Special INSTI

20

Transcript of Special INSTI

• Built-in true human IgG Control• Use 50μL of EDTA-whole blood, fingerstick blood, serum or EDTA-plasma

• Rapid results at a glance (as little as 60 seconds)• Proven early antibody detection• Large scale Canadian clinical trial data shows a >99.5% combined accuracy• Easy to understand instructions• No timing required• No refrigeration required

To ensure the integrity and quality of every test, INSTI® is manufactured in a state of the art facility with a fully implemented Certified Quality Management System (ISO 13485). Our staff of professionals in chemistry, molecular biology, biochemistry, engineering and highly skilled technicians adhere to strict process controls. Our facility is compliant with all regulations for the manufacture of medical devices.All raw materials and processed kit components are thoroughly inspected and tested to ensure they meet strict quality guidelines. Checks at each step of manufacturing ensure continued quality control. Rigorous in-process testing using EDTA-whole blood, plasma and serum is performed during each production run.All INSTI® solutions are proprietary formulations allowing us to control quality in our own laboratories. Our research and development team continues to develop new diagnostic tests.

Special INSTI® Features

Commitment to Quality

A D V A N C E D

D I A G N O S T I C

TECHNOLOGY

FAST• Result in 60 seconds

SIMPLE• All reagents in ready-to-use bottles

• No measuring or drop counting

• No contamination

• No timing required

• No additional materials required

CONVENIENT• Utilises EDTA-whole blood, fingerstick

blood, serum or EDTA-plasma

ACCURATE• Excellent correlation with approved

laboratory methods for HIV antibody detection

SENSITIVE & SPECIFIC• Recombinant HIV-1 and HIV-2 proteins

offer a >99.5% combined accuracy

TRAINING PACK

2 3

Table of Contents Interpretation of Results

HIV SPOT INTERPRETATION

SYPHILIS SPOT INTERPRETATION

HIV & SYPHILIS SPOT INTERPRETATION

Strong Reactive

Always readresults withINSTI® logofacing you

Strong Reactive

Strong Reactive Weak Reactive

Weak Reactive

Weak Reactive

Indeterminate

Indeterminate

Negative

Negative

Control

Syphilis

HIV

Always readresults withINSTI® logofacing you

Always readresults withINSTI® logofacing you

Interpretation of Results/Images 3

Guidelines & Interpretation 4

Guidance for Fingerstick Sample 5

Lateral View of Membrane Unit 6

INSTI® Do’s and Don’ts & FAQ’s 7

FAQ’s & Summary and Intended Use 8

Troubleshooting 9

Quick Reference 10

True Human IgG Control Spot 11

Instructions for Use 12-15

Test Controls IFU 16

Bibliography 17-18

Clinician’s Briefing Notes 19

Mutliplex Joint Test

Four Simple Steps 20

Interpretation Of Results 21

Instructions for Use 22-26

Syphilis Test Control IFU 27-28

Self Test

Introduction 28

Self Test IFU 29

Control

HIV

Syphilis

Control

Syphilis detection is only available on the INSTI® Multiplex HIV-1/2 Syphils Ab Test.

4 5

Guidelines & Interpretation Guidelines for obtaining fingerstick sample for an INSTI® test

Please be advised that the appearance of the control dot on the blotted membrane units may be non-homogeneous in appearance, with patterns of lighter and darker colour intensities appearing within the intact dot area as illustrated above. Control dots with this non-homogeneous appearance are valid and should be interpreted as per the INSTI® Instructions for Use (package insert).

bioLytical has conducted extensive testing of INSTI® membrane units and concludes that the INSTI® membrane units displaying these non-homogeneous control dots continue to meet all Quality Control and performance specifications including sensitivity and specificity.

bioLytical has filed vigilance reports on this observation with the appropriate regulatory and competent authorities.

Colour intensity may be variable between the ring and the dot.

Non-homogeneous appearance of the control dot

1 Patient to wash hands thoroughly in warm soapy water.

2 With gloves on, milk fingers towards the tip while observing which finger is optimal - look for the one that has the deepest colour for the ultimate blood flow.

3 Holding the finger correctly as you use the lancet.

4 Angle the capillary tube horizontally. When collecting blood sample hold the capillary tube at an angle as shown in the picture. Capillary action will be assisted by gravity.

TRAINING PACK

6 7

Lateral view of membrane Do’s and Don’ts & Frequently Asked Questions

Lateral view of Absorbent PadDuring INSTI® Procedure

1

2

3

Sample diluent absorbs through membraneinto absorbent pad

Colour developerblends with sample inabsorbent pad

Clarifying solutionblends with the othersolutions in the pad

Over time, the solutions in the absorbent pad spread uniformly, and can stain the membrane from below, occasionally resulting in a faint shadow at the test spot.

Do’s

3 Read the entire package insert and become familiar with its contents prior to beginning the test.

3 Ensure all test components and any stored specimens are at room temperature.

3 Warm the fingertip before using the lancet, to ease blood flow.

3 Allow the alcohol from the swabbed fingertip to dry completely before applying the lancet.

3 After addition of blood sample to bottle #1, shake well and immediately pour into the membrane unit.

3 Shake bottle #2 to re-suspend the solution before pouring into the membrane unit.

3 Use local universal precaution guidelines for infectious materials in handling and disposing of specimens and used testing materials.

Don’ts

7Do not mix reagents from different lots.

7Do not use reagents or kits beyond the stated expiration date on the outer packaging.

7Do not use the test kit if there is any sign of damage to or previous opening of the sealed membrane pouch, or leakage in any of the reagent bottles.

7Do not conduct repeated sampling of fingerstick blood with the disposable transfer pipette. 50μL should be collected in one continuous flow.

7Do not dilute the specimens prior to adding to bottle #1 of the INSTI® test.

7Do not add the bottle #1 contents to the membrane unit without mixing well just before addition.

7Do not add specimen directly to INSTI® membrane unit.

7Do not introduce delays between any of the INSTI® procedural steps.

7Do not interpret the results if more than 5 minutes have elapsed after adding the Clarifying Solution (bottle #3) to the INSTI® membrane unit.

What is the accuracy of the INSTI® kit?Proven accuracy of >99.5%

Does INSTI® differentiate HIV-1 from HIV-2 antibodies?No. INSTI® contains antigens from HIV-1 and HIV-2 in the membrane unit and can detect antibodies to HIV-1 and/or HIV-2 in patients’ specimens, but it will not differentiate the two.

Is a reactive INSTI® test conclusive of HIV diagnosis?No. INSTI® is considered as a first-line test only, and all patients with reactive or indeterminate INSTI® results should be re-tested with an HIV confirmatory assay before final HIV antibody status can be determined. As in all assays for HIV antibody, false positives can occur infrequently with INSTI®.

Are INSTI® results as accurate as laboratory-based HIV testing procedures?Yes. INSTI® is a registered and approved medical device and has undergone extensive clinical trials that have demonstrated its accuracy in direct comparison to laboratory-based methods. Much of this data is published in the INSTI® package insert.

Does INSTI® detect HIV antibodies early in the course of infection?Yes. In commercial HIV seroconversion panels and ‘real life’ documented seroconversion cases, INSTI® is equivalent to the most sensitive laboratory-based assays in early antibody detection. However, patients who are in the pre-antibody “window period” of HIV infection may not be reactive in the INSTI® test.

