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West Africa Genomics Research West Africa Ge nomics Research (WAGER) Network Characterizing Fevers of Unknown Origin (FUO) Through Microbial Metagenomics PI: Christian Happi Co-PI: Onikepe Folarin WAGER 2013 WAGER Network Redeemer’s University, Nigeria AFRICAN UNIVERSITY PARTNERS Redeemer’s University, Nigeria INTERNATIONAL Irrua Specialist Teaching Hospital, RESEARCH/TRAINING Nigeria Technology PARTNERS The Broad Institute of Harvard Universite Cheik Anta Diop, Transfer/Tr and MIT, Boston, USA Senegal aining/Sup port University of Sierra Leone, Sierra Harvard University, USA Leone WAGER 2013 Tulane University, USA OVERALL AIM Employ the power of new technologies and analytic methods for the benefit of patients and community caretakers. WAGER 2013 Objectives • Develop a rapid, field deployable, low cost and flexible diagnostic system for: – FUO causing diseases – Known drug resistance markers for malaria – Novel pathogens detected through high throughput metagenomic sequencing. • Develop Capacity in West African Institutions WAGER 2013 Develop Capacity in Genomics in West African Institutions – Transfer of technology in the area of genomics – Train personnel in area of genomics – Support and promote cutting edge genomics research on health in West Africa WAGER 2013 Introduction and Significance • High prevalence of infectious diseases in developing countries • All infectious pathogenic disease have common symptoms • Diagnosis possible only by culturing or targeted molecular approaches • This is challenging and problematic especially in developing countries WAGER 2013 • Better tool such as Microbial Metagenomics is therefore required for pathogen discovery in fever of unknown origin (FUO). – Is the unbiased characterisation of microbial nucleic acids. – does not require culturing. – No cloning. – a priori knowledge of the infecting microbes. WAGER 2013 • Better tool such as Microbial Metagenomics is therefore required for pathogen discovery in fever of unknown origin (FUO). – Is the unbiased characterisation of microbial nucleic acids. – does not require culturing. – No cloning. – a priori knowledge of the infecting microbes. • Microbial metagenomics have the potential to transform our understanding the disease pathogens in sub SaharanWAGER 2013 Africa Preliminary Studies: Lassa Fever • Study of host genetic determinants of susceptibility to Lassa fever in Nigeria and Sierra Leone – 600 venous blood samples were collected from febrile patients with no known origin of fever – 600 venous blood samples were collected from afebrile healthy controls – Next generation sequencing was carried out on a subset WAGER 2013 Clinical characteristics of healthy controls (afebrile) and patients with unexplained fever (febrile). WAGER 2013 Classification of sample content. Individual reads were normalized to 100,000 per sample and split into major taxa using a BLASTn query of GenBank and MEtaGenome Analyzer (MEGAN). WAGER 2013 Eukaryotic viruses identified in fever of unknown origin samples. Contigs were assembled using Trinity and genome coverage was calculated using Geneious. WAGER 2013 The Malaria Experience • WHO reports 25% decline in malaria death • This is due to interventions to reduce transmission such as field-deployable molecular barcode assays for diagnosis – Useful in clinical settings – Surveillance of parasite populations – Drug susceptibility status – Distinguish recrudescence from re infection in drug trials – Monitor frequency and distribution of specific parasites in a population. WAGER 2013 The Malaria Experience • WHO reports 25% decline in malaria death • This is due to interventions to reduce transmission such as field-deployable molecular barcode assays for diagnosis – Useful in clinical settings – Surveillance of parasite populations – Drug susceptibility status – Distinguish recrudescence from re infection in drug trials – Monitor frequency and distribution of specific parasites in a population. Determination of the optimal technological platform for application in field is required for effective and useful molecular barcode tool WAGER 2013 Illumina Eco system • Robust • Field-deployable • Low-cost • Compact • High performance • performs qPCR in less than 40 minutes The Eco Real-Time PCR System (Eco) • Lowest qPCR machine in the market. Field-deployable genotyping tool to identify and track P. falciparum parasites both from patient samplesWAGER 2013 and in the laboratory. Results • 106 SNPs were selected for assay development • qPCR-HRM assay were carried out • 74 single amplicon of expected size • 65 accurate detection of possible alleles from DNAs from Brazil WAGER 2013 Milestones • Milestone 1 To develop FUO Diagnostic and Sequencing capacity at Redeemers University. Initial Training at the Broad Institute In country validation Development of SOP WAGER 2013 • Milestone 2. Collect and characterize ~400 cases/year of FUO and healthy controls from Nigeria, Sierra Leone, and Senegal. Prioritizing FUO samples into acute febrile illness or extended fever without a defined diagnosis. WAGER 2013 • Milestone 3. Perform next-generation sequencing on FUO cases. Viral nucleic acid isolation . Library Construction. Sequencing on MiSeq at Redeemer’s University and HiSeq at Harvard University. WAGER 2013 • Milestone 4. • Perform Bioinformatic Analysis to identify and characterize potential microbial causes of FUO Demultiplexing and quality control Cleaning of reads Processing and de novo assembly of high quality non- human/non-contaminant reads Identification of known pathogens Identification of unknown pathogens WAGER 2013 • Milestone 5 Develop, validate, and implement a qPCR-HRM diagnostic panel of previously identified FUO-causing organisms in West Africa and new microbes identified in this project. Identify Pathogen-Specific Genomic Markers Primer and Probe Design Primer Validation Validate Diagnostic Array Applicability Using a Pathogen Test Panel at Broad Institute In-country application WAGER 2013 Potential Pitfalls And Contingency Plans Pitfalls Plans Low viral copy number HiSeq sequencing to achieve greater depth and assembly into long contigs; Construction of full length viral genomes by PCR-walking. Short acute phase Collection of blood by the consulting doctors; Use Transplex kit to amplify minute quantities of RNA Low numbers of bacteria in bloodstream Extract nucleic acids from larger blood volume Deplete host RNA Reduce host DNA Degradation of nucleic acids in blood Rapid sample processing and placed in samples guanadinium thiocyanite as quickly as possible; Isolated nucleic acid to be stored in Liquid Nitrogen. WAGER 2013 WAGER Network Members Members Country Christian Happi Nigeria Onikepe Folarin Nigeria Danny Asogun Nigeria Daouda Ndiaye Universite Cheik Anta Diop, Senegal Sheik Kahn University of Sierra Leone, Sierra Leone Donald Grant University of Sierra Leone, Sierra Leone Pardis sabeti Harvard/MIT, USA Robert Garry Tulane University, USA WAGER 2013 Where are we? • Documentation Process/Protocol development/Ethical approval • Kick off Jan 2014 WAGER 2013 Proposed Timelines Mileston es Y1 Y2 Y3 Y4 Y5 Pers onne l tra ini ng and tra ns fer of Technol ogy to Re ede emer’s University, validation and SOP development Sampl e col lections i n t he Afri can s ites Next ge ne ra tion s eque nc ing : Viral nuc leic ac id i sol at ion ; Library Construction; Sequencing on MiSeq at Redeemer’s University and HiSeq at Harvard University. Bi oi nf orm atic Ana lys is to ide nt ify and chara cteri ze pot ent ial microbial causes of FUO Deve lop, va lida te, and impl ement a qP CR -HRM di agnos tic panel of previously identified FUO-causing organisms in West Africa and new microbes identified in this project WAGER 2013 Acknowledgement WAGER 2013 Thank You Thank You WAGER 2013.
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