Phar. Nhat Mang/ Roche Vietnam EVALUATION THE EFFECTIVENESS OF TESTING VIRAL LOAD COBAS® AmpliPrep / COBAS® TaqMan® HIV-1 Test v2.0 Phar. Nhat Mang/ Roche Vietnam

Outline Background Objectives Methods Results & Discussions Conclusions Recommendations

Outline Background Objectives Methods Results & Discussions Conclusions Recommendations

<50% of patients on treatment have access to viral load testing Current and goal of HIV epidemic 15 million people on treatment as of March 2015 Only 45% of people living with HIV know they are infected 38% of HIV positive patients receive treatment <50% of patients on treatment have access to viral load testing Goal of ending the AIDS epidemic by 2030 UNAIDS reports

Better treatment monitoring WHO guidelines recommend use of viral load Driving scale up in resource limited settings Better treatment monitoring Use of Viral Load instead of CD4 26 Million Number of people now eligible for ART, and increase of over 10 million 3 Million Number of deaths averted by 2025 if guidelines are implemented Guidelines updated in July 2013, now promote the use of VL testing instead of CD4. Also recommend starting treatment earlier and putting all HIV+ pregnant women on treatment for life HIV viral load assay is the important step to monitor the effectiveness of ARV treatment and also follow up the progression of HIV disease WHO guidelines 2013

Overview HIV Monitoring HIV-1 genomes to stay state-of-the-art HIV is a retrovirus and belongs to the sub-family of lentiviruses. HIV type 1 and HIV type 2 can both cause AIDS although the latter causes a milder and slower disease. HIV-1 is divided into three main groups: Main (M), Outilier (O) and non-M, non-O (N) Subtype D Subtype F Subtype H Subtype J Subtype K CRF_AE Subtype G Subtype C Subtype B Subtype A Group N Group O CRF_AG other CRFs HIV has an extremely high mutation rate which differs from region to region Roche’s Global Surveillance Program

The technique of viral load testing 3 techniques for Real time PCR bDNA (branched-chain DNA) NASBA (nucleic acid sequence based amplification) Real time PCR is the most common method achievement the main goals of accuracy/reliability of test results Sensitivity Specificity Wide detection range Ensures enhanced reliability of test results and more confidence in assessing viral loads

The technique of viral load testing Real time PCR method focus Single target technology Dual target technology DUAL TARGET Target 1 SINGLE TARGET DUAL TARGET Target 2 What is the better technology and most reliable in assessing viral loads ?

Outline Background Objectives Methods Results & Discussions Conclusions Recommendations

Comparative RNA Quantification of HIV-1 CAP/CTM HIV-1 v2.0 and Real-Time HIV-1 PCR Assays Objective 1: To compare the effectiveness of detection of Roche CAP/CTM HIV-1 v2.0 and Real-Time HIV-1 PCR Assays for the detection and quantification of HIV-1 RNA in plasma sample containing different HIV-1 groups and subtypes Objective 2: Reinforce the DUAL target technology to enhances reliability and minimizes the effects against natural mutations than SINGLE Target Mutations can lead to under-quantitation or non-detection of patient specimen

Outline Background Objectives Methods Results & Discussions Conclusions Recommendations

Viral load measurements for patients CAP/CTM HIV-1 v2.0 and Real-Time HIV-1 PCR Assays These PCR assays were implemented with CAP/CTM HIV-1 v2.0: COBAS AmpliPrep and COBAS TaqMan Real-Time HIV-1 : m2000sp/m2000rt CAP/CTM is a fully automated system, reduces vastly the risk of contamination, is faster and has high-throughput

Two (dual) targets: COBAS® AmpliPrep / COBAS® TaqMan® HIV-1 Test v2.0 Gag: Targeted in previous Roche HIV-1 viral load tests to drive correlation Large sequence database from Roche’s Global surveillance program Not a drug target LTR: Targeted in Roche MPx blood screening test Not a drug target A dual target builds redundancy into the assay; potentially avoids underquantification Cobas HIV-1 v2.0 chose gag & LTR region as assay’s target LOD : 20 copies/ml Arellano, E. Virus Genes 2007 34:111-6