TRAINING PACK

8 9

Frequently Asked Questions & Summary and Intended Use Troubleshooting

What kind of antigen is used?The INSTI® test utilises recombinant transmembrane proteins from HIV-1 and HIV-2. Use of these proteins overcome sensitivity and specificity problems associated with a test based on viral lysates or a combination ofcore antigen and other viral proteins.

Does the INSTI® kit have a built-in sample addition control?Yes, the procedural control for the INSTI® kit consists of a protein capable of capturing the IgG normally present in human blood components. IgG is present in blood components in both HIV negative and HIV positive human specimens. Captured IgG reacts with a chromatic agent to produce a visual sign (dot) on the control membrane.

Does the INSTI® kit only react with whole blood samples?No, you can use the INSTI® kit with EDTA-whole blood, EDTA-plasma, fingerstick, serum or plasma samples.

How long does it take to perform a single test?INSTI® preparation is minimal and results show up in as little as 60 seconds. Results are interpreted immediately after pouring the clarifying solution (bottle #3) into the membrane unit.

What is the stability of the test results in the test membrane unit?The test results will remain visible in the INSTI® membrane unit for an indefinite period after performing the test. Read within the first 5 minutes after the test. The results may fade over time.

What kind of storage conditions are needed?INSTI® tests should be stored at:

1. INSTI® HIV-1/HIV-2 Antibody Test kit: store at 2-30°C

2. INSTI® HIV-1 and HIV-2 Controls: Store frozen at -20°C or refrigerate at 2°C to 8°C for up to one year. Once thawed store at (2- 8°C). They remain stable until expiry (up to 1 year). Do not re-freeze once the bottles have been opened.

3. INSTI® Multiplex Test kits: store at 15-30°C

4. INSTI® Syphilis Controls: Store at 2 - 8°C for up to 28 days or at ≤-20°C for up to 1 year. Once the controls are thawed following storage at ≤-20°C, they remain stable for 28 days at 2 - 8°C. Do not re-freeze once the bot-tles have been opened.

Note - the storage conditions for INSTI® HIV-1/HIV-2 Antibody Test kits and INSTI® Multiplex Test kits are different. Also the storage conditions for HIV and Syphilis controls are different.

What is the shelf life?INSTI® tests presently have a shelf life of: 1. INSTI® HIV-1/HIV-2 Antibody Test kit: Shelf-life of 15 months2. INSTI® HIV-1 and HIV-2 Controls: Shelf-life of 12 months3. INSTI® Multiplex Test kit: Shelf-life of 12 months4. INSTI® Syphilis Controls: Shelf-life of 12 monthsAll products are made to order to ensure that no short-dated products are shipped.

What level of quality assurance do you practice in your manufacturing facility?bioLytical® Laboratories is an ISO 13485 certified manufacturing facility that has been inspected and approved by the FDA, European Notified Body and Health Canada for the manufacturing of medical devices. Each INSTI® test kit undergoes multiple quality assurance steps and procedures prior to being released for sale.

What are the special benefits of using INSTI®?• Results in 60 seconds• Proven accuracy of >99.5%• Proven early detection• Built in sample addition control• Easy to use and understand

Where can I learn more about bioLytical® Laboratories and INSTI®?For enquires or to learn more, please visit our website at: www.biolytical.com, insti.com or www.pasante.com

Not for donor screening

The INSTI® HIV-1/HIV-2 Antibody Test is a single use, rapid, flow-through in vitro qualitative immunoassay for the detection of antibodies to Human Immunodeficiency Virus Type 1 and Type 2 in human EDTA-whole blood, fingerstick blood, serum or EDTA-plasma.

The test is intended for use by trained personnel in medical facilities, clinical laboratories, emergency care situations, and physicians’ offices as a diagnostic test capable of providing results in less than one minute. Although suitable for near-patient or point-of-care (POC) testing, the INSTI® HIV Antibody Test is not suitable for home testing at this time.

All required pre and post-test counselling guidelines must be followed in each setting in which the INSTI® HIV Antibody Test is used. The assay is packaged as a kit containing an INSTI® Membrane Unit, Sample Diluent, Colour Developer, and Clarifying Solution with or without support materials (lancet, pipette and alcohol swab).

Invalid Results:

No dots: Usually an indication that substantially less than 50μL of specimen (blood, serum or plasma) was used. Could also be an indication that the INSTI® solutions were added in the wrong sequence. Testing should be repeated with a fresh kit.

Solid Colour: A uniform blue tint across the entire membrane which obscures the control and test dots is an indication that the reagents were not used in the correct sequence. Testing should be repeated.

Specks on the membrane: This could be caused by slight aggregation of the contents of bottle #2. If the control dot is clearly visible, test results are valid even with specks on the membrane. Specks can also appear on the membrane if the kit was used past the expiry date, in which case the results are not considered valid.

Test dot only: This might occur very rarely and may be an indication that the test procedure has not been followed properly or there was a manufacturing problem. Testing should be repeated, on a fresh kit.

Additional factors which may cause Invalid results:• The membrane unit has not been used immediately after opening.• Reagents from different kit lots are combined to perform the INSTI® test.• Using reagents in the wrong order.• Some reagents are spilled while pouring into the membrane unit.• The INSTI® kits were stored at temperatures outside the approved storage temperature range.• The membrane has been contaminated causing blockage of flow.

Indeterminate Results:

In some instances, test dots might appear as very faint shadows or blue rings. These are to be interpreted as Indeterminate results, and testing should be repeated. Consult the INSTI® package insert section on Interpretation of results for follow-up action.

Indeterminate or Invalid results may occur if:

1. More or less than 50μL of blood, serum or plasma is added to the test.2. Flow times for the reagents are slow or greater than 2 minutes in total.3. There are delays introduced in the procedural sequence once the sample is added to bottle #1.

Or delays between pours.4. The contents of bottle #1 and bottle #2 are not mixed immediately before pouring into the membrane unit.5. INSTI® kits are used beyond the expiry date.

TRAINING PACK

10 11

Quick Reference Guide True Human IgG Control Spot

Non-Reactive ReactiveInvalid Indeterminate

Built-In Sample Addition Control:

The INSTI® HIV-1/HIV-2 Antibody Test kit has a built-in sample addition control capture that demonstrates assay validity and adequate sample addition. A blue colour in the control spot indicates that the proper specimen was added and that the assay procedure was performed correctly. The control spot will be visible on all valid INSTI® tests.

With INSTI®, if the control dot appears, this indicates the following:

• The test has been performed correctly.• The flow through mechanism of INSTI® has worked properly.• The correct amount and type of specimen has been added to the membrane unit.• In the absence of a visible test dot, this is a valid non-reactive result.• Any test dot that is readily visible is a valid reactive result.

External Controls:Only INSTI® controls should be used with INSTI test kits. It is advisable that external control material, consisting of at least one HIV negative and one HIV positive serum or plasma specimens, be tested with INSTI®:

• At regular intervals or with every shipment of INSTI®.• For new INSTI® user verification.• When switching to new lot number of INSTI® test kits.• When the storage area falls outside 2-30˚C.

It is the responsibility of each user of the INSTI® HIV-1/HIV-2 Antibody Test kit to establish an adequate quality assurance program to ensure proper performance under their specific locations and conditions of use.

