IFA) and ELISA (Enzyme Linked Immunosorbant Assay), are classical methods used in detection of Leishmania specific antibody. rK39 dipstick method are also introduced for detection.
It’s immunochromatography strip which immobilized the antigen. MAJOR OBJECTIVE To Study the ANTI-LEISHMANIAL ACTIVITY of A NOVEL – COMPOUND and to DETERMINE The MODE of CELL Death by this compoundSPECIFIC OBJECTIVE To determine the IC50 of the drug both In-vitro & Ex-vivo. To measure the Level of TH1 (TNF-±) & TH2 (TGF-џ) cytokine. EXPERIMENTAL OUTLINECulture of Leishmania strain in M199 mediaCheck Cell Viability by Dye Exclusion Method & MTT Asaay(In Vitro Method)Determination of IC50 from the in-vitro asaayROS Generation Assay after treatmentDetermination of Mode of Cell Death by DNA Fragmentation AssayPresence of Phosphatidyl Serine on Outer-leaflet of the apoptotic cells by FACSExpression level of Stress Responsive Enzyme (Trypanothione Reductase & Trypanothione Synthetase)(Treated & Untreated cell Total RNA isolation cDNA preparation Check for expression of two stress responsive enzymes)Macrophage Infection Assay( Isolation of Starch induced PEC from mice Macrophage Cytotoxicity Assay by our compound Infection of macrophage with parasite Afterthat,treatment with compound and determine the parasite load after 72 hours of treatment)ELISA(TNF-± ; TGF-џ ; IL-10) REVIEW OF LITERATUREHistory of leishmaniasis dates back to first century A.D. As evidence of cutaneous and mucocutaneous leishmaniasis from pre-Incan pottery from Ecuador and Peru displayed depictions of skin lesions and facial deformities in a first century AD. During 15th and 16th century texts from the Inca period and from Spanish colonials mention “valley sickness”, “Andean sickness”, or “white leprosy”, which are likely to be CL, noted the presence of skin lesions on agricultural workers returning from the Andes. In the mid-18th century, describe the disease now known as visceral leishmaniasis, as kala-azar or black fever in Africa and India in 1756, Alexander Russell referred this disease as Aleppo boil as it leaves an ugly scar of livid colour after being cicatrised, which remains through life, and seldom give much pain.The disease became known as Leishmaniasis after William Leishman, a Glasgwegian doctor seving with the British Army in India, developed one of the earliest stains of Leishmania in 1901. Leishman discovered ovoid bodies in the spleen of a British soldier who was experiencing bouts of fever, muscular atrophy, anaemia and swelling of the spleen in Dum Dum, a town near Calcutta. In 1903, Leishman published his findings and described this illness as dum dum fever(Steverding, D., et. al., 2017). After few week of Leishman published his discovery, Charles Donovan also recognized these symptoms in other kala-azar patients. These amastigotes were known as Leishman-Donovan bodies and this species became known as, L. Donovani after examining the parasite by using Leishman’s stain. By linking this protozoan with kala-azar, Leishman and Donovan discovered the genus, Leishmania.(Gibson, M.E., et. al., 1983)LEISHMANIASIS- Leishmaniasis is a “vector-borne” disease. Its infection is transmitted by the blood sucking female sandflies of family Diptera: Psychodidae: Phlebotominae of the genera Phlebotomus and Lutzomyia. Dogs, rats, humans, cattle, and sloths can act as hosts for Leishmania parasites. Leshmania is endemic in more than 60 countries worldwide, including Southern Europe, the Middle East and North Africa, Central and South America and the Indian subcontinent. Leishmaniasis consists of four main clinical syndromes: cutaneous leishmaniasis; muco-cutaneous leishmaniasis (also known as espundia); visceral leishmaniasis (VL; also known as kala-azar); and post-kala-azar dermal leishmaniasis (PKDL) (Chappuis., et al.,2007).. TAXNOMIC HIERARCHY-The classification of Leishmania was monothetic Linnean classifications that were proposed between 1916 and 1961, based on extrinsic characters only. In 1908, Leishmania classification was suggested by Nicolle who separated L. infantum, the etiological agent of Mediterranean visceral leishmaniasis, from the causative agent of Indian kala azar. (Akhoundi M., et. al., 2016.) Kingdom : Protista Subkingdom : Protozoa Phylum : Sacromastigophora Class : Zoomastigophora Order : Kinetoplastida Genus : LeishmaniaEPIDEMIOLOGY OF VISCERAL LEISHMANIASIS- The disease mainly affects poor people in Asia, Africa and Latin America, and is associated with malnutrition, poor housing, weak immune system, population displacement and lack of resources. Out of 200 countries and territories reporting to WHO, 97 countries and territories are endemic for leishmaniasis in 2017. This includes 65 countries that are endemic for both VL and CL, 10 countries that are endemic for VL only and 22 countries that are endemic for CL only. Four additional countries have previously reported cases of VL or CL.Since 2013, the WHO Global Leishmaniasis programme has been reporting separately the number of new autochthonous cases, to monitor the trends in incidence, and the number of imported cases.