WHO Prequalification of In Vitro Diagnostics PUBLIC REPORT. Product: Alere q HIV-1/2 Detect WHO reference number: PQDx

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1 WHO Prequalification of In Vitro Diagnostics PUBLIC REPORT Product: Alere q HIV-1/2 Detect WHO reference number: PQDx Alere q HIV-1/2 Detect with product codes , and , manufactured by Alere Technologies GmbH, CE marked regulatory version, was accepted for the WHO list of prequalified in vitro diagnostics and was listed on 13 June The public report was amended on 2 September 2016, to reflect fulfillment of an outstanding commitment to WHO prequalification. Intended use: Alere q HIV-1/2 Detect is a qualitative nucleic acid amplification test for the detection of human immunodeficiency virus (HIV) type 1 groups M/N and O, and type 2 in human whole blood and plasma specimens. The test can be used in laboratory as well as nonlaboratory environments by trained health care or laboratory professionals or other health care workers receiving appropriate training. It can be used as an aid in the diagnosis of HIV infection in paediatric and adult individuals, or as an additional test when specimens have already been tested using alternative methods. Alere q HIV-1/2 Detect is not intended to be used as a donor screening test for HIV. In order to perform an Alere TM q HIV-1/2 Detect test the Alere TM q instrument is required. Alere q is a portable automated bench-top analyser for processing Alere q test cartridges. Assay description: Sample Handling and Processing Peripheral blood may be collected from the patient either through standard finger or heel prick sampling techniques or venous blood draw. The sample volume required to run a test is 25 μl. Finger or heel prick blood can be applied directly and immediately onto the Alere q HIV-1/2 Detect cartridge. When using EDTA anti-coagulated venous blood or plasma, the appropriate volume is transferred into the cartridge using a volumetric pipette or transfer capillary. Standard phlebotomy sample collection practices for obtaining both capillary and venous blood samples are to be followed. After applying the sample, the cartridge cap is snapped into place, eliminating the chance of sample spillage or contamination of the instrument. After closing the cap the cartridge is inserted into the Alere q and the test is initiated automatically. The steps described in the following subsections are performed automatically by the Alere q within the cartridge. RNA Isolation Page 1 of 57

2 The RNA isolation consists of following steps: a) Complete lysis of the sample based on chaotropic salts in order to release all nucleic acids including cell-associated HIV RNA and HIV RNA from plasma based particles. b) Hybridization of oligonucleotides complementary to specific sequences of the HIV- 1 and HIV-2 genome. These sequence specific capture oligonucleotides carry a 3 - terminal biotin-residue. c) All biotinylated capture oligonucleotides are captured onto the surface of Streptavidin-Sepharose particles, thus any HIV RNA bound to a captured oligonucleotide is captured on the Sepharose too. d) Washing of the Streptavidin-Sepharose particles to remove all contaminants that bind non-specifically to the particles, i.e. human nucleic acids, cellular and extracellular proteins, cell membrane fragments and low molecular weight molecules. After the washing steps, the remaining HIV RNA molecules are ready for reverse transcription (RT) followed by polymerase chain reaction (PCR). Reverse Transcription and Amplification RNA which is captured onto the surface of the Streptavidin-Sepharose particles cannot be detected directly. Therefore an amplification of HIV-specific nucleic acid sequences has to be performed. This is realized by PCR which allows an in vitro amplification of DNA sequences. Since most DNA polymerases do not synthesize DNA directly from RNA a reverse transcription of RNA into cdna is necessary. Reverse transcription is an isothermal reaction which is performed at a defined temperature. The same reverse primers as for the subsequent PCR amplification are used for RT. The DNA primers hybridize with their complementary sequence onto the RNA and form a DNA-RNA hybrid. The reverse transcriptase then transcribes the RNA into its complementary cdna by extending the oligonucleotide primer. The reverse transcription is followed by a denaturation step at a defined temperature in order to deactivate the reverse transcriptase activate the DNA polymerase and separate the RNA-DNA hybrid to make the newly formed cdna accessible for primer oligonucleotide binding and cyclic primer extension by PCR. Primers are short specific oligonucleotides that hybridize readily to their complementary sequences at the appropriate annealing temperature (annealing) and form the starting point for extension by a heat stable DNA polymerase. Primer annealing and the amplification of the DNA (elongation) by DNA polymerase are carried out at defined temperatures. The three steps (denaturation, annealing and elongation) describe one PCR cycle and are repeated 45 times. To facilitate simultaneous detection of more than one specific nucleic acid sequence a multiplex PCR is performed. Target amplification between HIV-1 group M/N and group O, and HIV-2 is facilitated by specific primer pairs. In addition the primer pairs allow for the amplification of internal process controls. Detection Page 2 of 57