» shake well and pour» allow to flow through

» shake well and pour» allow to flow through

» add 50μL(blood, serum, plasma)

» allow to flow through» read immediately (within 5 minutes)

Procedure Steps (1-4) are to be carried out in sequence with no delays. If there is any delay between Procedure Steps (1) and (2):a) Step 2 must be performed within 5 minutes after Step 1. b) Re-mix Sample Diluent (bottle #1) by inverting several times before pouring into the Membrane Unit.c) Immediately proceed to Steps 3 and 4.

Do not conduct repeated sampling involving multiple transfers of small amounts of blood from fingertip to Sample Diluent Bottle #1 as this may result in improper blood volumes.

To facilitate fingerstick blood collection, the fingertip at the collection point should be warm. Ensure that the fingertip is completely dry after the use of the Alcohol Swab, before applying the Lancet.

A Reactive Test result should be readily visible to the naked eye. Repeat the test if the results are unclear.

Following a reactive or indeterminate INSTI® test result, a venous blood sample must be drawn in a ‘lavender-top’ EDTA collection tube (for whole blood or plasma) or red-top tube (for serum), and forwarded to laboratory of HIV confirmatory testing.

Customer enquiries: call Pasante Healthcare Ltd on +44 (0) 1903 854717

TRAINING PACKHIV

(1) (2) (3) (4)

Control ControlTest

ControlTestTest

12 13

Instructions for Use Instructions for Use

HIV-1/HIV-2 Antibody Test

Single-use rapid assay for the detection of antibodies toHuman Immunodeficiency Virus Type 1 (HIV-1) and Type 2 (HIV-2)

90-1015 - One INSTI® HIV-1/HIV-2 Antibody Test with support materials (for POC use)

Store at 2°C to 30°C K Sterilization using irradiation

Y Caution g Lot number

V In Vitro diagnostic medical device h Catalogue Number

i Consult instructions for use M Manufacturer

D Do not reuse C CE Mark

H Use by R22 – Harmful if swallowed

Store at 2°C – 30°C. For in vitro diagnostic use only. V

i It is recommended that the entire Instructions for Use be read prior to beginning the test procedure. Although the assay is designed to be simple to use, conformance with the test procedure is necessary to ensure accurate results.

INTENDED USE - Not for donor screening

The INSTI HIV-1/HIV-2 Antibody Test is a single use, rapid, flow-through in vitro qualitative immunoassay for the detection of antibodies to Human Immunodeficiency Virus Type 1 and Type 2 in human EDTA whole blood, fingerstick blood, serum or EDTA-plasma. The test is intended for use by trained personnel in medical facilities, clinical laboratories, emergency care situations, and physicians’ offices as a diagnostic test capable of providing results in less than one minute. Although suitable for near-patient or point-of-care (POC) testing, the INSTI HIV-1/HIV-2 Antibody Test is not intended for home testing. All required pre, and post-test counselling guidelines must be followed in each setting in which the INSTI HIV-1/HIV-2 Antibody Test is used. The assay is packaged as a kit containing INSTI Membrane Unit, Sample Diluent (Solution 1), Colour Developer (Solution 2), and Clarifying Solution (Solution 3) with support materials (lancet, pipette and alcohol swab).

SUMMARY

Acquired Immunodeficiency Syndrome (AIDS) is caused by at least two retroviruses, HIV-1 and HIV-2. HIV-1 and HIV-2 are similar in genomic structure, morphology and ability to cause AIDS.1 HIV is transmitted mainly by sexual contact, exposure to blood or blood products, or from an infected mother to her foetus. People with increased risk of HIV infection include haemophiliacs, intravenous drug-users and men having sex with men (MSM). HIV has been isolated from patients with AIDS, AIDS-related complex (ARC), and from persons at high risk of contracting AIDS.2-5 Antibodies specific for HIV envelope proteins are prevalent in sera from persons at high risk of contracting AIDS as well as in people with AIDS, or ARC.5-7 The presence of antibodies to HIV indicates previous exposure to the virus, but does not necessarily constitute a diagnosis of AIDS. The prevalence of antibodies to HIV in people not known to be at risk of acquiring HIV infection is unknown, but significantly less.5Absence of antibodies to HIV does not indicate that an individual is free of HIV-1 or HIV-2; HIV has been isolated fromseronegative individuals prior to seroconversion. Test specificity and sensitivity depend, amongst other factors, on: a) the selection of HIV antigens used for antibody detection, b) the classes of antibodies recognized by the detection conjugate, and c) complexity of the protocol used to perform the test.8 Non-specific reactions may be observed in some specimens. A reactive INSTI test result should be considered a preliminary result, with appropriate counselling provided in POC settings. Following a reactive rapid test result, a venous blood sample must be drawn in an EDTA collection tube (for wholeblood or plasma) or a tube with no anticoagulant (for serum), and forwarded to a laboratory for HIV confirmatory testing.

PRINCIPLES OF THE TEST

The INSTI HIV-1/HIV-2 Antibody Test is a manual, visually read, flow through immunoassay for the qualitative detection of HIV-1/HIV-2 antibodies in human blood, serum or plasma. The test consists of a synthetic filtration membrane positioned atop an absorbent material within a plastic cartridge, referred to as the INSTI Membrane Unit. The membrane has been specifically treated with HIV-1 and HIV-2recombinant proteins, which react with HIV-1/HIV-2 antibodies in the specimen to produce a distinct visual signal on the membrane. The membrane also includes a procedural control. The procedural control consists of a protein-A treated spot capable of capturing IgG antibodies normally present in blood and blood components. IgG antibodies react with a proprietary chromatic agent to produce a visual signal on the membrane. Since IgG antibodies are present in blood from normal or HIV positive human specimens, the control dot provides a visual signal when the test is run, indicating that the test was performed correctly. If the control dot does not appear, the test is considered invalid. In the case of the test spot, recombinant HIV-1 and HIV-2 proteins, embedded in the membrane, capture HIV specific antibodies, if present in the specimen. Antibodies captured in the test spot react with a proprietary chromatic agent to produce a visible signal on the membrane. The membrane unit is designed to filter, absorb, and retain the test specimen and all the test reagents in such a manner as to limit leakage and exposure of personnel to potentially infectious materials.Reagents required to conduct a test include Sample Diluent (Solution 1), Colour Developer (Solution 2) and Clarifying Solution (Solution 3). The test is performed by adding the blood, serum, or plasma specimen to the vial of Sample Diluent, which lyses the red blood cells. This specimen/diluent solution is then poured onto the well of the Membrane Unit. HIV-1/HIV-2 antibodies, if present in the specimen, are captured by proteins on the filtration membrane. Colour Developer is then added to the Membrane

Unit. The Colour Developer reacts with the captured antibodies to generate a distinct blue dot at the location of the control spot and, in the case that HIV-1/HIV-2 antibodies are present in the specimen, a blue dot also appears at the location of the test spot on the membrane. In the final step, the Clarifying Solution is then added to the membrane to decrease background colour in order to make the control and test dots more distinct.

