AREVIR-GenaFor-Meeting Bonn, 05.05.11 ________________ Comparison - - PowerPoint PPT Presentation

________________ Comparison of HIV-1 Viral Load samples between RealTime (Abbott) and COBAS TaqMan (Roche) ________________ Gudrun Naeth PZB AACHEN

• Blondelstr. 9 52062 Aachen

AREVIR-GenaFor-Meeting Bonn, 05.05.11

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Introduction Introduction

Low Level Viremia Comparision of Roche COBAS TaqMan vs.

Abbott m2000

Results

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Transient Transient low low level level viremia viremia during during ART ART

date ART HIV-1 RNA CD4 absolute CD4 relative 01.2001 AZT 3TC EFV 11.2001 AZT 3TC LPV/r 03.2002 <50 643 33 01.2003 TDF 3TC LPV/r 04.2004 <50 03.2005 47 01.2006 01.2007 <40 (det.) 641 37 01.2008 <40 (non det.) 617 31 02.2009 <40 (det.) 715 33 05.2009 59 617 31 07.2009 <40 641 37 11.2009 72 675 38 01.2010 40 659 37 05.2010 71 615 37

69

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Current Current Treatment Treatment Recomendations Recomendations

goal of therapy virologic failure <20-50 copies/ml >50 copies/ml

„evaluate virologic failure“

1 Deutsche AIDS-Gesellschaft e.V. (DAIG). Deutsch-Österreichische Leitlinien zur Antiretroviralen Therapie der HIV-1-Infektion (04.02.2010). 2 European AIDS Clinical Society (EACS). Guidelines. Clinical Management and Treatment of HIV-infected Adults in Europe. Version 5-3. 2011. 3 International AIDS Society (IAS). Recommendations. Antiretroviral Treatment of Adult HIV Infection. 2010. 4 Department of Health and Human Services (DHHS). Guidelines for the Use of Antiretroviral Agents in HIV-1 infected Adults and Adolescents (2011).

Europe2 2x >50 copies/ml International3 <50 copies/ml (PCR) <75 copies/ml (bDNA) 2x >50 copies/ml (PCR) 2x >75 copies/ml (bDNA) <50 copies/ml Germany1 2x >200 copies/ml <48 copies/ml International4

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HIV HIV-

• viral

viral load load measurements measurements and and resistance resistance analysis analysis

(PZB Aachen) (PZB Aachen)

Genotypic resistance analysis: reverse transcriptase and protease

number of measurments

n=910

61,2 % 61,1 %

<50 cop/ml <40 cop/ml

66,2 % 63,7%

<40 cop/ml <40 cop/ml

n=1.353 n=1.243

Braun P., 13 MAT, 2010 7.719 28.06.2010

71,6% <40 Kop/ml

40-100 copies/ml: 4,2% 101-1000 copies/ml: 5,9% >1000 copies/ml: 17,8%

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1 10 100 1.000 10.000 100.000 1.000.000

Nov 07 Jan 08 Feb 08 Mrz 08 Apr 08 Jun 08 Jan 09 Jan 10 Jan 11

HIV RNA [Kopien/ml] 200 400 600 800 1.000 CD4 [Zellen/µl]

antiretroviral therapy

Therapy Therapy success success = = viral viral load load below below detection detection limit limit?? ??

detection limit Abbott RealTime: 40 copies/ml Roche TaqMan: 20 copies/ml Siemens Versant kPCR: 37 copies/ml

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Abbott m2000 / Roche Abbott m2000 / Roche TaqMan TaqMan v 1.0 v 1.0

Automated COBAS AmpliPrep COBAS TaqMan Gag 1,0 mL 20-10.000.000 copies/mL Automated m2000sp m2000rt Pol IN 0,6 mL 40-10.000.000 copies/mL Cobas Ampli-Prep Cobas TaqMan HIV-1 assay Abbott Real Time HIV-1 assay

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Reproducibility and Precision