3 Detection of PCR product is based on Competitive Reporter Monitored Amplification technology utilizing an array of immobilized oligonucleotide probes and complementary fluorescently labelled reporter oligonucleotides in solution. In order to maximize initial signal intensity, reporters used in this reaction have fluorescence labels at the 5 and the 3 ends. Under suitable conditions the reporter will specifically hybridize to the immobilized probes. The reporter oligonucleotides are also complementary to a specific sequence of a target amplicon that is generated during PCR that compete with the immobilized probes for binding of the reporter oligonucleotides. At the onset of the amplification reaction, none or a few target molecules are present and thus the reporter is free to bind to its complementary probe on the array. In the presence of target template more target amplicons with a reporter specific binding site are synthesized as the amplification reaction proceeds. As amplicons accumulate the hybridization kinetics become more dependent on the amplicon concentration. The more amplicons are synthesized the more reporter binds to them. In addition, the solid support to which the oligonucleotide probe is attached introduces a diffusion barrier which significantly reduces the hybridization rate. Thus, generally, solution phase reactions are kinetically favoured to solid phase reactions. Therefore the amount of reporter hybridized to the complementary probe decreases proportionally to the formation of new amplicons. This decrease is observed until a plateau in the amplification reaction is reached. Measuring the change in signal intensity of each probe can be achieved by imaging the fluorescence pattern on the array during the amplification process. Fluorescence images are collected during the annealing phase of each amplification cycle. After acquiring the hybridization pattern an algorithm is applied that is able to identify and eliminate different noise signals from the data obtained by the array assisted real-time PCR. The algorithm then calculates the cycle threshold values from the resulting amplification kinetics determining the presence of the analyte. Test kit contents: Alere q HIV-1/2 Detect Cartridge Kit 10 x Cartridge Kit (product code ) Individually pouched test cartridges Guide x Cartridge Kit (product code ) Page 3 of 57

4 Instrumentation required: Item Quantity Equipment: Alere TM q (product code ) 1 Alternative Equipment Kits: Alere TM q Complete (product code ) -(containing product codes , , 1x per kit component , , ) Alere TM q Complete II (product code ) -(containing product codes , , 1x per kit component , , ) Consumables required but not provided: Item Finger Stick Sample Collection Kit (product code ) Neonatal Sample Collection Kit (product code ) Plastic Capillaries plain (product code ) Plastic Capillaries EDTA-K2 (product code ) Quantity x x 100 Items required but available separately: Item Quantity Alere TM USB Printer (product code ) 1 Alere TM Power Drum (product code ) 1 Alere TM Connectivity Pack I (product code ) 1 Alere TM Connectivity Pack II (product code ) 1 Printer Paper I (product code ) 10 Storage: The test kit should be stored at 4-30 C. Shelf-life upon manufacture: 9 months. Warnings/limitations: 1. WHO made suggestions to the instructions for use that will be amended in the forthcoming round of revision. 2. For warnings and limitations, please refer to current version of instructions for use. Page 4 of 57

5 Summary of WHO prequalification assessment for Alere q HIV-1/2 Detect Date Outcome PQ amended 2 September 2016 listed PQ listing 13 June 2016 listed Dossier review 18 December 2015 MR Site inspection(s) of quality management system 15 to 17 September MR 2015 Laboratory evaluation of performance and operational characteristics 14 August 2015 to 11 May 2016 MR MR: Meets requirements N/A: Not applicable Prioritization for prequalification Based on the established criteria, Alere q HIV-1/2 Detect was given priority for WHO prequalification. Product dossier assessment Alere Technologies GmbH submitted a product dossier for Alere q HIV-1/2 Detect as per the Instructions for compilation of a product dossier (PQDx_018 v1). The information (data and documentation) submitted in the product dossier was reviewed by WHO staff and external technical experts (assessors) appointed by WHO. Notwithstanding, certain aspects of the product dossier submitted for stringent regulatory review for the purposes of CE marking were reviewed by a technical expert during the site inspection. Following this review, a number of issues of concern for WHO are to be resolved as commitments for prequalification: 1. Revision to the instructions for use as per WHO requests by December WHO will follow-up on implementation of these commitments at the next re-inspection. Based on the product dossier screening and assessment findings, the product dossier for Alere q HIV-1/2 Detect meets WHO prequalification requirements. Manufacturing site inspection A comprehensive inspection was performed at the site of manufacture (Loebstedter Str , Jena, Germany) of Alere q HIV-1/2 Detect in 15 to 17 September 2015 as per the Information for manufacturers on prequalification inspection procedures for the sites of manufacture of diagnostics (PQDx_014 v1). The inspection found that the Page 5 of 57