Antigen Selection: The INSTI HIV-1/HIV-2 assay utilizes a combination of recombinant transmembrane proteins from HIV-1 (gp41) and HIV-2 (gp36). Use of these proteins overcomes sensitivity and specificity problems associated with tests based on viral lysates or a combination of core antigen and other viral proteins.9-13

Antibody Detection: The INSTI HIV-1/HIV-2 assay uses a unique reagent to detect antibodies to HIV-1/HIV-2. Although primarily designed to detect the IgG class of specific antibodies, the INSTI HIV-1/HIV-2 assay has been shown to detect antibodies in samples obtained early in infection, during seroconversion, and low titre anti-HIV-1 samples obtained later in infection.

Test Complexity: The INSTI HIV-1/HIV-2 assay was designed to reduce protocol complexity. The INSTI HIV-1/HIV-2 assay does not require sample preparation, accurate timing, or several steps, which include multiple washes and reagents. These requirements increase the complexity of an assay and lead to procedural errors which may adversely affect sensitivity and specificity. Total test time may vary slightly depending on specimen type; but results of valid tests are always clearly readable within one minute.

SPECIMEN COLLECTION AND STORAGE

1. For EDTA-whole blood, EDTA-plasma or serum specimens, follow venipuncture blood collection procedures using lavender-top EDTA anticoagulant tubes (for whole blood and plasma) or red-top (no anticoagulant) tubes for serum.

2. If plasma or serum is to be used, separate from the blood cells by centrifugation.3. Serum or EDTA-plasma may be stored at 2-8°C for up to 5 days, stored frozen at ≤ -20°C for 3

months, or stored frozen at ≤ -70°C for one year.4. Whole blood specimens collected in EDTA anticoagulant may be stored at 2-8°C and should be

tested within 48 hours. Do not heat or freeze whole blood specimens.5. Do not dilute prior to testing.

KIT COMPONENTS AND STORAGE

INSTI components should be stored at 2-30°C. For 90-1015 all kit components are individually packaged for single use only. Each test requires the following materials:

1. Membrane Unit, individually packaged, prepared with control (IgG capture) and test (gp41 and gp36 antigen) reaction spots. For single use only in the INSTI procedure.

2. Sample Diluent, Xn R22, Solution 1 vial, containing 1.5 ml of tris-glycine buffered solution containing cell lysis reagents, with adequate space for addition of blood, serum or plasma samples being tested with INSTI. Ready to use, no mixing or preparation required. Contains 0.1% sodium azide as a preservative, for single use only in the INSTI procedure. Stable to date and under storage conditions indicated on label.

3. Colour Developer, Xn R22, Solution 2 vial, containing 1.5 ml of a blue-coloured Borate buffered proprietary indicator solution designed to detect IgG in the control spot and specific HIV antibodies in the test spot. For single use only in the INSTI procedure. Ready to use, invert 2-3X immediately before use. Contains 0.1% sodium azide as a preservative. Stable to date and under storage conditions indicated on label.

4. Clarifying Solution, Xn R22, Solution 3 vial, containing 1.5 ml of a proprietary tris-glycine buffered clarifying solution designed to remove background staining from the membrane unit prior to reading the INSTI test results. Ready to use, no mixing or preparation required. For single use only in the INSTI procedure. Contains 0.1% sodium azide as a preservative. Stable to date and under storage conditions indicated on label.

SUPPORT MATERIALS DThe following materials are required when testing fingerstick whole blood and included with each kit:1. Single-use Alcohol Swab

2. Single-use Lancet K 0344

3. Single-use Pipette, 50μl

MATERIALS REQUIRED BUT NOT PROVIDED Personal protective equipment. Appropriate biohazard waste containers and disinfectants. Absorbent cotton balls for fingerstick or venipuncture wound closure.

For venipuncture blood collection and testing: Venipuncture apparatus if collecting blood samples. Appropriate blood collection tubes. Appropriate shipping containers. Precision pipette capable of delivering 50μl of sample.

MATERIALS AVAILABLE AS AN ACCESSORY TO THE KITINSTI HIV-1/HIV-2 Test Controls: Separate HIV-negative human serum substitute and HIV-1/HIV-2positive de-fibrinated human plasma control samples product no. 90-1036 are available from bioLytical Laboratories, for use in quality control procedures. Please refer to the section on Quality Control, following the Assay Procedure, and the INSTI HIV-1/HIV-2 Test Controls Instructions for Use.

WARNINGS

For in vitro diagnostic use only VIt is recommended that the entire Package Insert be read prior to beginning the test procedure. Although the assay is designed to be simple to use, conformance with the test procedure is necessary to ensure accurate results.

1. Do not mix reagents from different lots.2. Do not use reagents or kits beyond the stated expiration date.3. Do not use the Membrane Unit if the foil pouch has been previously opened or if the packaging

integrity has been compromised. Once the Membrane Unit has been opened, it must be used immediately.

4. Avoid microbial contamination of reagents.5. Y Sodium azide is present at 0.1% in all assay reagents. Sodium azide may react with lead or

copper plumbing to form highly explosive metal azides. If products containing sodium azide are discarded into a drain, flush with large amounts of water to prevent azide build-up. Check with local regulatory agencies to determine at what concentration sodium azide may cause a product to be regulated as hazardous waste.

6. The performance characteristics of the INSTI HIV-1/HIV-2 assay have not been established for body fluids other than EDTA whole blood, fingerstick blood, serum, and EDTA-plasma. The use of blood

collected in anticoagulants other than EDTA has not been validated. Insufficient data are available to interpret tests performed on other body fluids, pooled blood or pooled serum and EDTA-plasma, or products made from such pools.

7. Failure to use the recommended reagent and specimen volumes may result in leakage and/or overflow of liquids from the membrane unit.

8. If the kit is refrigerated, ensure it is brought to room temperature before performing the test. Use the INSTI HIV-1/HIV-2 Test Controls to ensure proper kit performance.

9. Y Patients that are on long term antiretroviral drug therapy may give a false negative INSTI HIV-1/HIV-2 test result.

10.Samples from patients with severe hypogammaglobulinemia conditions such as multiple myeloma may result in false negative or invalid results with INSTI.

11.Patients with elevated haemoglobin levels may test false negative with INSTI.15

PRECAUTIONS1. Y All specimens should be handled as if capable of transmitting infectious diseases. It is

recommended that BioSafety Level 2 practices, or equivalent regulations, be observed.14

2. Thoroughly wash hands after handling or performing this test.3. Do not smoke, eat, or drink in areas where specimens or kit reagents are being handled.4. Wear a lab coat and disposable gloves while handling kit reagents or specimens. Do not pipette by

mouth.5. Avoid contact with skin and eyes. If contact occurs, wash affected areas with water.6. Avoid forming aerosols.7. Y Dispose of all specimens and materials used to perform the test as if they contained infectious

agents. The preferred method of disposal is sterilization by autoclaving for a minimum of one hour at 121°C followed by incineration. Liquid waste not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 0.5% sodium hypochlorite (a solution containing 10% household bleach). Allow at least 30 minutes for decontamination to be completed. Do not autoclave solutions that contain bleach.

8. Spills should be cleaned up and decontaminated in accordance with the user facility’s established procedures for handling biohazardous spills.

ASSAY PROCEDURENOTE: All Membrane Units must be used immediately once opened. All reagents should be dispensed evenly in the center of the well.

Sampling Fingerstick Blood:

1. Gather support materials (swab, lancet, pipette), one sealed test pouch containing INSTI Membrane Unit, and one vial each of the Sample Diluent, Colour Developer, and Clarifying Solution for each test to be performed.