Two real-time PCR assays with international standards and serial dilutions of a patient sample

• 40,000 IU/ml = 24,000 copies/ml
• Three dilutions: 1x102, 5x102, and 1x103 copies/ml
• 20-fold measurement
• 100,000 IU/ml = 60,000 copies/ml
• Five dilutions: 1x102, 2.5x102, 5x102, 1x103, and 2.5x103 copies/ml
• 20-fold measurement for lowest viral load and 10-fold for other dilutions

PEI-Standard (Paul-Ehrlich-Institut) WHO-Standard (World-Health-Organisation) Patient Sample (PZB Aachen)

• Seven dilutions: 5x101, 1x102, 1x103, 1x104, 5x104, 1x105 and 5x105 copies/ml
• 10-fold measurement

Braun P., Ehret R., Wiesmann F, et al. Comparison of four commercial quantitative HIV-1 assays. Clin Chem Lab Med; 2007; 45: 93-99

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20-fold measurement

PEI-Standard

Reproducibility and Precision

1x102 5x102 1x103

B

R2=0,72

CAP/CTM v 1.0

1x102 5x102 1x103

A

R2=0,91

m1000/m2000rt

32% CV 16% CV 16% CV 56% CV 49% CV 41% CV

Braun P., Ehret R., Wiesmann F, et al. Comparison of four commercial quantitative HIV-1 assays. Clin Chem Lab Med; 2007; 45: 93-99

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WHO-Standard

Reproducibility and Precision

20 (10)-fold measurement

1x102 2,5x102 5x102 1x103 2,5x103 1x102 2,5x102 5x102 1x103 2,5x103

C D

R2=0,96 R2=0,88 Nominal level [copies/ml] Nominal level [copies/ml] Measurement [copies/ml] Measurement [copies/ml]

m1000/2000rt CAP/CTM v 1.0

23% CV 15% CV 21% CV 12% CV 14% CV 48% CV 22% CV 20% CV 47% CV 25% CV

Braun P., Ehret R., Wiesmann F, et al. Comparison of four commercial quantitative HIV-1 assays. Clin Chem Lab Med; 2007; 45: 93-99

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1,7 2,0 3,0 4,0 4,7 5,0 5,7 10 100 1000 10000 100000 1000000

Expected concentrations in log copies/ml

Measurement [copies/ml]

m2000rt m2000rt TaqMan TaqMan v 1.0 v 1.0

1,7 2,0 3,0 4,0 4,7 5,0 5,7 18% CV 11% CV 18% CV 9% CV 19% CV 34% CV 23% CV 36% CV 39% CV 37% CV

Reproducibility and Precision

Clinical Sample

10% CV 28% CV

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Correlation on Clinical Samples m2000rt versus TaqMan v 1.0

TaqMan minus m2000rt Differences (mean) in log10 copies/mL = 0,22 R2: 0,937

TaqMan [log copies/mL] m2000rt [log copies/mL]

Braun P., Ehret R., Wiesmann F, et al. Comparison of four commercial quantitative HIV-1 assays. Clin Chem Lab Med; 2007; 45: 93-99

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Comparison between Roche TaqMan (version 2) and Abbott m2000 Evaluation: Low Evaluation: Low level level viremia viremia

Slides not available Results in press, 2011

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Retrospective analysis of plasma samples (detectable and undetectable viral load) with RealTime and TaqMan v. 2.0

5 Groups (10 patients, 5 measuring points) Group 1: VL <40 copies/ml (not detected) Group 2: VL <40 copies/ml (detected not + detected) Group 3: VL <40 copiesn/ml (not detected) Group 4: VL >40 – 100 copies/ml Group 5: VL >40 – 200 copies/ml

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Retrospective Analysis

Date Previous value m2000 CTM 6 24.08.2010 41 63 116 17.08.2009 87 87 206 19.02.2009 79 73 192 20.10.2008 193 91 219 18.07.2008 120 97 168 Example

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Thank you for your attention!