HCV-DRAG, Tuesday Nov 17, 2015

Overview of Hepatitis C virus assays

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COBAS® AmpliPrep/COBAS® TaqMan® HCV Test

Overview molecular HCV analyses Viral load and genotyping

Versant HCV Genotype 2.0 Assay (INNO-LiPA)

Overview molecular HCV analyses sequence analysis

RNA isolation RT-PCR Plasma

Population sequencing

Cloning

Ultradeep/next gen seq

Phenotyping Amplimers

Sensitivity for detection mutations: -Population sequencing: ~10-25% -Clonal analysis: ~5% -Ultra Deep Sequencing: ~1%

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HCV genotyping for confirmation of standard assays, e.g., HCV-LiPA

NS5B small (~325 bp)

BLAST Search: homology score

Phylogenetic analysis

HCV (sub)type

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HCV Genotyping: Phylogenetic tree 1a._.6001.EF407422 (0.0205)

1a.US.BID-V105.EU155215 (0.0151)

1a.DE.BID-V35.EU155380 (0.0160)

1a._.7065.EF407455 (0.0248)

P176M0344. (0.0206)

1a.CH.BID-V227.EU482853 (0.0240)

P176M0251B2. (0.0125)

P176M0358B2. (0.0143)

1c.IN.AY051292.AY051292 (0.0242)

1c.IN.Khaja1.AY651061 (0.0262)

1b._.MD27.AF207768 (0.0247)

1b.RU.N589.AY587844 (0.0227)

1b.US.BID-V369.EU155257 (0.0217)

1b.US.BID-V352.EU155306 (0.0296)

1b.JP.MD1-911.AF165046 (0.0303)

1b._.MD32.AF207773 (0.0323)

1b._.MD29.AF207770 (0.0285)

1b.JP.HCV-KT1.AB426117 (0.0303)

1b.JP.MD8-1114.AF165060 (0.0202)

1b.US.BID-V370.EU482839 (0.0397)

5a.GB.EUH1480.Y13184 (0.0369)

5a.ZA.SA13.AF064490 (0.0225)

2a._.G2AK1.AF169003 (0.0297)

2a._.MD2A-2.AF238482 (0.0234)

2a._.MD2A-4.AF238483 (0.0270)

2a._.MD2A-1.AF238481 (0.0320)

2a.JP.JCH-6.AB047645 (0.0244)

2c._.BEBE1.D50409 (0.0688)

2k.MD.VAT96.AB031663 (0.0766)

2b._.JPUT971017.AB030907 (0.0415)

2b._.MD2B-1.AF238486 (0.0316)

2b.JP.HC-J8.D10988 (0.0278)

3a._.CB.AF046866 (0.0378)

3a._.NZL1.NC_009824 (0.0204)

3a.DE.HCVCENS1.X76918 (0.0248)

P176M0359B2. (0.0198)

3a._.K3A.D28917 (0.0430)

3b.IN.RG245.AY515261 (0.0451)

3b.JP.HCV-Tr.D49374 (0.0499)

3k.ID.JK049.D63821 (0.0448)

P176M0063A2. (0.0084)

P176M0293. (0.0035)

P176M0305. (0.0658)

4a._.F7157.DQ418788 (0.0142)

4a._.L835.DQ418789 (0.0155)

4a._.F753.DQ418787 (0.0283)

4a.EG.ED43.Y11604 (0.0416)

4d._.03-18.DQ418786 (0.0734)

7a.CA.QC69.EF108306 (0.1749)

6h.VN.VN004.D84265 (0.0891)

6j.TH.C-0667.DQ835761 (0.0207)

6j.TH.Th553.DQ835769 (0.0209)

6i.TH.C-0159.DQ835762 (0.0260)

6i.TH.Th602.DQ835770 (0.0274)

6m.TH.B4/ 92.DQ835767 (0.0216)

6m.TH.C-0208.DQ835763 (0.0170)

6m.TH.C-0185.DQ835765 (0.0132)

6m.TH.C-0192.DQ835766 (0.0105)