Y CAUTION! The amount of sample (fingerstick blood) is critical. To ensure that the proper amount of blood is achieved, follow these instructions carefully:

2. Massage the finger to allow the blood to move to the surface (fingertip will become pink). Use heating pad if available to warm the hand. Hand must be positioned at waist level or lower.

3. Wipe the fingertip with the alcohol swab.4. As soon as the finger is dry, twist and remove the protective insert from the lancet. Press the finger

firmly at the point just below where the lancet will be applied. With the other hand, place the lancet on the side of the fingertip and press hard until it clicks. Immediately dispose the used lancet into a proper sharps container.

5. As the blood droplet forms, hold the pipette horizontally and touch the tip of the pipette to the blood sample. Capillary action automatically draws the sample to the fill line and stops. If very little blood trickles out of the puncture, gently apply intermittent pressure below the puncture site to obtain the required blood volume. If blood is inadequate, perform a second skin puncture using a new lancet.

Y CAUTION! Filling is automatic: Never squeeze the tube while sampling.

6. Transfer the blood held in the pipette to the Sample Diluent vial (Solution 1). Align the tip of the pipette with the Sample Diluent vial and squeeze the bulb to dispense the sample. NOTE: If the sample will not expel, hold the pipette vertically and slide a finger over (without pressing) the vent hole, then squeeze the bulb. Recap the vial and mix by inversion. Follow General Procedure after Sampling, below.

Sampling EDTA Whole Blood, serum, EDTA-plasma and Test Controls:

1. Bring specimens to room temperature and mix each specimen thoroughly prior to use. Do not heat or repeatedly freeze/thaw specimens.

2. Gather one sealed test pouch containing INSTI Membrane Unit, and one vial each of the Sample Diluent, Colour Developer, and Clarifying Solution for each test to be performed.

3. Using a pipette, add 50μl of whole blood, serum, plasma, or kit controls (see Note) to the Sample Diluent vial. Recap the vial and mix by inversion. Y Adding an excessive amount of specimen may cause the device to overflow or leak.

NOTE: In POC settings, for INSTI kit controls, it is important to use a 50μl pipette device to add the control material to the Sample Diluent vial. Do not use the disposable single-use pipette provided for finger stick blood collection.

General Procedure after Sampling:

1. Tear open the pouch and remove the Membrane Unit without touching the center well. Place the unit on a level surface. For sample identification purposes the tab of the Membrane Unit may be labeled with the patient’s name or number.

NOTE: At this point, it is important that the following steps be performed immediately and in sequence.

2. Mix the Sample Diluent-specimen mixture by inverting several times and pour the entire contents to the center of the Membrane Unit well. (NOTE: Do this within 5 minutes after the specimen has been added to the Sample Diluent vial). The sample should be absorbed through the membrane in less than 30 seconds; however, absorption times will vary slightly depending upon sample type.

3. Resuspend the Colour Developer by slowly inverting to mix the solution thoroughly until the reagent is evenly suspended and add the entire contents to the center of the Membrane Unit well. The coloured solution should flow through completely in about 20 seconds.

4. Open the Clarifying Solution and add the entire contents to the center of the Membrane Unit well. This will lighten the background colour and facilitate reading. Immediately read the result while the membrane is still wet. Do not read the results if more than 5 minutes has elapsed following the addition of Clarifying Solution.

QUALITY CONTROL

Kit Controls:

The INSTI HIV-1/HIV-2 Antibody Test has a built-in IgG capture procedural control that demonstrates assay validity and adequate sample addition. A blue colour on the control dot indicates that the proper specimen was added and that the assay procedure was performed correctly. The control dot will appear on all valid INSTI tests. (Refer to Interpretation of Results, below.)

INSTI HIV-1/HIV-2 Test Controls are available separately for use only with the INSTI HIV-1/HIV-2Antibody Test. The controls are used to verify test performance and interpretation of results. Kit controls should be run under the following circumstances:

for new INSTI operator verification prior to performing testing on patient specimens when switching to a new lot number of INSTI test kits whenever a new shipment of kits is received when temperature during storage of the kit falls outside of 2°-30°C when the temperature of the test area falls outside of 15°-30°C at regular intervals as determined by the user facility.

Refer to the INSTI HIV-1/HIV-2 Test Controls instructions for use for additional information on the use of these reagents. It is the responsibility of each user of the INSTI HIV-1/HIV-2 Antibody Test to establish an adequate quality assurance program to ensure proper performance under their specific locations and conditions of use.

INTERPRETATION OF RESULTS Do not read the results if more than 5 minutes have elapsed following the addition of

Clarifying Solution. If using the control samples provided by bioLytical, all Positive Controls must be reactive

with INSTI and all Negative Controls must be non-reactive with INSTI. Controls that produce incorrect or invalid results must be re-tested with INSTI. If results are still incorrect or invalid, inform bioLytical Laboratories immediately.

NON-REACTIVE ► One blue dot that is clearly discernable above any background tint should appear on the membrane. This is the procedural Control Dot and shows that the test has been performed correctly. The control dot location is indicated by the letter C printed on the Membrane Unit. No reaction should be visible at the test spot, located below the control. A non-reactive result indicates that antibodies to HIV-1/HIV-2 were not detected in the specimen.

REACTIVE ► Two blue dots that are discernable above any background tint indicate that the specimen contains HIV-1/HIV-2 antibodies. One dot may be darker than the other. A sample giving this pattern is considered a preliminary reactive. Following a reactive rapid test result, a venous blood sample must be drawn in a lavender-top EDTA collection tube (for whole blood or plasma) or red-top tube (for serum), and forwarded to a laboratory for HIV confirmatory testing.

HIV-1/HIV-2 Antibody Test

Single-use rapid assay for the detection of antibodies toHuman Immunodeficiency Virus Type 1 (HIV-1) and Type 2 (HIV-2)

90-1015 - One INSTI® HIV-1/HIV-2 Antibody Test with support materials (for POC use)

Store at 2°C to 30°C K Sterilization using irradiation

Y Caution g Lot number

V In Vitro diagnostic medical device h Catalogue Number

i Consult instructions for use M Manufacturer

D Do not reuse C CE Mark

H Use by R22 – Harmful if swallowed

Store at 2°C – 30°C. For in vitro diagnostic use only. V

i It is recommended that the entire Instructions for Use be read prior to beginning the test procedure. Although the assay is designed to be simple to use, conformance with the test procedure is necessary to ensure accurate results.

INTENDED USE - Not for donor screening

The INSTI HIV-1/HIV-2 Antibody Test is a single use, rapid, flow-through in vitro qualitative immunoassay for the detection of antibodies to Human Immunodeficiency Virus Type 1 and Type 2 in human EDTA whole blood, fingerstick blood, serum or EDTA-plasma. The test is intended for use by trained personnel in medical facilities, clinical laboratories, emergency care situations, and physicians’ offices as a diagnostic test capable of providing results in less than one minute. Although suitable for near-patient or point-of-care (POC) testing, the INSTI HIV-1/HIV-2 Antibody Test is not intended for home testing. All required pre, and post-test counselling guidelines must be followed in each setting in which the INSTI HIV-1/HIV-2 Antibody Test is used. The assay is packaged as a kit containing INSTI Membrane Unit, Sample Diluent (Solution 1), Colour Developer (Solution 2), and Clarifying Solution (Solution 3) with support materials (lancet, pipette and alcohol swab).

