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 – 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: • 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 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 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 onnel tra ining and tra ns fer of Technologyt o Re ede emer’s University, validation and SOP development Sample collections i n the Afri can s ites Next ge ne ra tion seque nc ing : Viral nucleic ac id isolation ; Library Construction; Sequencing on MiSeqer’s at Redeem University and HiSeq at Harvard University.

Bioinf orm atic Ana lys is to ide ntify and chara cteri ze potential microbial causes of FUO Deve lop, va lida te, and implement a qP CR -HRM diagnostic panel of previously identified FUO-causings inorganism West Africa and new microbes identified in this project

WAGER 2013 Acknowledgement

WAGER 2013 Thank You Thank You WAGER 2013