P176M0354. (0.0497)

P176M0363. (0.0565)

6n.CN.KM42.DQ278894 (0.0231)

6n.TH.D86/ 93.DQ835768 (0.0244)

6k.CN.KM41.DQ278893 (0.0311)

6k.CN.KM45.DQ278891 (0.0342)

6k.VN.VN405.D84264 (0.0775)

6l.US.537796.EF424628 (0.0350)

P176M0331A2. (0.0252)

P176M0332A2. (0.0185)

P176M0339. (0.0143)

6b._.Th580.NC_009827 (0.1139)

P176M0337. (0.0213)

P176M0353. (0.0204)

P176M0346. (0.0278)

P176M0345. (0.0347)

P176M0352. (0.0321)

P176M0342. (0.0367)

P176M0343. (0.0512)

6.CN.GZ52557.DQ278892 (0.1203)

6g.HK.HK6554.DQ314806 (0.0290)

6g.ID.JK046.D63822 (0.0273)

6e.CN.GX004.DQ314805 (0.0346)

P176M0340. (0.0240)

P176M0347. (0.0295)

P176M0334A2. (0.0330)

P176M0338. (0.0403)

P176M0355A3. (0.0403)

P176M0336. (0.1361)

6c.TH.Th846.EF424629 (0.0878)

6d.VN.VN235.D84263 (0.0873)

6f.TH.C-0044.DQ835760 (0.0061)

6f.TH.C-0046.DQ835764 (0.0058)

P176M0351. (0.0861)

6q.CA.QC99.EF424625 (0.0272)

P176M0350. (0.0204)

P176M0349. (0.0273)

6p.CA.QC216.EF424626 (0.0365)

P176M0341. (0.0439)

P176M0333A2. (0.0638)

6o.CA.QC227.EF424627 (0.0951)

6t.VN.TV241.EF632069 (0.0295)

6t.VN.TV249.EF632070 (0.0344)

6t.VN.VT21.EF632071 (0.0249)

P176M0348. (0.0876)

1a

1c 1b

5a 2a 2c

2b 3a

3b 3k 4a 4d 6a-t

Clinical samples (boxed) are compared to a large reference set of all HCV genotypes

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Antiviral treatments

NS3 protease/helicase NS5B RNA polymerase NS5A

5’RACE

Availability of PCR assays or Sanger and/or NGS

Target GT1 GT2 GT3 GT4 GT5 GT6

5’UTR-core + + + + + - NS3-4a + + + + + + NS5a + + + + + + NS5B + + + + + + 5’UTR RACE + - - + - -

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HCV Amplification & Sequencing

• No prior subtyping required, only HCV type (except for GT6)

minimal amplification bias

• Using high-fidelity enzymes (Roche Transcriptor & Expand)

minimal polymerase errors

• Detection limit: 1,000-10,000 IU/ml

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Deep sequencing

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Illumina MiSeq

Data analysis and reporting pipeline

Lab report (extensive & complex)

Clinical site report (interpretation)

Total RNA sequencing HCV

First strand cDNA synthesis Second strand cDNA synthesis SPIA amplifciation

Ovation® RNA-Seq System v2 Library preparation

Data analysis: sequence annotation

Total RNA

Illumina sequencing

RNA isolation

Plasma

Extract HCV-matching reads

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Summary DDL HCV services

• Viral load testing • Genotyping HCV

– Siemens Versant HCV Genotyping LiPA 2.0 – NS5B (samples failing standard typing methods)

• Sequencing – GT1 to 6, NS3-4A, NS5A, NS5B covered (type-specific) – Population – Deep sequencing (Illumina MiSeq)

• Target-specific amplicons • Total RNA/full genome

– Clonal

• HCV replicon-based phenotyping • Data management and tailor-made reporting

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Key issues

• Intended use of assays

• Quality assurance

– Standardization, Validation & Proficiency testing

• Reporting complex data

– Need for consensus (procedures & nomenclature)

– FDA requirements (references)

• Clinical interpretation

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