Collaborative Trials in HIV-1, HBV, HCV and CMV Resistance Testing - - PowerPoint PPT Presentation

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AREVIR-Meeting Cologne, 03-04 May 2019

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Collaborative Trials in HIV-1, HBV, HCV and CMV Resistance Testing and Genotyping

• M. Kammel2,4, A. Zimmermann2, V. Lindig2, H.-P. Grunert3, H. Zeichhardt1,2,3,4

and

• M. Schütze5 , R. Ehret5 and M. Obermeier5,

1Professor of Virology (i.R.)

Charité - Universitätsmedizin Berlin

2IQVD GmbH - Institut für Qualitätssicherung in der Virusdiagnostik 3GBD Gesellschaft für Biotechnologische Diagnostik mbH, Berlin 4INSTAND e.V., Düsseldorf 5Medizinisches Infektiologiezentrum Berlin

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Disclosure

• Prof. Dr. Heinz Zeichhardt is share-holder
• f

GBD Gesellschaft für Biotechnologische Diagnostik mbH, Berlin and IQVD GmbH - Institut für Qualitätssicherung in der Virusdiagnostik, Berlin

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• INSTAND e.V.
• INSTAND External Quality Assessment (EQA) Schemes for Virus Genome

Detection: (383) HIV-1- Drug Resistance Determination of Protease and Reverse Transcriptase Inhibitors => Estimation of susceptibility according S/I/R-scheme (384) HIV-1- Drug Resistance Determination of Integrase and Determination of Tropism (396) Hepatitis B Virus Genotyping (397) Hepatitis B Virus Resistance Determination => Harmonization of the interpretation algorithms (375) Hepatitis C Virus Geno- / Subtyping (399) Hepatitis C Virus Resistance Determination (349) Cytomegalovirus Resistance Determination

• Summary

Outline

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INSTAND EQA schemes performed in cooperation with

(383, 384) HIV-1 resistance and tropism determination

NRZ für Retroviren: O.T. Keppler, J. Eberle, L. Gürtler Friedrich-Alexander-Universität Erlangen-Nürnberg: K. Überla, K. Korn IMD Medizinisches Versorgungszentrum Frankfurt: M. Stürmer Medizinisches Infektiologiezentrum Berlin: M. Obermeier, M. Schütze Uniklinik Köln: F. Klein, R. Kaiser, E. Heger, E. Knops Universitätsklinikum Frankfurt: V. Kempf, H. F. Rabenau, A. Berger

(396) HBV genotyping

Paul-Ehrlich-Institut: M. Nübling, M. Chudy, J. Kreß NRZ für HBV u. HDV: D. Glebe, C. Schüttler, H. Slanina, W. Gerlich, J. Ziebuhr

(397) HBV resistance determination

NRZ für HBV u. HDV: D. Glebe, C. Schüttler, H. Slanina, W. Gerlich, J. Ziebuhr

(375) HCV geno-/ subtyping

NRZ für Hepatitis C Viren: S. Ross Universitätsklinikum Düsseldorf: J. Timm, O. Adams, N. Lübke

(399) HCV resistance determination

NRZ für Hepatitis C Viren: S. Ross Universitätsklinikum Düsseldorf: J. Timm, O. Adams, N. Lübke

(349) CMV resistance determination

Universitätsklinikum Ulm,Institut für Virologie Konsiliarlabore für Cytomegalievirus (CMV): Universitätsklinikum Ulm: T. Stamminger (ab 01/2018), D. Michel,

• T. Mertens (bis 12/2017)

Universitätsklinikum Tübingen: T. Iftner (ab 04/2018), K. Hamprecht,

• G. Jahn (bis 03/2018)

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International 14 Organisations / Scientific Societies National 28 Organisations / Scientific Societies

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INSTAND e.V.

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INSTAND e.V.

EQAS in virus diagnostics - 2018:

• > 1500 participating laboratories
• from > 50 countries worldwide

(71)

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IQVD INSTAND EQA Schemes (71 Schemes) in Virus Immunology and Genome Detection 2019

Serology and Antigen Detection Virus Genome Detection and Typing

HIV-1/2 Herpes simplex Viruses HIV-1 (RNA) HIV-2 (RNA) Measles Rubella Mumps Viruses HIV-1 p24 Ag Varicella Zoster Virus HIV-1 Resistance + Tropism Adenoviruses HTLV-1/2 Epstein Barr Virus Hepatitis A Virus Norovirus Rotavirus Hepatitis A Virus

• Resp. Sync. Virus Ag

Hepatitis B Virus + Genotyping + Resistance Coronavirus

• incl. MERS CoV

Hepatitis B Virus, Prg. I Influenza A and B Ag

+A/H1N1 pdm 2009 +A/H5N1+A/H7N9

Hepatitis C Virus + Genotyping + Resistance Enteroviruses + Enterovirus (WHO/RKI) Hepatitis B Virus, Prg. II Rubella Virus Measles Virus Mumps Virus Hepatitis D Virus Hepatitis E Virus Human Rhinoviruses

• Resp. Syncytial Virus
• Hum. Metapneumovirus

Parainfluenza viruses Hepatitis C Virus TBE Virus Cytomegalovirus + Resistance Influenza A and B Ag

+A/H1N1 pdm 2009 +A/H5N1+A/H7N9

Hepatitis D Virus Hantavirus Epstein-Barr Virus BK Virus JC Virus Hepatitis E Virus Dengue Virus Chikungunya Virus Zika Virus Herpes simplex Viruses Dengue Virus West Nile Virus Chikungunya Virus Zika Virus Parvovirus B19 Rabies Virus Varicella Zoster Virus

• Hum. Papilloma Viruses

Cytomegalovirus BSE (PrPsc) (2002-2007) Parvovirus B19 Rabies Virus NEW - Multiplex: Respiratory viruses (1 + 2) NEW - Multiplex: Gastrointestinal viruses

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• A. Baillot

Niedersächsisches Landesgesundheitsamt, Hannover

• N. Bannert

Robert Koch-Institut, Berlin

• S. Becker / C. Keller / M. Eickmann

Philipps Universität Marburg, Nat. Konsiliarlab. Filoviren

• C.-T. Bock / S. Niendorf / S. Jacobsen / A. Mas-Marques

Robert Koch-Institut, Berlin; Nat. Konsiliarlab. Noroviren; Nat. Konsiliarlab. Rotaviren

• S. Diedrich / S. Böttcher

Robert Koch-Institut, Berlin; NRZ Poliomyelitis und Enteroviren Regionales Referenzlabor der WHO/EURO für Poliomyelitis

• U. Dittmer / S. Ross

Universitätsklinik Essen; NRZ HCV; Nat. Konsiliarlab. Tollwut

• G. Dobler / J. Bugert

Institut für Mikrobiologie der Bundeswehr, München; Nat. Konsiliarlab. FSME

• Ch. Drosten / J. Hofmann / V.M. Corman

Charité Universitätsmedizin Berlin, CCM;

• Nat. Konsiliarlab.Hantaviren; Nat. Konsiliarlab. Coronaviren;

Labor Berlin – Charité Vivantes GmbH

• R. Dürrwald / B. Biere

Robert Koch-Institut, Berlin; NRZ Influenza

• A.-M. Eis-Hübinger

Universität Bonn

• G. Enders / M. Enders

Labor Enders, Stuttgart

• J. Fuhrmann

MVZ Labor 28 GmbH, Berlin

• A. Gessner / B. Schmidt / J. Wenzel / A. Plentz

Universität Regensburg; Nat. Konsiliarlab. HAV und HEV

• S. Günther / J. Schmidt-Chanasit / P. Emmerich

Bernhard-Nocht-Institut, Hamburg; NRZ trop. Infektionserreger, WHO CC

• G. Harms-Zwingenberger

Institut für Tropenmedizin, Berlin

• H. Hengel / D. Huzly / M. Panning

Universitätsklinikum Freiburg; Nat. Konsiliarlab. HSV u. VZV

• J. Hugget / D. O’Sullivan

LGC, National Measurement Laboratory, London, UK

• T. Iftner / K. Hamprecht

Universitätsklinikum Tübingen; Nat. Konsiliarlab. CMV

• A. Karl / K. Frank / K. Gubbe

DRK-Blutspendedienst Nord-Ost, Plauen

• V. Kempf / H. Rabenau / A. Berger

Universitätsklinikum Frankfurt

• O.T. Keppler / J. Eberle / L. Gürtler / H. Nitschko

Ludwig-Maximillians-Universität München, NRZ Retroviren

• F. Klein /U. Wieland /S. Silling /R. Kaiser /E. Heger /E. Knops Uniklinik Köln; NRZ Papillom- und Polyomaviren
• H. Knechten / P. Braun / F. Wiesmann / G. Naeth

