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Identification of Plasmodium Falciparum MSP-1 Variants in Malaria sequential infections YAA Reuben Mangi (I56/ 81536/ 2012) Bsc. University of Nairobi (2012) Supervisors Dr. Isabella Oyier, Wellcome- Trust, Kenya Medical Research Institute (KEMRI), Kilifi Dr. George Obiero, Centre for Bioinformatics and Biotechnology (CEBIB) University of Nairobi Introduction Malaria is an intermittent and remittent fever disease caused by a species in the genus Plasmodium. Malaria thrives in the tropical areas of Asia, Africa, and Central & South America, where it infects millions of people. Figure 1. Malaria Victim( National Geographic) There are approximately 219 million malaria cases and mortality rates of approximately 1 million cases a year in the world. 28 million Kenyans living in malaria risk regions and majority being children. Introduction Cont.. • The erythrocytic stages of the parasite life cycle are responsible for all symptoms and pathologies of the disease. • The symptoms are; fever, headache, malaise, fatigue, muscular pains, and occasionally nausea, vomiting and diarrhoea. Figure 2 Malaria Parasites Amid Red Blood Cells (National Geographic) • Sequestered RBCs adhere to the blood vessels resulting to destruction and blockage of vessels leading to hypoxia and toxemia in vital tissues and organs. Life cycle of Plasmodium falciparum The parasite undergoes a complex life cycle that requires both a human host and a female Anopheles mosquito. Figure 3. Life cycle of P. falciparum (Nature) Molecular basis of invasion The surface is comprised of Glycosyl Phosphatidyl Inositol (GPI) anchored membrane proteins(Ligands) and their associated partners. These include Merozoite Surface proteins(MSP), Apical Membrane Antigen1(AMA-1), The Reticulocyte Binding Homologue proteins (RBLs), Erythrocyte Binding like proteins (EBLs). Other peripheral proteins involved in primary contact also exist. Well characterized EBLs are, EBA-175, EBA-140 and their receptors are glycophorin A, Glycophorin C respectively. Figure 4: Fine structure of Plasmodium falciparum Merozoite (Cowman and Crabb 2006) Invasion of Erythrocytes by Plasmodium falciparum Merozoite Figure 5: Diagram of RBC invasion -(Cowman and Crabb 2006) A process that involves is multi-step sequence of initial recognition, reorientation and entry. The contact requires interactions of merozoite surface proteins (MSP) and specific receptors on the host cell. Immunity to Invasion by Merozoites Clinical immunity to malaria is slow to develop and short lived. One reason for this is the extensive diversity found in Plasmodium antigens. Merozoites are among the parasite stages targeted by immune system. The merozoite stage represents an attractive target for controlling malaria due to its uniqueness towards its association with the disease clinical symptoms. The merozoite surface proteins(MSP) are the major targets of immune attack by antibodies. The well characterized MSP is MSP-1. Other MSP gene families exist. Plasmodium falciparum MSP-1 gene • Located at the middle of chromosome 9 • Organized into blocks • Its polymorphic and genetically diverse The product of this is MSP-1, a high-molecular-weight precursor which is then processed into several fragments . At the time of red cell invasion, only the 19-kDa C-terminal fragment remains on the surface. and is carried into the invaded erythrocytes. Since MSP-1, can elicit immune responses in natural malaria infections, and is genetically diverse containing multiple polymorphisms in P. falciparum it will be considered in sequential infections in this study. Research Question Are there Plasmodium falciparum MSP-1 polymorphic differences in sequential clinical malaria infections Hypothesis There are no Plasmodium falciparum MSP-1 polymorphic variants in sequential cases of clinical malaria infections. General Objective Determine MSP-1 19kDa genetic variants in parasite obtained from individuals with multiple malaria sequential infections. Specific Objectives • Identify MSP-119kDa gene polymorphisms in sequential clinical malaria infections. • Determine allelic changes in individuals across the sequential infections. • Determine the population proportion of allele frequencies between infections. Justification • Malaria parasite resistant to drugs. • Vector resistant to insecticides. • Developing a vaccine towards malaria would be the most practical way to arrest this quagmire. However, efforts that are underway have been unsuccessful. • This work will determine the frequency of MSP-119kDa gene polymorphisms and allele prevalence in individuals with multiple sequential infections and contribute to the knowledge of MSP-1 diversity which is useful towards the development of malaria vaccine merozoite candidates. Methodology Study design: This will be Longitudinal study where MSP-1 variable will be measured in all subjects in the cohort Study site The lab work will be conducted at Centre for Biotechnology and Bioinformatics (CEBIB) molecular labs in Nairobi and at ILRI. Study population The target group will be composed of 33 children aged between 0 and 5 years who had been followed up weekly with over 9 malaria infection episodes after visiting a dispensary in Junju, Kilifi County. Ethical clearance Ethical clearance for this study has already been obtained from the KEMRI ethics review board for integrated studies of natural immunity to malaria. . Amplification and sequencing of MSP-119 kDa genes PCR • Optimization • Gene specific primers for MSP-1 19KDa, High Fidelity PCR Taq (Roche) and Controls (3D7) • Gel electrophoresis Samples run in batches Gel preparation using Cyber Safe® and view by Gel Doc BigDye PCR • • • Sequencing Amplicons will be purified using ethanol precipitation. Cycle sequencing PCR. Analysis by DNA analyser machine (Applied Biosystems) Data Analysis • • • Quality checking and contig assembly using BioEdit, DNASTAR, CLC bio softwares. Alignment to identify Nucleotide polymorphisms Gene polymorphisms frequencies by R Timeline Budget Acknowledgements: Parwos Abraham: CEBIB admin Colleagues Identification of Plasmodium Falciparum MSP-1 Variants in Malaria sequential infection YAA Reuben Mangi (I56/ 81536/ 2012) Bsc. University of Nairobi (2012) THANK YOU