Single target: Real-Time HIV -1 int : Targeted in Abbott HIV-1 viral load tests A drug target and over 42 mutations associated with drug resistance to Integrase Inhibitors A dual target builds redundancy into the assay; potentially avoids underquantification Figure: Young T P et al. J. Clin. Microbiol. 2011;49:1631-1634 Abbott Package Insert Reigadas et al. J Antimicrob Chemother. 2013 Apr;68(4):969-72 Simon et al. Lancet. 2006 Aug 5;368(9534):489-504 Real-Time HIV-1 choice the INT (integrase) region (pol gene) as the assay’s target LOD : 40 copies /ml Ceccherini-Silberstein F et al. Characterization and structural analysis of HIV-1 integrase conservation. AIDS Rev. 2009 Jan-Mar; 11: 17-29. Latallaide M et al. Natural polymorphism of the HIV-1 integrase gene and mutations associated with integrase inhibitor resistance. Antivir Ther. 2007; 12: 563-570. Reigadas et al. J Antimicrob Chemother. 2013 Apr;68(4):969-72 Simon et al. Lancet. 2006 Aug 5;368(9534):489-504

Outline Background Objectives Methods Results & Discussions Conclusions Recommendations

Comparative RNA Quantification of HIV-1 CAP/CTM HIV-1 v2.0 and Real-Time HIV-1 PCR Assays 260 plasma sample from HIV-1 group M Roche the median pVL value : 3.08 log10 copies/ml Abbott the median pVL value : 2.81 log10 copies/ml Confirmed that the higher value of the CAP/CTM v2.0 assay Sire JM, et al. J Acquir Immune Defic Syndr. 2011 Mar;56(3):239-43. PubMed PMID: 21164353.

Comparative RNA Quantification of HIV-1 CAP/CTM HIV-1 v2.0 and Real-Time HIV-1 PCR Assays A difference greater than 0.5 log10 copies/ml is considered clinically relevant 20% significantly lower by Abbott than COBAS® AmpliPre/COBAS TaqMan® HIV-1 Test, v2.0 Sire JM, et al. J Acquir Immune Defic Syndr. 2011 Mar;56(3):239-43. PubMed PMID: 21164353.

Comparative RNA Quantification of HIV-1 CAP/CTM HIV-1 v2.0 and Real-Time HIV-1 PCR Assays 84 samples below LOD of Abbott technique (<40 copies/ml) 55 samples below LOD of CAP/CTM v2.0 (<20 copies/ml) 12 samples between 20-40 copies/ml of CAP/CTM v2.0 17 samples above 40 copies/ml of CAP/CTM v2.0 ( range 41-897 copies/ml) A difference of individual result for two techniques Negative by Abbott Negative by CAP/CTM v2.0 Positive by CAP/CTM v2.0 Sire JM, et al. J Acquir Immune Defic Syndr. 2011 Mar;56(3):239-43. PubMed PMID: 21164353.

Integrase region of the pol gene* Roche 2013 4/27/2017 What impact does this have on patients? HIV resistance in a patient likely failing treatment Bryan – this is real for patients Integrase region of the pol gene* HIV resistance in a patient likely failing treatment may not be detected with the Abbott HIV Test Sire JM, et al. J Acquir Immune Defic Syndr. 2011 Mar;56(3):239-43. PubMed PMID: 21164353.

More Literature demonstrates…underquantitation! The Abbott assay appeared to underestimate viral load 40-50% of the time compared to 2nd WHO Standard Janse van Rensburg 30 samples not quantified by the Abbott RealTime HIV-1 assay were quantitated by COBAS® AmpliPrep/COBAS TaqMan® HIV-1 Test, v2.0 Paba Three samples (1%) underquantified by Abbott assay by more than 1 log10 relative to COBAS® AmpliPrep/COBAS TaqMan® HIV-1 Test, v2.0 Bryan Pas Abbott underquants 10% of the samples (n=50, >1 log10 (cp/mL) = 5) Church

Outline Background Objectives Methods Results & Discussions Conclusions Recommendations

Comparative RNA Quantification of HIV-1 Stay One Step Ahead with the HIV-1 dual target assay Objective 1: Higher values obtained with CAP/CTM test v2.0 test relative to the Abbott test Higher sensitivity of CAP/CTM test v2.0 assay (20 copies) compared with the Abbott test The higher meaningful for clinicians and patient because the CAP/CTM test v2.0 test may show low-level viral load in patients whose viral load is undetectable with other methods Objective 2: Abbott – Single target might be impaired by mutations in the integrase target region. Beside that Roche CAP/CTM dual target builds redundancy into the assay, potentially avoids underquantification Ensures enhanced reliability of test results and more confidence in assessing viral loads

Outline Background Objectives Methods Results & Discussions Conclusions Recommendations

Value of result …… COBAS® AmpliPrep / COBAS® TaqMan® HIV-1 Test v2.0 Provides diagnostic accuracy of test results even if mutations occur in one of the two regions Dual target minimizes the effects against MUTATIONS and also represents an important step forward Is fully traceable to WHO international standards and CE-IVD certificated

Doing now what patients need next Thank you Doing now what patients need next