Labor Knechten, Aachen

• U.G. Liebert / M. Maier

Universität Leipzig

• P. Maes

KU Leuven, Belgium

• A. Mankertz / S. Santibanez

Robert Koch-Institut, Berlin; NRZ Masern, Mumps und Röteln

• T. Stamminger / D. Michel

Universitätsklinikum Ulm; Nat. Konsiliarlab. CMV

• S. Modrow

Universität Regensburg; Nat. Konsiliarlab. Parvoviren

• S. Nick / H. Scheiblauer

Paul-Ehrlich-Institut, Langen; Prüflabor für IVD

• M. Nübling / M. Chudy / S.A. Baylis / J. Kreß

Paul-Ehrlich-Institut, Langen, WHO CC

• M. Obermeier / R. Ehret / M. Schütze

Medizinisches Infektiologiezentrum, Berlin

• J. Reiche / R. Dürrwald

Robert Koch-Institut, Berlin; Nat. Konsiliarlab. RSV, Parainfluenzaviren, HMPV

• T. Schulz / A. Heim / W. Puppe / C. Schmitt

Medizinische Hochschule Hannover; Nat. Konsiliarlab. Adenoviren

• S. Smola / J. Rissland

Universitätsklinikum des Saarlandes

• J. Timm / O. Adams / N. Lübke

Universitätsklinikum Düsseldorf

• K. Überla / K. Korn

Universitätsklinikum Erlangen

• B. Weißbrich

Universität Würzburg

• J. Ziebuhr / D. Glebe / C. Schüttler / S. Slanina / W. Gerlich

Universität Gießen; NRZ HBV und HDV

INSTAND Expert Laboratories (total: 38)

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EQA schemes (383, 384) for HIV-1 Resistance Determination and Tropism Determination

• first EQA schemes: November/December 2014 (pilot EQASs)
• performed 1 x per year
• number of participants increased from 9 (pilot EQASs 2014) to 42 (term Nov/Dec 2018)
• cooperation partners:

Nationales Referenzzentrum für Retroviren Ludwig-Maximilians-Universität München Max-von-Pettenkofer Institut Klinische Virologie

• Prof. Dr. O. T. Keppler,
• Prof. Dr. J. Eberle,
• Prof. Dr. L. Gürtler

Friedrich-Alexander-Universität Erlangen-Nürnberg Universitätsklinikum Erlangen Institut für Klinische und Molekulare Virologie

• Prof. Dr. K. Überla,
• Dr. K. Korn

IMD Medizinisches Versorgungszentrum, Frankfurt PD Dr. M. Stürmer Medizinisches Infektiologiezentrum Berlin

• Dr. M. Obermeier,
• M. Schütze

Uniklinik Köln Institut für Virologie

• Prof. Dr. F. Klein,
• Dr. R. Kaiser,
• Dr. E. Heger
• Dr. E. Knops

Universitätsklinikum Frankfurt Institut für Medizinische Virologie

• Prof. Dr. V. Kempf,
• Prof. Dr. H. F. Rabenau,
• Prof. Dr. A. Berger

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EQA Schemes (383,384) for HIV-1 Resistance Determination and Tropism Determination Overview of Parameters

EQA Scheme Virus Genome Detection – HIV-1 Drug Resistance Determination – (383) Standard Program for Protease and Reverse Transcriptase Inhibitors Parameters for drug resistance determination of protease and reverse transcriptase inhibitors (10) Evaluation of the sequence quality (20) Determination of resistance-associated mutations (30) Interpretation of antiretroviral drug resistance / drug sensitivity EQA Scheme Virus Genome Detection – HIV-1 Drug Resistance Determination – (384) additional Program for Integrase Inhibitors and Tropism Determination Parameters for drug resistance determination of integrase inhibitors (10) Evaluation of the sequence quality (20) Determination of resistance-associated mutations (30) Interpretation of antiretroviral drug resistance / drug sensitivity Parameters for genotypic determination of tropism (40) Evaluation of sequence quality (50) Determination of V3-loop amino-acid sequence (60) Interpretation of tropism

Evaluation in consecutive steps according to genotypic HIV-1 resistance determination

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HIV-1 Resistance panels for PR and RT Inhibitors (383) plus INT Inhibitors and Tropism Determination (384)

(383) HIV-1 Resistance Standard Program (384) HIV-1 Resistance Additional Program Source Concen- tration Subtype PR inhibitors RT inhibitors INT inhibitors Tropism Mixture wt / mut Susceptibility: S / I / R X4 R5 Virus (in plasma) ≥ 10 000 copies/ml A X n.a. B X X X X n.a. C X X n.a. D X n.a. F X X X n.a. G X X n.a. Plasmid (in water) n.a. B X X 0 / 100 X X 50 / 50

Different HIV-1 viruses (cell culture, inactivated), plasmids and plasmid mixtures were used in the EQA schemes for HIV-1 resistance determination

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EQA Schemes (383,384) for HIV-1 Resistance Determination and Tropism Determination Overview of Parameters

EQA Scheme Virus Genome Detection – HIV-1 Drug Resistance Determination – (383) Standard Program for Protease and Reverse Transcriptase Inhibitors Parameters for drug resistance determination of protease and reverse transcriptase inhibitors (10) Evaluation of the sequence quality (20) Determination of resistance-associated mutations (30) Interpretation of antiretroviral drug resistance / drug sensitivity EQA Scheme Virus Genome Detection – HIV-1 Drug Resistance Determination – (384) additional Program for Integrase Inhibitors and Tropism Determination Parameters for drug resistance determination of integrase inhibitors (10) Evaluation of the sequence quality (20) Determination of resistance-associated mutations (30) Interpretation of antiretroviral drug resistance / drug sensitivity Parameters for genotypic determination of tropism (40) Evaluation of sequence quality (50) Determination of V3-loop amino-acid sequence (60) Interpretation of tropism

Evaluation in consecutive steps according to genotypic HIV-1 resistance determination

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Sequence Quality: a Brief Guide

From sequence to quality: objectives

• Rough estimate for feedback to participants
• Need of a ‘reference’ to compare submitted sequences

Caution: there is no actual reference sequence known in advance, because there is no guarantee for having the actual HIV target sequence – each sample is treated individually But: thanks to Next Generation Sequencing in the laboratories of our cooperation-partners we can characterize the samples today better than all trials before Steps: 1. Multiple Sequence Alignment 2. Consensus sequence calculation 3. Calculation of participants sequence divergence 4. Definition of cutoff-threshold

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Sequence Quality: Initial Thoughts

Phylogenetic tree reported sequences samples 383001, 383002, 383003, 383004

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Sequence Quality: Multiple Sequence Alignment

• each sample is processed in separate alignment to reference HXB2
• editing and cutting of participants sequences if necessary
• Algorithms:

− ClustalX − Tcoffee

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Sequence Quality: Consensus Sequence Calculation

• performed for each sample
• aim: generate a consensus consisting only of [ACGT]
• ther IUPAC-bases are scored in their respective composition

− e.g. Y scores 50% C and 50% T − D scores 33% A, 33% G and 33% T

• most dominant base is calculated for each position

C C T C A G A

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Sequence Quality: Participants Sequence Divergence

• performed with every submitted sequence of each participant
• difference between consensus and submitted sequence is evaluated:

− IUPAC-bases are scored according their composition

Consensus sequence A T C A T C C C A G T A A G G G T A Participants sequence A T C A T C M C A G T T A G G G T A Divergence score (s) ½ 1

• final calculation: nucleotide substitutions per 100 basepairs

Δ = 100 𝑡𝑡𝑡 𝑡𝑗

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Sequence Quality: Participants Sequence Divergence

• performed with every submitted sequence of each participant
• difference between consensus and submitted sequence is evaluated:

− IUPAC-bases are scored according their composition

Consensus sequence A T C A T C C C A G T A A G G G T A Participants sequence A T C A T C M C A G T T A G G G T A Divergence score (s) ½ 1

• final calculation: nucleotide substitutions per 100 basepairs

Δ = 100 𝑡𝑡𝑡 𝑡𝑗

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• problems:

− if focusing on the standard deviation of each individual sample we experienced a high fluctuation and following inconsistencies in interpretation

Sequence Quality: Cutoff-Threshold Definition

• crucial to separate sequences
• f good and bad quality with a

single parameter cutoff here?

