Next-generation Sequencing for Drug resistant TB: Webinar Series, February 5, 2019

Implementing WGS and Culture-free NGS: An Overview of Challenges and Solutions

Timothy C. Rodwell MD, PhD, MPH & the FIND NGS TeamSenior Scientific Officer (FIND)

Associate Professor of Medicine (UCSD)

Topics to be covered

Benefits of Culture-free Sequencing for Drug Susceptibility

Testing (DST)

The Target Product Profile for NGS Diagnosis of DR-TB

NGS Workflow challenges using primary clinical samples

Bioinformatics challenges

Regulatory challenges

Implementing NGS for Diagnosis of Drug-resistant TB

Phenotypic Drug Susceptibility Testing

Culture 6-9 WEEKS Drug Susceptibility Testing

Phenotypic Drug Susceptibility Testing

Slow Significant hands-on time

Significant biosafety hazards Requires specialized laboratories

Min potential for costs reductions Not optimal for national/global networking

Cx-Free Next Gen Sequencing Drug Susceptibility Testing

NGS Drug Susceptibility Testing

Rapid Huge potential for automation

Minimal biosafety hazards Benchtop workflow

Rapidly decreasing costs (batching, tech,

reagents)

Designed for national/global networking

2-3 DAYS

Minimal acceptable characteristics

Direct from sputum sample (culture-free, no BSL3)

Setting: Central TB Reference Laboratory (Level 3)

• Optimal: District hospital (Level 2)

Detect primary 1st and 2nd line drug resistance mutations (XDR+PZA)

• Open design – expandable to new mutations (e.g. Delamanid, Bedaquiline) and additional

features (surveillance, Tx monitoring)

Cost should be <60 USD (library prep + seq) per test (WGS 100 USD)

LOD: tNGS - 5,000 Mtb genomes (Scanty AFB ~103 genomes/mL); WGS - 2x105 Mtb genomes

• Optimal: 100 genomes

Heteroresistance detection: 500-R/4500-S (10%)

• Optimal: 1-R/99-S

https://www.who.int/tb/publications/2018/WHO_technical_guide_nextgen_sequencing/en/

Target Product Profile for detection of resistance associated mutations in

Mycobacterium tuberculosis complex utilizing Next Generation Sequencing

1. Uncertainties in predicting DR phenotypes from genotypes

2. Difficulty of sequencing from direct clinical samples

3. Complexity of workflows and high costs

4. Expertise required for analysis and clinical interpretation

5. Lack of regulatory standards and guidance

Challenges to Global Implementation of NGS for DR TB Diagnosis

Source: https://academic.oup.com/jac/article/66/7/1417/786529

Predicting XDR-TB Phenotypes with mutations in M. tuberculosis

Overcoming the Challenges

Drug resistance is acquired through spontaneous mutation

• No horizontal gene transfer

Clonal expansion

Mutations in <15 genes confer >90% of M/XDR-TB (high sensitivity)

• MICs

• Mixed populations

Expression of resistance is reliable (high specificity)

A DNA Signal to Noise Issue

Image Source: DNA molec image: https://www.huffingtonpost.com/anne-wojcicki/science-education-fuels-i_b_3149540.html

Sequencing from Direct Clinical Samples - Challenge

Image Source: Global Laboratory Initiative (GLI)

Sequencing from Direct Clinical Samples - Solutions

Enriching the TB DNA Signal

WGS Targeted NGS

Early MGIT Cx ( adds 1-5 days)

Multiplex PCR (1-4 hours)Differential lysis (in development)

RNA Baiting (adds sig cost & time)

Technological Complexity of NGS - Challenge

Instruments shown are for demonstration purposes only and not intended to indicate endorsement

Stand-Alone or Cloud-Based

Software & KnowlegebaseDNA Extraction Instrument

(automated)

Sequencer

(automated) Library Preparation

(manual)

Sputum sample

DNA extraction Library preparationTargeted

sequencingData analysis & interpretation

Standardized Result Report

Technological Complexity of NGS - Solutions

Instruments shown are for demonstration purposes only and not intended to indicate endorsement

Cost of Goods

Targeted NGS - $50-$100 per sample

WGS - $150-$200 per sample

Cost of NGS – Challenges and Solutions

DNA Extraction Instrument

(automated)