SUMMARY

Acquired Immunodeficiency Syndrome (AIDS) is caused by at least two retroviruses, HIV-1 and HIV-2. HIV-1 and HIV-2 are similar in genomic structure, morphology and ability to cause AIDS.1 HIV is transmitted mainly by sexual contact, exposure to blood or blood products, or from an infected mother to her foetus. People with increased risk of HIV infection include haemophiliacs, intravenous drug-users and men having sex with men (MSM). HIV has been isolated from patients with AIDS, AIDS-related complex (ARC), and from persons at high risk of contracting AIDS.2-5 Antibodies specific for HIV envelope proteins are prevalent in sera from persons at high risk of contracting AIDS as well as in people with AIDS, or ARC.5-7 The presence of antibodies to HIV indicates previous exposure to the virus, but does not necessarily constitute a diagnosis of AIDS. The prevalence of antibodies to HIV in people not known to be at risk of acquiring HIV infection is unknown, but significantly less.5Absence of antibodies to HIV does not indicate that an individual is free of HIV-1 or HIV-2; HIV has been isolated fromseronegative individuals prior to seroconversion. Test specificity and sensitivity depend, amongst other factors, on: a) the selection of HIV antigens used for antibody detection, b) the classes of antibodies recognized by the detection conjugate, and c) complexity of the protocol used to perform the test.8 Non-specific reactions may be observed in some specimens. A reactive INSTI test result should be considered a preliminary result, with appropriate counselling provided in POC settings. Following a reactive rapid test result, a venous blood sample must be drawn in an EDTA collection tube (for wholeblood or plasma) or a tube with no anticoagulant (for serum), and forwarded to a laboratory for HIV confirmatory testing.

PRINCIPLES OF THE TEST

The INSTI HIV-1/HIV-2 Antibody Test is a manual, visually read, flow through immunoassay for the qualitative detection of HIV-1/HIV-2 antibodies in human blood, serum or plasma. The test consists of a synthetic filtration membrane positioned atop an absorbent material within a plastic cartridge, referred to as the INSTI Membrane Unit. The membrane has been specifically treated with HIV-1 and HIV-2recombinant proteins, which react with HIV-1/HIV-2 antibodies in the specimen to produce a distinct visual signal on the membrane. The membrane also includes a procedural control. The procedural control consists of a protein-A treated spot capable of capturing IgG antibodies normally present in blood and blood components. IgG antibodies react with a proprietary chromatic agent to produce a visual signal on the membrane. Since IgG antibodies are present in blood from normal or HIV positive human specimens, the control dot provides a visual signal when the test is run, indicating that the test was performed correctly. If the control dot does not appear, the test is considered invalid. In the case of the test spot, recombinant HIV-1 and HIV-2 proteins, embedded in the membrane, capture HIV specific antibodies, if present in the specimen. Antibodies captured in the test spot react with a proprietary chromatic agent to produce a visible signal on the membrane. The membrane unit is designed to filter, absorb, and retain the test specimen and all the test reagents in such a manner as to limit leakage and exposure of personnel to potentially infectious materials.Reagents required to conduct a test include Sample Diluent (Solution 1), Colour Developer (Solution 2) and Clarifying Solution (Solution 3). The test is performed by adding the blood, serum, or plasma specimen to the vial of Sample Diluent, which lyses the red blood cells. This specimen/diluent solution is then poured onto the well of the Membrane Unit. HIV-1/HIV-2 antibodies, if present in the specimen, are captured by proteins on the filtration membrane. Colour Developer is then added to the Membrane

Unit. The Colour Developer reacts with the captured antibodies to generate a distinct blue dot at the location of the control spot and, in the case that HIV-1/HIV-2 antibodies are present in the specimen, a blue dot also appears at the location of the test spot on the membrane. In the final step, the Clarifying Solution is then added to the membrane to decrease background colour in order to make the control and test dots more distinct.

Antigen Selection: The INSTI HIV-1/HIV-2 assay utilizes a combination of recombinant transmembrane proteins from HIV-1 (gp41) and HIV-2 (gp36). Use of these proteins overcomes sensitivity and specificity problems associated with tests based on viral lysates or a combination of core antigen and other viral proteins.9-13

Antibody Detection: The INSTI HIV-1/HIV-2 assay uses a unique reagent to detect antibodies to HIV-1/HIV-2. Although primarily designed to detect the IgG class of specific antibodies, the INSTI HIV-1/HIV-2 assay has been shown to detect antibodies in samples obtained early in infection, during seroconversion, and low titre anti-HIV-1 samples obtained later in infection.

Test Complexity: The INSTI HIV-1/HIV-2 assay was designed to reduce protocol complexity. The INSTI HIV-1/HIV-2 assay does not require sample preparation, accurate timing, or several steps, which include multiple washes and reagents. These requirements increase the complexity of an assay and lead to procedural errors which may adversely affect sensitivity and specificity. Total test time may vary slightly depending on specimen type; but results of valid tests are always clearly readable within one minute.

SPECIMEN COLLECTION AND STORAGE

1. For EDTA-whole blood, EDTA-plasma or serum specimens, follow venipuncture blood collection procedures using lavender-top EDTA anticoagulant tubes (for whole blood and plasma) or red-top (no anticoagulant) tubes for serum.

2. If plasma or serum is to be used, separate from the blood cells by centrifugation.3. Serum or EDTA-plasma may be stored at 2-8°C for up to 5 days, stored frozen at ≤ -20°C for 3

months, or stored frozen at ≤ -70°C for one year.4. Whole blood specimens collected in EDTA anticoagulant may be stored at 2-8°C and should be

tested within 48 hours. Do not heat or freeze whole blood specimens.5. Do not dilute prior to testing.

KIT COMPONENTS AND STORAGE

INSTI components should be stored at 2-30°C. For 90-1015 all kit components are individually packaged for single use only. Each test requires the following materials:

1. Membrane Unit, individually packaged, prepared with control (IgG capture) and test (gp41 and gp36 antigen) reaction spots. For single use only in the INSTI procedure.

2. Sample Diluent, Xn R22, Solution 1 vial, containing 1.5 ml of tris-glycine buffered solution containing cell lysis reagents, with adequate space for addition of blood, serum or plasma samples being tested with INSTI. Ready to use, no mixing or preparation required. Contains 0.1% sodium azide as a preservative, for single use only in the INSTI procedure. Stable to date and under storage conditions indicated on label.

3. Colour Developer, Xn R22, Solution 2 vial, containing 1.5 ml of a blue-coloured Borate buffered proprietary indicator solution designed to detect IgG in the control spot and specific HIV antibodies in the test spot. For single use only in the INSTI procedure. Ready to use, invert 2-3X immediately before use. Contains 0.1% sodium azide as a preservative. Stable to date and under storage conditions indicated on label.

4. Clarifying Solution, Xn R22, Solution 3 vial, containing 1.5 ml of a proprietary tris-glycine buffered clarifying solution designed to remove background staining from the membrane unit prior to reading the INSTI test results. Ready to use, no mixing or preparation required. For single use only in the INSTI procedure. Contains 0.1% sodium azide as a preservative. Stable to date and under storage conditions indicated on label.