• r here?

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Sequence Quality: Cutoff-Threshold Definition

• problems:

− if focusing on the standard deviation of each individual sample we experienced a high fluctuation and following inconsistencies in interpretation

• cutoff definition:

− triple of the standard deviation of all participants divergences proved to be robust − meaning in normally distributed values we suppose roughly 99.7% of samples should pass

• crucial to separate sequences
• f good and bad quality with a

single parameter cutoff here!

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Sequencing Quality: Improvements over the Years

• sequencing quality improved: standard deviation of

divergence decreased − 2014: 0.0202 − 2015: 0.0560 − 2016: 0.0063 − 2017: 0.0062 − 2018: 0.0033 − threshold of 6.5 nt/100bp was applied for evaluation of the sequence quality in 2014, 2015 and 2016 − threshold of 6.5 nt/100bp was reduced to 2.3 nt/100bp in 2017

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EQA schemes (383 and 384) for HIV-1 Resistance Determination: Evaluation of reported sequence ranges

Consensus sequence: generated by an alignment of all sequences submitted by the participants Protease:

AA 10 AA 90

Reverse Transcriptase:

AA 41 AA 238

Integrase:

AA 49 AA 263 AA 318 AA 1 AA 1

Evaluated resistance related sequence range:

evaluated sequence range additional sequence range with resistance associated mutations AA Amino Acid

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EQAS HIV-1 Resistance Determination of PR and RT Inhibitors (Term Nov/Dec 2017) – Sequence Quality (TC10)

• 3

2 7 12 17 22 27 32 37 42 1 51 101 151 201 251 301

EQA participant amino acid position

Reported reverse transcriptase sequence segments spl. 383015

1 = TN7 2 = TN9 3 = TN25 4 = TN33 5 = TN54 6 = TN128 7 = TN236 8 = TN308 9 = TN400 10 = TN427 11 = TN769 12 = TN909 13 = TN976 14 = TN1056 15 = TN1191 16 = TN1888 17 = TN2147 18 = TN2278 19 = TN2362 20 = TN2497 21 = TN3571 22 = TN3803 23 = TN4297 24 = TN4721 25 = TN5303 26 = TN5577 27 = TN5969 28 = TN6950 29 = TN8705 30 = TN30006 31 = TN30616 32 = TN30674 33 = TN31281 34 = TN31626 35 = TN33478 36 = TN41387 37 = TN47494 38 = TN50180 39 = TN57844 Codon 41 Codon 238 Codon 318

Codon 41 Codon 238 Codon 318

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EQAS HIV-1 Resistance Determination of Integrase Inhibitors (Terms Nov/Dec 2017 and 2018) – Sequence Quality (TC10)

• 2

3 8 13 18 23 28 33 38 1 51 101 151 201 251

EQA participant amino acid position

Nov/Dec 17, integrase, sample 384007

1 = TN7 2 = TN9 3 = TN25 4 = TN33 5 = TN54 6 = TN128 7 = TN308 8 = TN400 9 = TN427 10 = TN539 11 = TN769 12 = TN909 13 = TN1056 14 = TN1888 15 = TN2147 16 = TN2278 17 = TN2362 18 = TN2497 19 = TN3571 20 = TN3803 21 = TN4297 22 = TN5303 23 = TN5969 24 = TN6950 25 = TN8705 26 = TN29286 27 = TN30006 28 = TN30616 29 = TN31281 30 = TN31626 31 = TN33478 32 = TN41387 33 = TN49871 34 = TN50174 35 = TN57844 Codon 49 Codon 263

Codon 49 Codon 263

Reported integrase sequence segements:

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EQAS HIV-1 Resistance Determination of Integrase Inhibitors (Terms Nov/Dec 2017 and 2018) – Sequence Quality (TC10)

• 2

3 8 13 18 23 28 33 38 1 51 101 151 201 251

EQA participant amino acid position

Nov/Dec 17, integrase, sample 384007

1 = TN7 2 = TN9 3 = TN25 4 = TN33 5 = TN54 6 = TN128 7 = TN308 8 = TN400 9 = TN427 10 = TN539 11 = TN769 12 = TN909 13 = TN1056 14 = TN1888 15 = TN2147 16 = TN2278 17 = TN2362 18 = TN2497 19 = TN3571 20 = TN3803 21 = TN4297 22 = TN5303 23 = TN5969 24 = TN6950 25 = TN8705 26 = TN29286 27 = TN30006 28 = TN30616 29 = TN31281 30 = TN31626 31 = TN33478 32 = TN41387 33 = TN49871 34 = TN50174 35 = TN57844 Codon 49 Codon 263

Codon 49 Codon 263

• 2

3 8 13 18 23 28 33 38 1 51 101 151 201 251

EQA participant amino acid position

Nov/Dec 18, integrase, sample 384010

1 = TN7 2 = TN9 3 = TN25 4 = TN33 5 = TN54 6 = TN128 7 = TN308 8 = TN400 9 = TN427 10 = TN539 11 = TN769 12 = TN909 13 = TN1056 14 = TN1888 15 = TN2147 16 = TN2278 17 = TN2362 18 = TN2497 19 = TN3571 20 = TN4297 21 = TN4721 22 = TN5303 23 = TN5969 24 = TN6950 25 = TN8705 26 = TN8736 27 = TN29286 28 = TN30006 29 = TN30616 30 = TN31281 31 = TN31626 32 = TN33478 33 = TN41387 34 = TN49871 35 = TN50174 36 = TN58150 Codon 49 Codon 263

Codon 49 Codon 263

Reported integrase sequence segements:

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Sequence Quality: Detecting variants

example of Sanger-Sequence with variant

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Sequence Quality: The NGS-Approach

...PQIYMDDHTRE...

Standard Sanger-sequencing

...PQIYVDDHTRE... ...PQIYMDDHTRE... ...PQIYMDDHTRE... ...PQIYMDDHTRE... ...PQIYMDDHTRE... ...PQIYMDDHTRE...

Ultra-deep-sequencing

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Sequence Quality: variant analyses by NGS prior to the EQA scheme – sample 383019

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Sequence Quality: variant analyses by NGS during the EQA scheme – sample 383019

• sample 383019 contained a resistant variant
• frequencies observed by our cooperation-partners via NGS:
• K103N: 35%, 62%, 35,4%, 37%, 50,4% (mean 44%)
• K219E: 43%, 51,8%, 65%, 80%, 66,3% (mean 61,2%)
• Participant results:

30/37 participants stated K103N or K103KN 31/37 participants stated K219E or K219EK

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Quasispecies Reconstruction of sample 383019

QuRE: Prosperi, Mattia C. F., and Marco Salemi. ‘QuRe: Software for Viral Quasispecies Reconstruction from next-Generation Sequencing Data’. Bioinformatics 28, no. 1 (1 January 2012): 132–33. https://doi.org/10.1093/bioinformatics/btr627.

