Kathleen England TB Diagnostics Advisor MSF-Access Campaign

The Future of DST Next Generation Sequencing

McGill Advanced TB Diagnostic Course June 17-19, 2019

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Evolution of Genomics

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DOI: 10.1097/WCO.0000000000000374

CRISPR

Advancing technology and reducing cost

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Science Innovation OLD “Sanger” Dideoxy-chain termination

NGS Fragmentation synthesis

Newest – Nanopore Technology Changes in electric current

Sequencing Applications

Approaches: Whole genome: complete DNA sequence, culture isolate (Surveillance) Target-based: selected genes, direct specimens (Clinical) 5

The NGS Movement

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• Platforms & Methods • Utility & workflows • Graded Mutations • Accuracy • Implementation

For DR-TB

Paradigm shift

Culture Based Testing Molecular Based Testing (Phenotypic) (Genotypic) cDST or pDST mDST or gDST

Research using Seq

Measured bacterial growth in the presence at a critical concentration of an anti-TB drug which correlates with a poor clinical outcome

Mutations linked to resistant phenotypes.

Solid/Liquid (MGIT) DST and MIC plate-based technologies

LPA, CBNAAT, DNA Chip or Array-based technologies

New Tools

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• Precision testing • Less infrastructure • Faster time to results • Direct specimens • Decentralization

Markers of resistance to anti-TB drugs

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Anti-TB Drug Gene Targets Isoniazid katG, inhA , ndh, aphC, oxyR, mshA, furA Rifampicin rpoB Ethambutol embB, aftA, embA, embC, ubiA Pyrazinamide pncA, rpsa, panD Streptomycin rpsl, rrs, gidB Amikacin/Kanamycin rrs, eis, whibB Capreomycin rrs, tlyA, eis, whibB Fluroquinolones gyrA, gyrB, mfpA, pstB, lfrA, corD Eth/Prothionamide ethA, ethR, inhA, ndh, mshA, furA p-aminosalycyclic acid thyA, dfiA, folC, ribD Cycloserine/Terizidone alr, ald, ddl, cycA Linezolid rrl, rplC Clofazamine mmpR (Rv0678) Bedaquiline mmpR (Rv0678), atpE, mmpL5, mmpS5, pepQ Delamanid ddn, fdg1, fbiA, fbiB, fbiC

• Know where to identify resistance- conferring mutations • (BOLD) Most common genes

Rifampicin resistance studies

Others outside of rpoB (81bp): V146F and I572F Silent Mutations: F506,T508,Q510,L511,Q513, F514,T525, A532, L533, P535

513, 526, 531 > 90% RR

cases

10 Slide courtesy of P. Miotto (2016)

Fluoroquinolone resistance studies

81% FQ mutations in QRDRs

gyrA Main Loci: 90, 91, 94 Non FQ-R: E21Q, S95T, G668D,G247S,A384V Silent: I614I, A830A

gyrB Main Loci: 461, 494, 499 Silent: T221T, V265V, A334A

11 Slide courtesy of D.Cirillo (2017)

The overall pooled sensitivity: 91% (87–94) for rpoB (rifampicin) 86% (74–93) for katG, inhA, and fabG promoter combined (isoniazid) 54% (39–68) for pncA (pyrazinamide) 85% (77–91) for gyrA/gyrB combined (ofloxacin/levofloxacin) 88% (81–92) for gyrA/gyrB combined (moxifloxacin)

Demonstrated a correlation in the estimated prevalence of drug resistance by sequencing and the estimated prevalence by phenotypic testing.

Zignol et al. (2018) Lancet Infect Dis.

WHO WGS DR-TB surveillance

Building an encyclopedia of mutations

Miotto et al. (2017) ERJ:50

Confidence grading>>

Grading: High Moderate Minimal None

C.Gilpin, Sequencing Webinar 2019

Prediction accuracy when including all mutations, even low confidence

Still accumulating data for LZD, CFZ, BDQ, DLM, etc

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Increasing knowledge to grade & interpret variants

customized microtitre plates

NGS Illumina

Bedaquiline

Delamanid

Clofazimine

Linezolid

Ethionamide

PAS

Levofloxacin

Moxifloxacin

Kanamycin

Amikacin

Capreomycin

Pyrazinamide

Ethambutol

Rifabutin

Rifampicin

Isoniazid

• 100,000 isolates: MDR, pre/XDR (6600 currently) • Perform WGS / MICs • Link data: pDST, MIC, genetic variants

for grading and interpretation

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Target- based (tNGS): Genoscreen Deeplex MycTB

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• Target amplification from direct sputum sediment = FAST

• A 24-plex amplicon preparation before sequencing = FOCUSED

• Identifies species, genotype, and drug resistance profiling for 18 genes targets = FUNDAMENTAL

24 gene targets amplified

Only 24 genes sequenced Analysis/ Report

Deeplex MycTB – Sample Sequencing Report

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Translate output into useful

clinical reporting (sample template)

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Progress toward global standardization

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• Living database/analysis tool (WHO ReSeq, 2019) • Analytical evaluation end-to-end solutions (FIND, 2019) • Clinical trials using end-to-end solutions (FIND, 2020) • Guidance/Recommendations for Routine Use (WHO, 2021) • Quality Assurance Measures (SRLN, 2021)

Living database with a cloud-based analysis tool

Sputum Result

2-3 days $40-50/test

Utility for programs

CONSIDERATIONS: pDST, preference for oral

route, DR prevalence, previous treatment,

tolerability, drug-drug interactions, severe forms

STANDARDIZED SHORTER MDR-TB REGIMEN 4-6 Amk-M-Pto-Cfz-Z-Hhd-E / 5 M-Cfz-Z-E

TB-POSITIVE Rif-R

(Pre/ XDR-TB)

TREATMENT FOR DRUG SUSCEPTIBLE TB

(2HRZE/4HR)

RAPID DIAGNOSTIC (XPERT MTB/RIF)

LPA-SL PRECISION TREATMENT

LONGER MDR-TB REGIMEN SOCIAL SUPPORT & COUNSELLING

CENTRAL DATABASE FEED-BACK LEARNING LOOP FOR

ADJUSTMENT OF DX, RX, APPROACHES

DRTB- SURVEILLANCE

DRUG PROCUREMENT

Mario C. Raviglione Global Health

Adapted from

TARGETED SEQUENCING ACCESSIBLE

DIAGNOSIS (upfront in specific settings)

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Considerations

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• Benefit to testing? • Implement or outsource testing? • Strategic plan, budget, commitment • Algorithm- Who to test and when? • Pre-implementation preparations? • Anticipated challenges? • Cost implications? • Desired impact or change?

Can we move to sequencing for DRTB? “Absolutely” - Yes we can!

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