SUPPORT MATERIALS DThe following materials are required when testing fingerstick whole blood and included with each kit:1. Single-use Alcohol Swab

2. Single-use Lancet K 0344

3. Single-use Pipette, 50μl

MATERIALS REQUIRED BUT NOT PROVIDED Personal protective equipment. Appropriate biohazard waste containers and disinfectants. Absorbent cotton balls for fingerstick or venipuncture wound closure.

For venipuncture blood collection and testing: Venipuncture apparatus if collecting blood samples. Appropriate blood collection tubes. Appropriate shipping containers. Precision pipette capable of delivering 50μl of sample.

MATERIALS AVAILABLE AS AN ACCESSORY TO THE KITINSTI HIV-1/HIV-2 Test Controls: Separate HIV-negative human serum substitute and HIV-1/HIV-2positive de-fibrinated human plasma control samples product no. 90-1036 are available from bioLytical Laboratories, for use in quality control procedures. Please refer to the section on Quality Control, following the Assay Procedure, and the INSTI HIV-1/HIV-2 Test Controls Instructions for Use.

WARNINGS

For in vitro diagnostic use only VIt is recommended that the entire Package Insert be read prior to beginning the test procedure. Although the assay is designed to be simple to use, conformance with the test procedure is necessary to ensure accurate results.

1. Do not mix reagents from different lots.2. Do not use reagents or kits beyond the stated expiration date.3. Do not use the Membrane Unit if the foil pouch has been previously opened or if the packaging

integrity has been compromised. Once the Membrane Unit has been opened, it must be used immediately.

4. Avoid microbial contamination of reagents.5. Y Sodium azide is present at 0.1% in all assay reagents. Sodium azide may react with lead or

copper plumbing to form highly explosive metal azides. If products containing sodium azide are discarded into a drain, flush with large amounts of water to prevent azide build-up. Check with local regulatory agencies to determine at what concentration sodium azide may cause a product to be regulated as hazardous waste.

6. The performance characteristics of the INSTI HIV-1/HIV-2 assay have not been established for body fluids other than EDTA whole blood, fingerstick blood, serum, and EDTA-plasma. The use of blood

collected in anticoagulants other than EDTA has not been validated. Insufficient data are available to interpret tests performed on other body fluids, pooled blood or pooled serum and EDTA-plasma, or products made from such pools.

7. Failure to use the recommended reagent and specimen volumes may result in leakage and/or overflow of liquids from the membrane unit.

8. If the kit is refrigerated, ensure it is brought to room temperature before performing the test. Use the INSTI HIV-1/HIV-2 Test Controls to ensure proper kit performance.

9. Y Patients that are on long term antiretroviral drug therapy may give a false negative INSTI HIV-1/HIV-2 test result.

10.Samples from patients with severe hypogammaglobulinemia conditions such as multiple myeloma may result in false negative or invalid results with INSTI.

11.Patients with elevated haemoglobin levels may test false negative with INSTI.15

PRECAUTIONS1. Y All specimens should be handled as if capable of transmitting infectious diseases. It is

recommended that BioSafety Level 2 practices, or equivalent regulations, be observed.14

2. Thoroughly wash hands after handling or performing this test.3. Do not smoke, eat, or drink in areas where specimens or kit reagents are being handled.4. Wear a lab coat and disposable gloves while handling kit reagents or specimens. Do not pipette by

mouth.5. Avoid contact with skin and eyes. If contact occurs, wash affected areas with water.6. Avoid forming aerosols.7. Y Dispose of all specimens and materials used to perform the test as if they contained infectious

agents. The preferred method of disposal is sterilization by autoclaving for a minimum of one hour at 121°C followed by incineration. Liquid waste not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 0.5% sodium hypochlorite (a solution containing 10% household bleach). Allow at least 30 minutes for decontamination to be completed. Do not autoclave solutions that contain bleach.

8. Spills should be cleaned up and decontaminated in accordance with the user facility’s established procedures for handling biohazardous spills.

ASSAY PROCEDURENOTE: All Membrane Units must be used immediately once opened. All reagents should be dispensed evenly in the center of the well.

Sampling Fingerstick Blood:

1. Gather support materials (swab, lancet, pipette), one sealed test pouch containing INSTI Membrane Unit, and one vial each of the Sample Diluent, Colour Developer, and Clarifying Solution for each test to be performed.

Y CAUTION! The amount of sample (fingerstick blood) is critical. To ensure that the proper amount of blood is achieved, follow these instructions carefully:

2. Massage the finger to allow the blood to move to the surface (fingertip will become pink). Use heating pad if available to warm the hand. Hand must be positioned at waist level or lower.

3. Wipe the fingertip with the alcohol swab.4. As soon as the finger is dry, twist and remove the protective insert from the lancet. Press the finger

firmly at the point just below where the lancet will be applied. With the other hand, place the lancet on the side of the fingertip and press hard until it clicks. Immediately dispose the used lancet into a proper sharps container.

5. As the blood droplet forms, hold the pipette horizontally and touch the tip of the pipette to the blood sample. Capillary action automatically draws the sample to the fill line and stops. If very little blood trickles out of the puncture, gently apply intermittent pressure below the puncture site to obtain the required blood volume. If blood is inadequate, perform a second skin puncture using a new lancet.

Y CAUTION! Filling is automatic: Never squeeze the tube while sampling.

6. Transfer the blood held in the pipette to the Sample Diluent vial (Solution 1). Align the tip of the pipette with the Sample Diluent vial and squeeze the bulb to dispense the sample. NOTE: If the sample will not expel, hold the pipette vertically and slide a finger over (without pressing) the vent hole, then squeeze the bulb. Recap the vial and mix by inversion. Follow General Procedure after Sampling, below.

Sampling EDTA Whole Blood, serum, EDTA-plasma and Test Controls:

1. Bring specimens to room temperature and mix each specimen thoroughly prior to use. Do not heat or repeatedly freeze/thaw specimens.

2. Gather one sealed test pouch containing INSTI Membrane Unit, and one vial each of the Sample Diluent, Colour Developer, and Clarifying Solution for each test to be performed.

3. Using a pipette, add 50μl of whole blood, serum, plasma, or kit controls (see Note) to the Sample Diluent vial. Recap the vial and mix by inversion. Y Adding an excessive amount of specimen may cause the device to overflow or leak.

NOTE: In POC settings, for INSTI kit controls, it is important to use a 50μl pipette device to add the control material to the Sample Diluent vial. Do not use the disposable single-use pipette provided for finger stick blood collection.

General Procedure after Sampling:

1. Tear open the pouch and remove the Membrane Unit without touching the center well. Place the unit on a level surface. For sample identification purposes the tab of the Membrane Unit may be labeled with the patient’s name or number.

NOTE: At this point, it is important that the following steps be performed immediately and in sequence.

2. Mix the Sample Diluent-specimen mixture by inverting several times and pour the entire contents to the center of the Membrane Unit well. (NOTE: Do this within 5 minutes after the specimen has been added to the Sample Diluent vial). The sample should be absorbed through the membrane in less than 30 seconds; however, absorption times will vary slightly depending upon sample type.

3. Resuspend the Colour Developer by slowly inverting to mix the solution thoroughly until the reagent is evenly suspended and add the entire contents to the center of the Membrane Unit well. The coloured solution should flow through completely in about 20 seconds.