Phylogenetic Reconstruction of quasispecies by QuRE software using NGS data of MIB

• marks populations

harboring K103N

38,5 % 64,8 % 16,8 % 6,1 % 12,3 %

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EQA Schemes (383,384) for HIV-1 Resistance Determination and Tropism Determination Overview of Parameters

EQA Scheme Virus Genome Detection – HIV-1 Drug Resistance Determination – (383) Standard Program for Protease and Reverse Transcriptase Inhibitors Parameters for drug resistance determination of protease and reverse transcriptase inhibitors (10) Evaluation of the sequence quality (20) Determination of resistance-associated mutations (30) Interpretation of antiretroviral drug resistance / drug sensitivity EQA Scheme Virus Genome Detection – HIV-1 Drug Resistance Determination – (384) additional Program for Integrase Inhibitors and Tropism Determination Parameters for drug resistance determination of integrase inhibitors (10) Evaluation of the sequence quality (20) Determination of resistance-associated mutations (30) Interpretation of antiretroviral drug resistance / drug sensitivity Parameters for genotypic determination of tropism (40) Evaluation of sequence quality (50) Determination of V3-loop amino-acid sequence (60) Interpretation of tropism

Evaluation in consecutive steps according to genotypic HIV-1 resistance determination

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EQA schemes (383,384) for HIV-1 Resistance Determination: Determination of Resistance Associated Mutations

Target value for resistance associated mutations: generated by comparison of the consensus sequence with the HIV-1 subtype B reference sequence. Example: HIV-DB analysis - mutations EQAS November/December 2016 sample 383012 (consensus sequence):

M46I I54V V82A T215Y M184V V108I A98G M41L

NNRTI resistance mutations NRTI resistance mutation NRTI resistance mutations

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Source Plasmid G10178 - HIV-1 Subtype B (with Partial Resistance against PR and RT Inhibitors) – Success Rates in 4 EQA Schemes

Success rates EQA scheme term Source plasmid / sample No. Dil. Parameter 10 Evaluation of sequence quality Parameter 20 Determination of resistance associated mutations Parameter 30 Interpretation of antiretroviral drug resistance / drug sensitivity Dec 14/Jan 15 G10178/383004 n.a. 27/27 (100%) 28/28 (100%) 28/28 (100%) Dec 15/Jan 16 G10178/383006 n.a. 32/35 (91.4%) 33/37 (89.2%) 24/37 (64.9%) Nov/Dec 16 G10178/383012 n.a. 37/39 (94.9%) 38/40 (95.0%) 37/40 (92.5%) Nov/Dec 18 G10178/383020 n.a. 36/37 (97.3%)* 36/37 (97.3%)* 30/37 (81.1%)* G10178: plasmid in water * preliminary data

Sample Property: Plasmid with sequence of HIV-1 subtype B with partial resistance against PR and RT inhibitors (plasmid in water) Resistance Associated Mutations: Protease: L10I, M46I, I54V, A71T, V82A; Reverse transcriptase: M41L, A98G, V108I, M184V, T215Y

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Source Plasmid G10178 - HIV-1 Subtype B (with Partial Resistance against PR and RT Inhibitors) – Participants’ Drug Sensitivity Statements

EQA Dec 14/Jan 15

• Spl. 383004

EQA Dec 15/Jan 16

• Spl. 383006

EQA Nov/Dec 16

• Spl. 383012

EQA Nov/Dec 18

• Spl. 383020

not stated

I intermediate

S susceptible R resistant

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IQVD

NRTI = Nucleoside and nucleotide reverse- transcriptase inhibitors NNRTI = Non- nucleoside reverse- transcriptase inhibitors PI = Protease inhibitors INI = Integrase inhibitors 3TC = Lamivudin EFV = Efavirenz ATV = Atazanavir* ATV/r = Atazanavir (Ritonavir boosted) DTG = Dolutegravir ABC = Abacavir ETR = Etravirin DRV/r = Darunavir (Ritonavir boosted) EVG = Elvitegravir AZT = Zidovudin NVP = Nevirapin FPV/r = Fosamprenavir (Ritonavir boosted) RAL = Raltegravir d4T = Stavudin* RPV = Rilpivirin IDV/r = Indinavir* (Ritonavir boosted) ddI = Didanosin* LPV/r= Lopinavir (Ritonavir boosted) FTC = Emtricitabin NFV = Nelfinavir* TDF = Tenofovir SQV/r = Saquinavir (Ritonavir boosted) TPV/r = Tipranavir (Ritonavir boosted)

Antiretroviral drugs Update from EQA term 2017 to 2018

* antiretroviral drugs no longer prescribed in the EU

SLIDE 37

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

Source Plasmid G10178 - HIV-1 Subtype B (with Partial Resistance against PR and RT Inhibitors) – Participants’ Drug Sensitivity Statements

EQA Dec 14/Jan 15

• Spl. 383004

EQA Dec 15/Jan 16

• Spl. 383006

EQA Nov/Dec 16

• Spl. 383012

EQA Nov/Dec 18

• Spl. 383020

not stated

I intermediate

S susceptible R resistant

2018

SLIDE 38

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

Source Plasmid G10178 - HIV-1 Subtype B (with Partial Resistance against PR and RT Inhibitors) – Algorithms and Target Values

EQA Nov/Dec 18

• Spl. 383020

PR inhibitors RT inhibitors NNRTI RT inhibitors NRTI ATV ATV/r DRV/r FPV/r IDV/r LPV/r NFV SQV/r TPV/r EFV ETR NVP RPV 3TC ABC AZT d4T ddI FTC TDF HIV-GRADE R S I I I S S S S S R I I R I ANRS R S S R I S S I S S R R R R S HIV-DB R S I R R I I S I I R I I R I REGA I S I I I S I S I S R I I R S preliminary target value R/I S I/S I/R I/R S/I S/I S/I S/I S/I R I/R I/R R S/I

Success rates for all methods:

Parameter 10 Evaluation of the sequence quality Parameter 20 Determination of resistance-associated mutations Parameter 30 Interpretation of antiretroviral drug resistance / drug sensitivity 97.3% (36/37)* 97.3% (36/37)* 81.1% (30/37)*

Sample: 383020 Term November/December 2018 Sample property:

Plasmid with sequence of HIV-1 subtype B with partial resistance against PR and RT inhibitors (plasmid in water)

Antiretroviral Drugs Algorithms

not stated

I intermediate

S susceptible R resistant

*preliminary data

SLIDE 39

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

Source Plasmid G10178 - HIV-1 Subtype B (with Partial Resistance against PR and RT Inhibitors) – participants’ problems

Algorithm selection

EQA Nov/Dec 18

• Spl. 383020

PR inhibitors RT inhibitors NNRTI RT inhibitors NRTI ATV ATV/r DRV/r FPV/r IDV/r LPV/r NFV SQV/r TPV/r EFV ETR NVP RPV 3TC ABC AZT d4T ddI FTC TDF HIV-GRADE R S I I I S S S S S R I I R I ANRS R S S R I S S I S S R R R R S HIV-DB R S I R R I I S I I R I I R I REGA I S I I I S I S I S R I I R S preliminary target value R/I S I/S I/R I/R S/I S/I S/I S/I S/I R I/R I/R R S/I

geno2pheno I S I R S S S

• I
• R

R R R S

Antiretroviral Drugs Algorithms

not stated

I intermediate

S susceptible R resistant

applied algorithm beside others: HIV-GRADE applied algorithm beside others: geno2pheno

SLIDE 40

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

Source Plasmid G10178 - HIV-1 Subtype B (with Partial Resistance against PR and RT Inhibitors) – participants’ problems

algorithm S = susceptible I = intermediate R = resistant HIV-DB susceptible potential low-level resistance low-level resistance intermediate resistance high-level resistance HIV-GRADE susceptible limited susceptible intermediate resistance REGA susceptible GSS 1 susceptible GSS 1.5 intermediate resistant GSS 0.75 intermediate resistant GSS 0.5 intermediate resistant GSS 0.25 resistant GSS 0

Translation in the S/I/R scheme

SLIDE 41

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

• INSTAND e.V.
• INSTAND External Quality Assessment (EQA) Schemes for Virus Genome

Detection: (383) HIV-1- Drug Resistance Determination of Protease and Reverse Transcriptase Inhibitors => Estimation of susceptibility according S/I/R-scheme (384) HIV-1- Drug Resistance Determination of Integrase and Determination of Tropism (396) Hepatitis B Virus Genotyping (397) Hepatitis B Virus Resistance Determination => Harmonization of the interpretation algorithms (375) Hepatitis C Virus Geno- / Subtyping (399) Hepatitis C Virus Resistance Determination (349) Cytomegalovirus Resistance Determination

• Summary

Outline

SLIDE 42

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (396) Hepatitis B Virus Genotyping

• first EQA schemes: November/December 2016
• performed 1 x per year
• number of participants increased from 38 (2016) to 39 (2018)
• cooperation partners:

Paul-Ehrlich-Institut Bundesinstitut für Impfstoffe und biomedizinische Arzneimittel

• Dr. Michael Chudy, Dr. Julia Kreß

Nationales Referenzzentrum für Hepatitis-B-Virus u. Hepatitis-D-Virus Justus-Liebig-Universität Gießen, Institut für Medizinische Virologie