6 manufacturer had an acceptable quality management system and good manufacturing practices in place that ensured the consistent manufacture of a product of good quality. The manufacturer's responses to the nonconformities found at the time of the inspection were accepted on 2 May Based on the site inspection and corrective action plan review, the quality management system for Alere q HIV-1/2 Detect meets WHO prequalification requirements. Laboratory evaluation Alere q HIV-1/2 Detect assay is a qualitative nucleic acid amplification test for the detection of Human Immunodeficiency Virus (HIV) type 1 groups M/N and O and type 2 in human whole blood and plasma. This evaluation was performed on venous whole blood. A volume of 25µl of whole blood/specimen is needed to perform the assay. This type of assay does require laboratory equipment and can be performed in laboratories with limited facilities. Analytical specimens: The assay detected the following subtypes: A, B, C, D, F, AE, AG in whole blood. The total hit rate for 21 replicates in the precision study was 100%. The limit of detection was estimated to be 2937 IU/ml [95% Fiducial limits: ]; copies/ml [95% Fiducial Limits ]. No carry-over was detected. Clinical specimens: The reproducibility assessment showed a hit rate of 100%, while the reproducibility hit rate was determined to be 100%. In this limited performance evaluation, on a panel of 301 infant whole blood specimens we found, in specimens from infants aged less than 18 months, an initial sensitivity of 98.67% (95% CI: ) and an initial specificity of % (95% CI: ) compared to the reference results for all specimens tested. In specimens from individuals older than 15 years of age, we found an initial sensitivity of 90.00% (95% CI: ) and an initial specificity of % (95% CI: ). The final sensitivity and specificity for infant whole blood specimens were 99.33% (95% CI: ) and % (95% CI: ) respectively. In specimens from individuals older than 15 years of age, we found a final sensitivity of 94.00% (95% CI: ) and a final specificity of % (95% CI: ). In this study, the invalid rate was 5.58%. Page 6 of 57

7 Performance characteristics in comparison with an agreed reference standard Initial (95% CI) Final (95% CI) Sensitivity % Infant specimens: 98.67% ( ) 98.69% ( ) Adult specimens: 90.00% ( ) 94.00% ( ) Specificity % Infant specimens: Adult specimens: % ( ) % ( ) 100% ( ) % ( ) Invalid rate % 5.58% Additional performance characteristics Subtype detection 21/21 were correctly classified Limit of detection using WHO 3 rd 2937 IU/ml [95% Fiducial limits: ]; International Reference Standard copies/ml [95% Fiducial Limits ] Carry-over 0% Key operational characteristics Validated specimen types Number of steps Time to result Internal QC In-use stability of reagents Venous (EDTA) and capillary whole blood. EDTA plasma. 4 steps 0h:56 minutes (3-4 min hands-on time, 52 minutes on instrument) Assay Process Controls 1- Internal process controls for HIV-1 and HIV-2 2- Positive hybridization control 3- Negative hybridization control Reagents are all contained within the cartridge which is single use. Filled cartridges need to be processed immediately after sample loading. Page 7 of 57

8 Labelling 1. Labels 2. Instructions for use Page 8 of 57

9 1. Labels 1.1 Alere TM q HIV-1/2 Detect Cartridge Top Label Cartridge User Label Page 9 of 57

10 Primary Packaging Label Secondary Packaging Label Page 10 of 57

11 1.2 Consumables Finger Stick Sample Collection Kit Packaging Labels Neonatal Sample Collection Kit Packaging Labels Page 11 of 57

12 1.3 Instrument Kits Alere TM q Device Label Page 12 of 57

13 Packaging Label Page 13 of 57

14 1.3.2 Alere TM q Complete Packaging Label Page 14 of 57

15 1.3.3 Alere TM q Complete II Packaging Label Page 15 of 57

16 1.4 Optional Items Alere TM Power Drum Device Label Packaging Label Page 16 of 57

17 1.4.2 Alere TM Power Drum Component Labels Battery Module Battery Label Packaging Label Page 17 of 57

18 Alere TM Power Drum Module Device Label Packaging Label Page 18 of 57

19 1.4.3 Alere TM USB Printer Device Label Packaging Label Page 19 of 57

20 1.4.4 Alere TM Connectivity Packs Alere TM Connectivity Pack I Packaging Label Alere TM Connectivity Pack II Packaging Label Page 20 of 57

21 2. Instructions for use 2.1 Alere TM q HIV-1/2 Detect Page 21 of 57

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