Sequencer

(automated) Library Preparation

(manual)

• Compute intensive

• Requires high-level expertise

• QA/QC and validation

Ezewudo et al. Sci Rep. 2018 Oct 18;8(1):15382. doi: 10.1038/s41598-018-33731-1

Bioinformatics - Challenges

genTBPhyResSE

TB Profiler

Bioinformatics - Solutions

Source: Tornheim et al. Clin Infect Dis – in final revision

Standardized Clinical Reporting

Phenotypic Data

Clinical Data

SNPs

• Fully automated processing and analysis of Mtb sequences

• Integration of new genotypic, phenotypic & clinical data

• Iteratively improved interpretation and standardized reporting

for surveillance and clinical diagnosis

Bioinformatics - Solutions

• ReSeqTB pipeline and knowledgebase being transferred to cluod

under WHO authority (ReSeqWHO - April 2019)

• Platform for global DR TB Surveillance program

• Data analysis, management and reporting

• Knowledgebase – genotypic and phenotypic data

• Eventually diagnostics (2020)

• Country agreements on data-sharing already in place

• Interoperability and data-sharing with key TB databases

• CRyPTIC, EBI (2021)

• Living knowledgebase

• Periodic review of confidence-graded mutations by

international community of TB experts (1st Review 2020)

• Inform strategy and policy for NGS (ongoing)

ReSeqWHO

Bioinformatics - Solutions

The Regulatory Landscape

NGS-based Dx of DR-TB falls under the category of In Vitro

Diagnostics (IVD)

The first regulatory guidance for NGS IVDs was released by FDA in

2018 for Dx of Germline Diseases

EMA guidance regarding how NGS IVD solutions will be handled under

the new IVDR regulations is still evolving

Country-specific guidance on regulation of NGS IVD solutions is still

evolving

Regulatory Environment – Challenges

FIND has been working closely with WHO and global

partners to build a foundation of knowledge and evidence

to support WHO technical guides, guidelines and

eventually policy

• Existing Guides/Reports

- Interpreting mutations conferring resistance to TB

drugs

- NGS for detecting drug-resistance conferring

mutations

• Future

- WHO endorsed list of mutations for Dx DR-TB (2020)

- Policy for use of NGS for diagnosis of DR-TB (~2021)

Regulatory Environment – Solutions for the Global Market

FIND global market strategy for NGS as Dx for DR-TB

• Seek WHO endorsement of commercial end-to-end NGS solutions

• Submit WHO endorsed solutions to the Global Drug Facility (GDF)

catalog for listing and purchase in LMICs with Global Fund/domestic

support

Proposed path to GDF listing

• Complete analytical evaluation of existing commercial, culture-free, end-

to-end NGS solutions for Dx of DR-TB (2019)

• Conduct a clinical trial of end-to-end solutions meeting TPP criteria

(2020)

• Submit trial data to WHO for review (2021)

• Submit solutions receiving WHO endorsement to GDF catalog (~2021)

Instruments shown are for demonstration purposes only and not intended to indicate endorsement

Regulatory Environment – Solutions for the Global Market

Where we will succeed

1. WHO DR TB surveillance will be based largely on NGS

2. 1-3 sample-to-answer targeted NGS solutions for Dx DR TB potentially endorsed by WHO

3. Cost of goods (COGs) in high-throughput labs in LMICs: $40-$50 per sample

4. Time from sample-to-answer of ~48hrs

5. There will be a global network of NGS data for DR TB

By 2022

Where we will need to continue to work on

1. Low TB DNA yield in direct clinical sample NGS

2. Genetic markers of resistance for novel and repurposed TB drugs

3. Data sharing

THE FUTURE of Cx-free NGS for Surveillance and Diagnosis of DR TB

“Most people overestimate what they can do in one

year and underestimate what they can do in ten years.”

Bill Gates

THE FUTURE

Swiss Development and Coop eration;State Secretariat for Education, Research andInnov ation

Neth erlands Enterprise Ag ency

Funding and Partners

FIND TB NGS Team

Anita Suresh

Rebecca Colman

Swapna Uplekar

Claudia Denkinger

Ranga Sampath

Thank you