• Prof. Dr. Dieter Glebe, Dr. Christian Schüttler, Prof. Dr. Wolfram Gerlich,
• Prof. Dr. John Ziebuhr
• sample material: HBV positive plasma provided from Paul-Ehrlich-Institut

SLIDE 43

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (396) Hepatitis B Virus Genotyping Term November/December 2017 Results and Evaluation

Sample Sample properties Results considered as "correct" (target values) Success rates for each sample Test category 30 (Hepatitis B virus genotyping) Test category 40 (Hepatitis B virus genosubtyping) Test category 30 Test category 40 (not evaluated) 396009 HBV-positive plasma A A1 97.1% (34/35) 95.0% (19/20) 396010 HBV-positive plasma B B2 100% (35/35) 100% (20/20) 396011 HBV-positive plasma A A2 100% (35/35) 100% (20/20) 396012 HBV-positive plasma D D1 100% (35/35) 100% (20/20) Overall success rates for all samples in test categories 30 und 40 97.1% (34/35) 95.0% (19/20)

• High success rate
• Evaluation of Hepatitis B virus genosubtyping possible in 2018

SLIDE 44

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (396) Hepatitis B Virus Genotyping Term November 2018 - Results and Evaluation

Sample Sample properties Results considered as "correct" (target values) Success rates for each sample Parameter 30 (Hepatitis B virus genotyping) Parameter 35 (Hepatitis B virus genosubtyping ) Parameter 30 (Hepatitis B virus genotyping) Parameter 35 (Hepatitis B virus genosubtyping) 396013 HBV-positive plasma C C2* 92.1% (35/38) 13.0% (3/23)* 396014 HBV-positive plasma A A2 100% (38/38) 100% (23/23) 396015 HBV-positive plasma D D3 100% (38/38) 100% (23/23) 396016 HBV-positive plasma B B2** 100% (38/38) 60.9% (14/23)* Overall success rates for all samples in parameters 30 und 35 92.1% (35/38) 13.0% (3/23) *not evaluated

• High success rate in parameter 30
• Evaluation of Hepatitis B virus genosubtyping (parameter 35)

SLIDE 45

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (396) Hepatitis B Virus Genotyping Term November 2018 - Results and Evaluation Genosubtyping, Sample 396013 & 396016

=> genosubtyping results based on sequencing of only S-gene not sufficient

Sample: 396013 , expected result genosubtype C2 Reagent Testkit Total (N=23) below detection limit/negative (N=2) Genosubtype C1 (N=18) Genosubtype C2 (N=3) Quota (13.04%) in-house 23 2 18 3 13.04% 23 2 18 3 13.04% Sample: 396016 , expected result genosubtype B2 Reagent Testkit Total (N=23) below detection limit/negative (N=0) Genosubtype B1 (N=9) Genosubtype B2 (N=14) Quota (60.87%) in-house 23 9 14 60.87% 23 9 14 60.87%

correct

SLIDE 46

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

• INSTAND e.V.
• INSTAND External Quality Assessment (EQA) Schemes for Virus Genome

Detection: (383) HIV-1- Drug Resistance Determination of Protease and Reverse Transcriptase Inhibitors => Estimation of susceptibility according S/I/R-scheme (384) HIV-1- Drug Resistance Determination of Integrase and Determination of Tropism (396) Hepatitis B Virus Genotyping (397) Hepatitis B Virus Resistance Determination => Harmonization of the interpretation algorithms (375) Hepatitis C Virus Geno- / Subtyping (399) Hepatitis C Virus Resistance Determination (349) Cytomegalovirus Resistance Determination

• Summary

Outline

SLIDE 47

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (397) Hepatitis B Virus Resistance Determination

• first EQA scheme: November/December 2016
• performed 1 x per year
• number of participants increased from 32 (2016) to 33 (2018)
• cooperation partner:

Nationales Referenzzentrum (NRZ) für Hepatitis-B-Virus u. Hepatitis-D-Virus Justus-Liebig-Universität Gießen, Institut für Medizinische Virologie

• Prof. Dr. Dieter Glebe, Dr. Christian Schüttler, D. H. Slanina,
• Prof. Dr. Wolfram Gerlich, Prof. Dr. John Ziebuhr
• sample material: Plasmids provided by NRZ für HBV und HDV Gießen
• riginated from a library of Nationalen Referenzzentrum, Gießen, group of Prof. Protzer,

München, and group of Prof. Nassal, Freiburg

• evaluated parameters:

(15) Determination of the HBV genotype (20) Determination of resistance-associated mutations (30) Interpretation of genotypic resistance

SLIDE 48

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (397) Hepatitis B Virus Resistance Determination - Term Nov/Dec 2016 Results and Evaluation

Sample Sample properties Genotype/ subgenotype Results considered as "correct" (target values) Success rates for each sample Test category 20 (Determination of resistance- associated mutation) Test category 30 (Interpretation of genotypic resistance) Test category 20 Test category 30

397005 plasmid* M204V

Lamivudine: R Telbivudine: R Adefovir: S Entecavir: I/R Tenofovir: S 90.6% (29/32)

62.5% (20/32)

genotype: D/D3 397006 plasmid* L180M, M204V

Lamivudine: R Telbivudine: R Adefovir: S Entecavir: I/R Tenofovir: S 90.6% (29/32) 81.3% (26/32)

genotype: D/D3 397007 plasmid* A181V, N236T

Lamivudine: R Telbivudine: R Adefovir: R Entecavir: S Tenofovir: I/R 87.5% (28/32) 68.8% (22/32)

genotype: D/D3 397008 plasmid* no resistance- associated mutations

Lamivudine: S Telbivudine: S Adefovir: S Entecavir: S Tenofovir: S 81.3% (26/32) 81.3% (26/32)

genotype: C/C1

Overall success rates for all samples in test categories 20 und 30

78.1% (25/32) 68.8% (22/32)

* Plasmids were kindly provided by „Nationalen Referenzzentrum für Hepatitis-B-und D-Viren“ Gießen and were originated from a library of Nationalen Referenzzentrum, Gießen, group of Prof. Protzer, München, and group of Prof. Nassal, Freiburg. (Nassal et al., Journal of Virology, 1992).

SLIDE 49

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (397) Hepatitis B Virus Resistance Determination – Interpretation results and algorithms Plasmid with M204V mutation

2a

EQA Nov/Dec 16,

• Spl. 397005

not stated S sensitive (S)

I intermediate (I)

R resistant (R)

Plasmid (mutation M204V): evaluation of interpretation of genotypic resistance (30) for Entecavir correct I

intermediate

accepted

R resistant

false

S sensitive

ad hoc group:

• J. Hofmann

Charité Universitätsmedizin Berlin, CCM; Labor Berlin – Charité Vivantes GmbH

• A.-M. Eis-Hübinger

Universität Bonn

• H. Rabenau / A. Berger

Universitätsklinikum Frankfurt

• R. Kaiser /E. Heger /E. Knops

Uniklinik Köln; NRZ Papillom- und Polyomaviren

• U.G. Liebert / M. Maier

Universität Leipzig

• M. Obermeier

Medizinisches Infektiologiezentrum, Berlin

• K. Korn

Universitätsklinikum Erlangen

• D. Glebe / C. Schüttler / S. Slanina / W. Gerlich

Universität Gießen; NRZ HBV und HDV

• M. Kammel / H.-P. Grunert / A. Zimmermann / H. Zeichhardt INSTAND, IQVD, GBD

SLIDE 50

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (397) Hepatitis B Virus Resistance Determination – Interpretation results and algorithms Plasmid with M204V mutation

EQA Nov/Dec 17,

• Spl. 397011

EQA Nov/Dec 18,

• Spl. 397014

2a 2b 2c 2d

EQA Nov/Dec 16,

• Spl. 397005

nucleos(t)ide analoga Adefovir Lamivudine Telbivudine Tenofovir Entecavir HIV-GRADE S R R S I geno2pheno S R R S I not stated S sensitive (S)

I intermediate (I)

R resistant (R)

Plasmid (mutation M204V): evaluation of interpretation of genotypic resistance (30) for Entecavir correct I

intermediate

accepted

R resistant

false

S sensitive

SLIDE 51

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (397) Hepatitis B Virus Resistance Determination - Success rates 2016 – 2018 Plasmid with M204V mutation

