Next Generation Sequencing - TeachEpi · Next Generation Sequencing . McGill Advanced TB Diagnostic...
Transcript of Next Generation Sequencing - TeachEpi · Next Generation Sequencing . McGill Advanced TB Diagnostic...
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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