Evaluation of Alere q HIV12 VL Plasma Amilcar

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 1

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: o Faster progression to AIDS o Higher vertical transmission o Higher sexual transmission o When ART is started VL responds much faster than CD 4 or Clinical Parameters 2

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 3

WHO Guidelines on ART 4

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 5

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 812 samples per 8 hs working day; • Importantly, the result is available for the health professional during the patient visit and allows for immediate action 6

Alere q Cartridge Alere q Analyzer Cartridge holds all reagents needed for running the test § § Handling and Processing RNA Isolation Reverse Transcription and Amplification Detection and Quantification 7

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 8

Alere q HIV-1/2 VL plasma Preliminary Results - Limit of Detection Lo. D Summarized for HIV-1 M, HIV-1 O and HIV-2 Analyte Limit of Detection (95 % CI) HIV-1 group M 358 cp/m. L (291 cp/m. L, 475 cp/m. L) HIV-1 group O 232 cp/m. L (189 cp/m. L, 302 cp/m. L) HIV-2 352 cp/m. L (284 cp/m. L, 464 cp/m. L) HIV-1 M/N HIV-2 HIV-1 O Number of valid run per point: 84 (minimum) and 93 (maximum) 9

Alere q HIV-2 Viral Load 8. 0 HIV-1 + HIV-2 7. 0 VL (Log cp/m. L) 6. 0 5. 0 4. 0 3. 0 2. 0 1. 0 0. 0 HIV-1 HIV-2 N=5 HIV-1 HIV-2 N=3 HIV-1 HIV-2 N=8 10

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; 11

Alere q evaluation: South Africa Sensitivity and specificity against Roche CAPCTM At 1000 cp/ml Source: S. Carmona (AIDS 2016) 12

Alere q evaluation: Brazil Sensitivity and specificity against Abbott M 2000 Abbott x Alere q Lin. Reg. (N=123) Alere q VL (Log cp/m. L) 6. 0 5. 0 4. 0 Agmt Q 3. 0 Agmt ND 2. 0 Agmt NQ 1. 0 Misc f. Pos 0. 0 Misc f. Neg 0. 0 1. 0 2. 0 3. 0 4. 0 Abbott VL (Log cp/m. L) 5. 0 6. 0 Sensitivity Specificity % (95% CI) 95. 4 (87. 1 – 99. 0) 91. 4 (81. 0 – 97. 1) N = 65 N = 58 13

VL of patients on ART and (suspected) virological failure 6 Abbott - 1 st visit VL (Log cp/m. L) 5 4 3 Alere q Lo. D 2 1 0 -1 2 3 4 5 6 7 Patient # 8 9 10 6. 0 5. 0 4. 0 VL (Log cp/m. L) 1 3. 0 2. 0 1. 0 0. 0 Abbott 3. 0 Abbott Alere q 2. 0 Alere q 1. 0 Patients/Visits 0. 0 Patients/Visits 14

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. 15

Collecting finger stick capillary blood using a microtainer 16

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 (Lo. D) for HIV-1, HIV-2 and HIV -1 group O is in the same range; • Alere q Lo. D for HIV-1 is 358 cp/m. L, below the 1, 000 cp/m. L 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 17

Acknowledgments: UFRJ: Diana Mariani UNIRIO: Isabelle Vasconcellos, Cassia Alves , Luis Ribeiro, Marcelo Velho Alere Brazil: Luis Felipe Gonzales 18