Plasmid - HBV genosubtype D3, mutation M204V – success rates in 3 EQA schemes Success rates EQA scheme term Source plasmid / Sample No. Parameter 15 (Determination of the HBV genotype) Parameter 20 (Determination of resistance-associated mutation) Parameter 30 (Interpretation of genotypic resistance) Nov/Dec 16 Plasmid/397005

• 29/32 (90.6%)

20/32 (62.5%) Nov/Dec 17 Plasmid/397011 28/30 (93.3%) 31/33 (93.9%) 29/33 (87.9%) Nov/Dec 18 Plasmid/397014 29/30 (96.7%)* 32/43 (97.0%)* 31/33 (96.7%)* Plasmid: plasmid in water * preliminary data

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AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (397) Hepatitis B Virus Resistance Determination – Success rates 2016 - 2018

EQA term

Overall success rates for all samples

Parameter 15 (Determination of the HBV genotype) Parameter 20 (Determination of resistance-associated mutation) Parameter 30 (Interpretation of genotypic resistance)

Nov/ Dec 2016

• 78.1%

(25/32) 68.8% (22/32) Nov/ Dec 2017 93.3% (28/30) 87.9% (29/33) 63.6% (21/33) Nov/ Dec 2018 83.3%* (25/30)* 78.8%* (26/33)* 78.8%* (26/33)* *preliminary data

SLIDE 53

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (397) Hepatitis B Virus Resistance Determination - Term Nov/Dec 2018 Escape mutations

Sample No. 397013 397014 397015 397016 expected HBsAg escape mutation

• G145R
• reported

HBsAg escape mutations 4 x P127T / 33 analyses 9 x P127T / 33 analyses 18 x G145R 1 x P127I / 33 analyses 8 x P127T 1 x P127T, S143L 1 x A128AV / 33 analyses

HIV-GRADE geno2pheno

SLIDE 54

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (397) Hepatitis B Virus Resistance Determination - Term Nov/Dec 2018 Escape mutations

HIV-GRADE geno2pheno

Sample No. 397013 397014 397015 397016 expected HBsAg escape mutation

• G145R
• reported

HBsAg escape mutations 4 x P127T / 33 analyses 9 x P127T / 33 analyses 18 x G145R 1 x P127I / 33 analyses 8 x P127T 1 x P127T, S143L 1 x A128AV / 33 analyses

SLIDE 55

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

• INSTAND e.V.
• INSTAND External Quality Assessment (EQA) Schemes for Virus Genome

Detection: (383) HIV-1- Drug Resistance Determination of Protease and Reverse Transcriptase Inhibitors => Estimation of susceptibility according S/I/R-scheme (384) HIV-1- Drug Resistance Determination of Integrase and Determination of Tropism (396) Hepatitis B Virus Genotyping (397) Hepatitis B Virus Resistance Determination => Harmonization of the interpretation algorithms (375) Hepatitis C Virus Geno- / Subtyping (399) Hepatitis C Virus Resistance Determination (349) Cytomegalovirus Resistance Determination

• Summary

Outline

SLIDE 56

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (375) Hepatitis C Virus Geno- / Subtyping

• first EQA scheme: September 2006
• performed 1 x per year
• number of participants increased from 94 (2006) to 123 (2018)
• cooperation partners:

Nationales Referenzzentrum für Hepatitis C Viren, Institut für Virologie, Universitätsklinikum Essen: Prof. Dr. Stefan Ross Universitätsklinikum Düsseldorf, Institut für Virologie:

• Prof. Dr. J. Timm, Prof. Dr. O. Adams, Dr. N. Lübke
• sample material provided by NRZ für Hepatitis C Viren, Prof. Dr. S. Ross
• from EQA scheme September 2015:

Differentiation between subtypes 1a and 1b necessary for HCV genotype 1 positive samples

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AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (375) Hepatitis C Virus Geno- / Subtyping Term September 2018 Results and Evaluation

Sample Sample properties Results considered as "correct" (target values) Success rates for each sample Test category 30 (Hepatitis C virus geno/-subtyping) Test category 30 375041 HCV-positive serum 3 / 3a 98.4% (126/128) 375042 HCV-positive serum 1a 96.9% (124/128) 375043 HCV-positive serum 1b$ 92.2% (118/128) 375044 HCV-positive serum 2 / 2b 97.7% (125/128) 375046 HCV-positive serum 2 / 2a 99.2% (127/128) Overall success rates for all samples in test category 30 89.4% (110/123)

$ Sample 375043 (subtype 1b): In test category 30, some participants could not determine the subtype by using the test of one manufacturer (Abbott - RealTime

HCV Genotype II). These inconsistent results have not been evaluated for this EQA scheme (without disadvantage for the certificate of successful participation). The Nationales Referenzzentrum für HCV (Essen) and the manufacturer have been informed.

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AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA scheme (375) Hepatitis C Virus Geno- / Subtyping Term September 2018 Applied Methods: Sample 375043

• 30. Hepatitis C Virus Geno- / Subtyping

Sample property: hepatitis C virus subtype 1b

Test results in total total considered as "correct" results

• ther results

Success rate subtype 1b genotype 1 subtype 1a

• ther genotype/

subtype 128 118 8 1 1 92.2% Methods for the analyses of the geno-/subtype / manufacturer - name of test kit total considered as "correct" results

• ther results

Success rate subtype 1b genotype 1 subtype 1a

• ther genotype/

subtype Hybridization (71) 55 53 1 1 96.4% Siemens - VERSANT HCV-Genotyp-2.0 (LiPA) 51 49 1 1a 96.1% NLM – Gen-C 2.0 3 3 100.0% AB Analytica – Ampliquality HCV Type Plus 1 1 100.0% Sequencing (72) 33 33 100.0% in house 32 32 100.0% Vela Diag. – Sentosa SQ HCV Genotyp. Assay 1 1 100.0% Other method (99) 40 32 7 1 80.0% Abbott - RealTime HCV Genotype II 17 9 7b 1b 52.9% Roche – Cobas HCV GT 16 16 100.0% in house 3 3 100.0% Sacace - HCV Genotype Plus Real-TM 2 2 100.0%

• ther manufacturer

2 2 100.0%

b Sample 375043 (subtype 1b): In test category 30, some participants could not determine the subtype by using the test of one manufacturer (Abbott - RealTime HCV Genotype II). These

inconsistent results have not been evaluated for this EQA scheme (without disadvantage for the certificate of successful participation). The Nationales Referenzzentrum für HCV (Essen) and the manufacturer have been informed.

SLIDE 59

AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

• INSTAND e.V.
• INSTAND External Quality Assessment (EQA) Schemes for Virus Genome

Detection: (383) HIV-1- Drug Resistance Determination of Protease and Reverse Transcriptase Inhibitors => Estimation of susceptibility according S/I/R-scheme (384) HIV-1- Drug Resistance Determination of Integrase and Determination of Tropism (396) Hepatitis B Virus Genotyping (397) Hepatitis B Virus Resistance Determination => Harmonization of the interpretation algorithms (375) Hepatitis C Virus Geno- / Subtyping (399) Hepatitis C Virus Resistance Determination (349) Cytomegalovirus Resistance Determination

• Summary

Outline

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AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (399) Hepatitis C Virus Resistance Determination

• first EQA schemes: November/December 2016
• performed 1 x per year
• number of participants between 17 and 20
• cooperation partners:

Nationales Referenzzentrum für Hepatitis C Viren, Institut für Virologie, Universitätsklinikum Essen: Prof. Dr. Stefan Ross Universitätsklinikum Düsseldorf, Institut für Virologie:

• Prof. Dr. J. Timm, Prof. Dr. O. Adams, Dr. N. Lübke
• sample material provided by cooperation partners
• evaluated parameters:

(15 / 45) Determination of the HCV genotype (20 / 50) Determination of resistance-associated mutations (30 / 60) Interpretation of genotypic resistance

• for the NS5a-gene region (parameters 15, 20 and 30)
• for the NS3-gene region (parameters 45, 50 and 60)

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AREVIR-Meeting Cologne, 03-04 May 2019

IQVD

EQA Scheme (399) Hepatitis C Virus Resistance Determination – Term November/December 2016 Results and Evaluation

