Evaluation of Alere q HIV-1/2 VL Plasma Amilcar Tanuri Orlando C. Ferreira Jr. Molecular Virology Laboratory Universidade Federal do Rio de Janeiro, Rio de Janeiro Brazil

The importance of viral load (VL) for ART Monitoring VL is an excellent indicator of HIV replication and efficacy of ARV treatment High VL is associated with: Faster progression to AIDS Higher vertical transmission Higher sexual transmission When ART is started VL responds much faster than CD4 or Clinical Parameters

Use of VL to Monitor Antiretroviral Therapy WHO Guideline recommend VL to monitor ARV treatment WHO defines virological failure as 2 consecutive results greater than 1,000 copies/ml over 3 months Centralized testing is problematic in Low Income Countries Transportation of specimens as well as returning of results Turn around time can be problematic for intervention measures (pregnant women) Around 40% of results are not returned to patient’s files

WHO Guidelines on ART

Dried Blood Spots (DBS) for VL DBS has been used in the past for Early Infant Diagnosis (EID) For VL DBS collection by finger stick can be problematic because of variable blood volume in the spots (for EID this is not so critical) WHO has evaluated the performance of DBS for VL and consider specificity and sensitivity >85% acceptable

POC VL is a important technology for the availability of VL in Low Income Countries The Alere q platform is a interesting device to use in low income countries; Dependant on the assay, test results range from approx. 60 minutes and one machine can run from 8-12 samples per 8hs working day; Importantly, the result is available for the health professional during the patient visit and allows for immediate action

Cartridge holds all reagents needed Alere q Cartridge Alere q Analyzer Cartridge holds all reagents needed for running the test Luis o que voce quer colocar aqui mais sobre a tecnologia Handling and Processing RNA Isolation Reverse Transcription and Amplification Detection and Quantification

Alere q – Filling the Cartridge Fill the cartridge with 50 µL plasma Check for sufficient sample loading Close the cartridge cap Amplification & Target Detection Competitor Monitored Amplification (CMA) and Displacement of Fluorescence Background

Alere q HIV-1/2 VL plasma Preliminary Results - Limit of Detection LoD Summarized for HIV-1 M, HIV-1 O and HIV-2 Analyte Limit of Detection (95 % CI) HIV-1 group M 358 cp/mL (291 cp/mL, 475 cp/mL) HIV-1 group O 232 cp/mL (189 cp/mL, 302 cp/mL) HIV-2 352 cp/mL (284 cp/mL, 464 cp/mL) HIV-1M/N HIV-2 HIV-1 O Number of valid run per point: 84 (minimum) and 93 (maximum)

Alere q HIV-2 Viral Load N = 5 N = 3 N = 8

Study Protocol A cohort of 450 patients under ARV (first line) therapy are followed in the outpatient clinics of the HUGG, Rio de Janeiro; At every visit a plasma sample is collected and stored for future analysis From this cohort, 26 patients had virological failure after 18 months since beginning of ARV therapy;

Alere q evaluation: South Africa Sensitivity and specificity against Roche CAPCTM At 1000 cp/ml Source: S. Carmona (AIDS2016)

Alere q evaluation: Brazil Sensitivity and specificity against Abbott M2000 95.4 (87.1 – 99.0) 91.4 (81.0 – 97.1) N = 65 N = 58

VL of patients on ART and (suspected) virological failure

How to obtain small amounts of plasma volume without the need of venous puncture? To apply Alere q to (small) clinical settings it is important to develop a simple procedure to obtain 50 µL of plasma from finger pricking! An adequate lancet to finger prick; Collect blood in a microtainer containing EDTA; A small centrifuge to obtain plasma; A power source for the centrifuge.

Collecting finger stick capillary blood using a microtainer

Final Remarks Alere q HIV-1/2 VL plasma assay meets the acceptable sensitivity and specificity established by WHO for DBS VL assays; Sens.: 95.4% (95% CI 87.1 – 99.0) Spec.: 91.4% (95% CI 81.0 – 97.1) The limit of detection (LoD) for HIV-1, HIV-2 and HIV-1 group O is in the same range; Alere q LoD for HIV-1 is 358 cp/mL, below the 1,000 cp/mL defined by WHO as cutoff for ART virological failure 50 µl plasma sample volume is feasible to obtain in point of care settings, making Alere q a good candidate to monitor patients under ARV treatment

Isabelle Vasconcellos, Acknowledgments: UFRJ: Diana Mariani UNIRIO: Isabelle Vasconcellos, Cassia Alves , Luis Ribeiro, Marcelo Velho Alere Brazil: Luis Felipe Gonzales