Sample

Sample properties Genotype/ subgenotype (Gene region) Results considered as "correct" (target values) Success rates for each sample 2016 Test category 20 (Determination of resistance-associated mutation) Test category 30 (Interpretation of genotypic resistance) Test category 20 Test category 30

399005

patient serum, 1 : 154 diluted

D168A

Asunaprevir: R Boceprevir: S/R Grazoprevir: R Paritaprevir: R Simeprevir: R Telaprevir: S/I

88.2% (15/17) 82.4% (14/17)

genotype: 1/1b (NS3)

Overall success rates in test categories 20 und 30 (mutation in gene region NS3)

88.2% (15/17) 82.4% (14/17)

399006

patient serum, 1 : 64 diluted

Y93H

Daclatasvir: R Elbasvir: R Ledipasvir: R Ombitasvir: R

87.5% (14/16) 87.5% (14/16)

genotype: 1/1b (NS5A)

399007

patient serum, 1 : 55 diluted

M28T, Q30R

Daclatasvir: R Elbasvir: R Ledipasvir: R Ombitasvir: R

81.3% (13/16) 81.3% (13/16)

genotype: 1/1a (NS5A)

399008

patient serum, 1 : 62 diluted

no resistance- associated mutations

Daclatasvir: S Elbasvir: S Ledipasvir: S Ombitasvir: S

81.3% (13/16) 87.5% (14/16)

genotype: 1/1b (NS5A)

399009

patient serum, 1 : 77 diluted

Y93H

Daclatasvir: R Elbasvir: R Ledipasvir: R Ombitasvir: R

75.0% (12/16) 75.0% (12/16)

genotype: 1/1b (NS5A)

Overall success rates for all samples in test categories 20 und 30 (mutations in gene region NS5A)

68.8% (11/16) 68.8% (11/16)

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IQVD

Protease inhibitors

drug EQA schemes 2016 EQA schemes 2017 EQA scheme 2018

Boceprevir x x Simeprevir x x Telaprevir x x Asunaprevir x x x Grazoprevir x x x Paritaprevir x x x Glecaprevir x Voxilaprevir x

NS5A inhibitors

drug EQA scheme 2016 EQA scheme 2017 EQA scheme 2018

Ombitasvir x x x Daclatasvir x x x Elbasvir x x x Ledipasvir x x x Velpatasvir x x Pibrentasvir x

EQA Scheme (399) HCV Resistance Determination – updates of requested drug susceptibilities

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IQVD

EQA Scheme (399) Hepatitis C Virus Resistance Determination – Term November/December 2018 Results and Evaluation

Sample

Sample properties (Gene region) Results considered as "correct" (target values) Success rates for each sample 2018 Parameter 15 / 45 (Determination of the HCV genotype) Parameter 20 / 50 (Determination of resistance-associated mutation) Parameter 30 / 60 (Interpretation of genotypic resistance) Parameter 15 / 45 Parameter 20 / 50 Parameter 30 / 60

399014

patient serum, 1 : 34 diluted

1 /1b Y56F

Asunaprevir: S Glecaprevir: S Grazoprevir: I Paritaprevir: S Voxilaprevir: S

100% (16/16) 87.5% (14/16) 81.3% (13/16)

(NS3)

399015

patient serum, 1 : 54.2 diluted

1 / 1a Q89K, N174S

Asunaprevir: S Glecaprevir: S Grazoprevir: S Paritaprevir: S Voxilaprevir: I

100% (16/16) 75.0% (12/16) 87.5% (14/16)

(NS3)

Overall success rates in parameters 45, 50 und 60 (mutation in gene region NS3)

100% (16/16) 68.8% (11/16) 81.3% (13/16)

399016

patient serum, 1 : 75 diluted

1 / 1b L31M, Y93H

Daclatasvir: R Elbasvir: R Ledipasvir: R Ombitasvir: R Pibrentasvirs: S Velpatasvir: R

100% (17/17) 82.4% (14/17) 76.5% (13/17)

(NS5A)

399017

patient serum, 1 : 414 diluted

3 / 3a Q30K

Daclatasvir: R Elbasvir: R Ledipasvir: R Ombitasvir: not licensed§ Pibrentasvir: I Velpatasvir: I

88.2% (15/17) 76.5% (13/17) 76.5% (13/17)

(NS5A)

Overall success rates for all samples in parameters 15, 20 und 30 (mutations in gene region NS5A)

88.2% (15/17) 76.5% (13/17) 70.6% (12/17)

§ The participants stated that Ombitasvir is not licensed for treatment of HCV genotype 3.

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IQVD

EQA Scheme (399) Hepatitis C Virus Resistance Determination – Success rates 2016 - 2018

NS3-gene region

EQA term Overall success rates for all samples Parameter 45 (Determination of the HCV genotype) Parameter 50 (Determination of resistance-associated mutation) Parameter 60 (Interpretation of genotypic resistance)

Nov/ Dec 2016

• 88.2%

(15/17) 82.4% (14/17) Nov/ Dec 2017

• 80.0%

(16/20) 70.0% (14/20) Nov/ Dec 2018 100%* (16/16)* 68.8%* (11/16)* 81.3%* (13/16)*

NS5A-gene region

EQA term Overall success rates for all samples Parameter 15 (Determination of the HCV genotype) Parameter 20 (Determination of resistance-associated mutation) Parameter 30 (Interpretation of genotypic resistance)

Nov/ Dec 2016

• 68.8%

(11/16) 68.8% (11/16) Nov/ Dec 2017

• 78.9%

(15/19) 73.7% (14/19) Nov/ Dec 2018 88.2%* (15/17)* 76.5%* (13/17)* 70.6%* (12/17)* *preliminary data

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IQVD

• INSTAND e.V.
• INSTAND External Quality Assessment (EQA) Schemes for Virus Genome

Detection: (383) HIV-1- Drug Resistance Determination of Protease and Reverse Transcriptase Inhibitors => Estimation of susceptibility according S/I/R-scheme (384) HIV-1- Drug Resistance Determination of Integrase and Determination of Tropism (396) Hepatitis B Virus Genotyping (397) Hepatitis B Virus Resistance Determination => Harmonization of the interpretation algorithms (375) Hepatitis C Virus Geno- / Subtyping (399) Hepatitis C Virus Resistance Determination (349) Cytomegalovirus Resistance Determination

• Summary

Outline

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EQA Scheme (349) Cytomegalovirus Resistance Determination

• first EQA scheme: November/December 2016
• performed 1 x per year
• number of participants between 17 and 20
• cooperation partners:

Universitätsklinikum Ulm,Institut für Virologie Konsiliarlabor für Cytomegalievirus (CMV) Schwerpunkt: CMV-Infektionen bei immunsupprimierten Personen

• Prof. Dr. Thomas Stamminger (ab 01/2018), Prof. Dr. Detlef Michel,
• Prof. Dr. Thomas Mertens (bis 12/2017)

Universitätsklinikum Tübingen, Institut für Medizinische Virologie und Epidemiologie der Viruskrankheiten, Nationales Konsiliarlaboratorium für Cytomegalievirus (CMV), Schwerpunkt: kongenitale/postnatale CMV-Infektionen

• Prof. Dr. T. Iftner (ab 04/2018), Prof. Dr. Klaus Hamprecht, Prof. Dr. Gerhard Jahn (bis 03/2018)
• evaluated test categories:

(20 / 50) Determination of resistance-associated mutations (30 / 60) Interpretation of genotypic resistance

• for the UL97-gene region (parameters 20 and 30)
• for the UL54-gene region (parameters 50 and 60)

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EQA Scheme (349) Cytomegalovirus Resistance Determination - Term November/December 2018 Results and Evaluation

UL97

Sample Sample properties Genotype/ subgenotype Results considered as "correct" (target values) Success rates for each sample Parameter 20 / 50 (Determination of resistance-associated mutation) Parameter 30 / 60 (Interpretation of genotypic resistance) Parameter 20 / 50 Parameter 20 / 50

349013 CMV isolate (EDTA blood) C603W Ganciclovir / Valganciclovir: R

100% (18/18) 100% (18/18)

cell culture supernatant diluted 1 : 100 in plasma 349014 CMV isolate (EDTA blood) C607Y Ganciclovir / Valganciclovir: R

100% (18/18) 100% (18/18)

cell culture supernatant diluted 1 : 400 in plasma 349016 CMV isolate (EDTA blood) no resistance-associated mutations Ganciclovir / Valganciclovir: S

100% (18/18) 100% (18/18)

cell culture supernatant diluted 1 : 3 750 in plasma 349016 CMV isolate (EDTA blood) L595S Ganciclovir / Valganciclovir: R

100% (18/18) 100% (18/18)

cell culture supernatant diluted 1 : 118 in plasma

Overall success rates in parameters 20 und 30 (mutation in gene region UL97)

100% (18/18) 100% (18/18)

UL54

349014 CMV isolate (EDTA blood) P522S Ganciclovir / Valganciclovir : R Cidofovir / Brincidofovir: R Foscarnet: S

100% (12/12) 100% (12/12)

cell culture supernatant diluted 1 : 100 in plasma 349015 CMV isolate (EDTA blood) no resistance-associated mutations Ganciclovir / Valganciclovir : S Cidofovir / Brincidofovir: S Foscarnet: S

100% (12/12) 100% (12/12)

cell culture supernatant diluted 1 : 400 in plasma 349016 CMV isolate (EDTA blood) V715M Ganciclovir / Valganciclovir : S Cidofovir / Brincidofovir: S Foscarnet: R

100% (12/12) 100% (12/12)

cell culture supernatant diluted 1 : 3 750 in plasma

Overall success rates in parameters 50 und 60 (mutation in gene region UL54)

100% (12/12) 100% (12/12)

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EQA Scheme (349) Cytomegalovirus Resistance Determination – Success rates 2016 - 2018

UL54-gene region

EQA term Overall success rates for all samples Parameter 50 (Determination of resistance-associated mutation) Parameter 60 (Interpretation of genotypic resistance)

Nov/ Dec 2016

• Nov/ Dec 2017
• Nov/ Dec 2018

100%* (12/12)* 100%* (12/12)*

UL97-gene region

EQA term Overall success rates for all samples Parameter 20 (Determination of resistance-associated mutation) Parameter 30 (Interpretation of genotypic resistance)

Nov/ Dec 2016 82.4% (14/17) 94.1% (16/17) Nov/ Dec 2017 75.0% (12/16) 87.5% (14/16) Nov/ Dec 2018 100%* (18/18)* 100%* (18/18)* *preliminary data

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EQAS Resistance Determination Participants’ Problems

• translation of drug sensitivities into 3-level scheme (S/I/R)
• insufficient sequence quality
• incomplete sequence data
• missing statements of Interpretation of resistance / sensitivity to

antiretroviral drugs

• reporting of none resistance associated mutations
• algorithm selection
• drug mix-up during interpretation

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Summary

Reliable genotyping and antiviral drug resistance determination is of major importance for

• correct selection and evaluation of therapeutic regimes
• successful treatment of patients
• saving money for the health care system
• comparison and harmonization of algorithms

INSTAND EQA schemes for antiviral drug resistance determination

are suitable for analyses of

• sequence quality
• detection of resistance associated

mutations

• interpretation of antiviral drug

resistance considering different algorithms are well proven tools for

• evaluation of competence of the

laboratories

• training of laboratory staff
• evaluation of performance of test

systems

• vigilance of the market (post-

marketing surveillance)

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Partners in the EQA Network

Berlin

Teams of GBD Gesellschaft für Biotechnologische Diagnostik mbH, Berlin and IQVD GmbH - Institut für Qualitätssicherung in der Virusdiagnostik, Berlin

and Düsseldorf

INSTAND-Team and 38 INSTAND Expert Laboratories incl. Robert Koch-Institut Paul-Ehrlich-Institut National Reference and Coniliary Labs Physikalisch-Technische Bundesanstalt (PTB)

and

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• A. Baillot

Niedersächsisches Landesgesundheitsamt, Hannover

• N. Bannert

Robert Koch-Institut, Berlin

• S. Becker / C. Keller / M. Eickmann

Philipps Universität Marburg, Nat. Konsiliarlab. Filoviren

• C.-T. Bock / S. Niendorf / S. Jacobsen / A. Mas-Marques

Robert Koch-Institut, Berlin; Nat. Konsiliarlab. Noroviren; Nat. Konsiliarlab. Rotaviren

• S. Diedrich / S. Böttcher

Robert Koch-Institut, Berlin; NRZ Poliomyelitis und Enteroviren Regionales Referenzlabor der WHO/EURO für Poliomyelitis

• U. Dittmer / S. Ross

Universitätsklinik Essen; NRZ HCV; Nat. Konsiliarlab. Tollwut

• G. Dobler / J. Bugert

Institut für Mikrobiologie der Bundeswehr, München; Nat. Konsiliarlab. FSME

• Ch. Drosten / J. Hofmann / V.M. Corman

Charité Universitätsmedizin Berlin, CCM;

• Nat. Konsiliarlab.Hantaviren; Nat. Konsiliarlab. Coronaviren;

Labor Berlin – Charité Vivantes GmbH

• R. Dürrwald / B. Biere

Robert Koch-Institut, Berlin; NRZ Influenza

• A.-M. Eis-Hübinger

Universität Bonn

• G. Enders / M. Enders

Labor Enders, Stuttgart

• J. Fuhrmann

MVZ Labor 28 GmbH, Berlin

• A. Gessner / B. Schmidt / J. Wenzel / A. Plentz

Universität Regensburg; Nat. Konsiliarlab. HAV und HEV

• S. Günther / J. Schmidt-Chanasit / P. Emmerich

Bernhard-Nocht-Institut, Hamburg; NRZ trop. Infektionserreger, WHO CC

• G. Harms-Zwingenberger

Institut für Tropenmedizin, Berlin

• H. Hengel / D. Huzly / M. Panning

Universitätsklinikum Freiburg; Nat. Konsiliarlab. HSV u. VZV

• J. Hugget / D. O’Sullivan

LGC, National Measurement Laboratory, London, UK

• T. Iftner / K. Hamprecht

Universitätsklinikum Tübingen; Nat. Konsiliarlab. CMV

• A. Karl / K. Frank / K. Gubbe

DRK-Blutspendedienst Nord-Ost, Plauen

• V. Kempf / H. Rabenau / A. Berger

Universitätsklinikum Frankfurt

• O.T. Keppler / J. Eberle / L. Gürtler / H. Nitschko

Ludwig-Maximillians-Universität München, NRZ Retroviren

• F. Klein /U. Wieland /S. Silling /R. Kaiser /E. Heger /E. Knops Uniklinik Köln; NRZ Papillom- und Polyomaviren
• H. Knechten / P. Braun / F. Wiesmann / G. Naeth

Labor Knechten, Aachen

• U.G. Liebert / M. Maier

Universität Leipzig

• P. Maes

KU Leuven, Belgium

• A. Mankertz / S. Santibanez

Robert Koch-Institut, Berlin; NRZ Masern, Mumps und Röteln

• T. Stamminger / D. Michel

Universitätsklinikum Ulm; Nat. Konsiliarlab. CMV

• S. Modrow

Universität Regensburg; Nat. Konsiliarlab. Parvoviren

• S. Nick / H. Scheiblauer

Paul-Ehrlich-Institut, Langen; Prüflabor für IVD

• M. Nübling / M. Chudy / S.A. Baylis / J. Kreß

Paul-Ehrlich-Institut, Langen, WHO CC

• M. Obermeier / M. Schütze

Medizinisches Infektiologiezentrum, Berlin

• J. Reiche / R. Dürrwald

Robert Koch-Institut, Berlin; Nat. Konsiliarlab. RSV, Parainfluenzaviren, HMPV

• T. Schulz / A. Heim / W. Puppe / C. Schmitt

Medizinische Hochschule Hannover; Nat. Konsiliarlab. Adenoviren

• S. Smola / J. Rissland

Universitätsklinikum des Saarlandes

• J. Timm / O. Adams / N. Lübke

Universitätsklinikum Düsseldorf

• K. Überla / K. Korn

Universitätsklinikum Erlangen

• B. Weißbrich

Universität Würzburg

• J. Ziebuhr / D. Glebe / C. Schüttler / S. Slanina / W. Gerlich

Universität Gießen; NRZ HBV und HDV

INSTAND Expert Laboratories (total: 38)

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