4. Open the Clarifying Solution and add the entire contents to the center of the Membrane Unit well. This will lighten the background colour and facilitate reading. Immediately read the result while the membrane is still wet. Do not read the results if more than 5 minutes has elapsed following the addition of Clarifying Solution.

QUALITY CONTROL

Kit Controls:

The INSTI HIV-1/HIV-2 Antibody Test has a built-in IgG capture procedural control that demonstrates assay validity and adequate sample addition. A blue colour on the control dot indicates that the proper specimen was added and that the assay procedure was performed correctly. The control dot will appear on all valid INSTI tests. (Refer to Interpretation of Results, below.)

INSTI HIV-1/HIV-2 Test Controls are available separately for use only with the INSTI HIV-1/HIV-2Antibody Test. The controls are used to verify test performance and interpretation of results. Kit controls should be run under the following circumstances:

for new INSTI operator verification prior to performing testing on patient specimens when switching to a new lot number of INSTI test kits whenever a new shipment of kits is received when temperature during storage of the kit falls outside of 2°-30°C when the temperature of the test area falls outside of 15°-30°C at regular intervals as determined by the user facility.

Refer to the INSTI HIV-1/HIV-2 Test Controls instructions for use for additional information on the use of these reagents. It is the responsibility of each user of the INSTI HIV-1/HIV-2 Antibody Test to establish an adequate quality assurance program to ensure proper performance under their specific locations and conditions of use.

INTERPRETATION OF RESULTS Do not read the results if more than 5 minutes have elapsed following the addition of

Clarifying Solution. If using the control samples provided by bioLytical, all Positive Controls must be reactive

with INSTI and all Negative Controls must be non-reactive with INSTI. Controls that produce incorrect or invalid results must be re-tested with INSTI. If results are still incorrect or invalid, inform bioLytical Laboratories immediately.

NON-REACTIVE ► One blue dot that is clearly discernable above any background tint should appear on the membrane. This is the procedural Control Dot and shows that the test has been performed correctly. The control dot location is indicated by the letter C printed on the Membrane Unit. No reaction should be visible at the test spot, located below the control. A non-reactive result indicates that antibodies to HIV-1/HIV-2 were not detected in the specimen.

REACTIVE ► Two blue dots that are discernable above any background tint indicate that the specimen contains HIV-1/HIV-2 antibodies. One dot may be darker than the other. A sample giving this pattern is considered a preliminary reactive. Following a reactive rapid test result, a venous blood sample must be drawn in a lavender-top EDTA collection tube (for whole blood or plasma) or red-top tube (for serum), and forwarded to a laboratory for HIV confirmatory testing.

TRAINING PACKHIV

14 15

Instructions for Use Instructions for Use

INVALID ► The test is invalid if any of the following occurs:A. There is no dot on the membraneB. The test dot appeared without the control dotC. Uniform tint across the membraneD. Only blue specks appear on the membrane

A B C D

NOTE: Invalid tests with fingerstick blood samples in POC settings should be repeated with a fresh sample using a new membrane unit, kit components and support materials. Invalid tests with EDTA whole blood, EDTA plasma or serum samples in laboratory settings should be repeated using a new membrane unit and kit components.

INDETERMINATE ► The test is indeterminate if a faint background ring appeared on the test area. Following an indeterminate INSTI test result, a venous blood sample must be drawn in a lavender-top EDTA collection tube (for whole blood or plasma) or red-top tube (for serum), and forwarded to a laboratory for HIV confirmatory testing.

Please note the following:

1. Following a reactive or indeterminate INSTI test result, a venous blood sample must be drawn in a lavender-top EDTA collection tube (for whole blood or plasma) or red-top tube (for serum), and forwarded to a laboratory for HIV confirmatory testing.

2. Depending on the antibody titer, a reactive specimen may be less intense in colour than the procedural control, or vice versa.

3. Only a blue spot of colour discernibly darker than the background colour should be interpreted as reactive or positive. In rare instances, a faint background ring may appear around the test spot; this should not be interpreted as a reactive result. Only tests exhibiting distinct fully formed blue test dot combined with a distinct fully formed blue control dot should be interpreted as reactive. Colour intensity may be variable within or between the dots.

4. An invalid result indicates that the test was performed incorrectly or there is a problem with the sample or device. The absence of a distinct control dot usually indicates that the sample volume was insufficient. An invalid test must be repeated.

5. A test resulting in a uniform blue tint across the entire membrane, thus obscuring the control and test spots, can occur when more than 60μL of whole blood is used and the flow through the assay membrane is obstructed.

6. An individual who has a non-reactive result but was involved in HIV-risk activity is likewise recommended to obtain additional testing over the next months.

7. To significantly reduce the risk of HIV transmission, it is advisable to refrain from high risk activities such as unprotected sex and needle sharing at all times.

LIMITATIONS OF THE TEST Flow Times

In some instances, samples may exhibit longer than normal flow times (from the time the Sample Diluent specimen mixture is poured in the membrane well to the time the Clarifying Solution has fully flowed through the membrane). This is due to variable factors such as cellular components, especially with whole blood. In instances of long flow times, a faint shadow in the form of a ring may appear at the test spot location, but this should not be interpreted as a reactive result. This should be considered as an indeterminate result. In these instances, a venous blood sample should be drawn in a lavender-top EDTA collection tube, and forwarded to a laboratory for HIV confirmatory testing.

The INSTI HIV-1/HIV-2 Antibody Test procedure and the interpretation of result must be followed closely when testing for the presence of antibodies to HIV in serum, plasma or whole blood.

Insufficient data are available to interpret tests performed on other body fluids, pooled blood or pooled serum and plasma, or products made from such pools; therefore, testing of these specimens is not recommended.

The INSTI HIV-1/HIV-2 Antibody Test has not been validated for detection of antibodies to HIV-1Group N subtypes.

The INSTI HIV-1/HIV-2 Antibody Test detects antibodies to HIV-1/HIV-2 and is useful in establishing infection with HIV. Because a variety of factors may cause non-specific reactions, a patient found to be positive using the INSTI HIV-1/HIV-2 assay should have an EDTA blood sample drawn for laboratory-based confirmatory testing. A person who has antibodies to HIV is presumed to be infected with the virus and appropriate counseling and medical evaluation should be offered. The presence of HIV antibodies indicates past exposure to HIV but is not a diagnosis of AIDS, which can only be made by a physician. However, a non-reactive test does not rule out past exposure to HIV. The risk of an asymptomatic person with repeated reactive serum developing AIDS is not known. The prevalence of HIV infection in various groups, as well as clinical and public health guidelines, are available in the CDC Morbidity and Mortality Report.8

PERFORMANCE CHARACTERISTICS

SENSITIVITY DETECTION OF ANTIBODIES TO HIV-1 IN SPECIMENS FROM INDIVIDUALS INFECTED WITH HIV-1

A multi-center prospective study was conducted to evaluate the clinical performance of the INSTI HIV Antibody Test. There were 483 subjects known to be HIV-1 positive, and 905 subjects with unknown HIV status. The subjects with unknown HIV status were tested with INSTI and by a composite reference method (comparator method) which consisted of an licensed/approved EIA with supplemental Western blot and PCR assays as required. The result of INSTI was compared to the known or determined HIV status of the subject.

In this study, all 517/517 true HIV antibody positive subjects were identified as reactive by the INSTI HIV-1/HIV-2 Antibody Test, resulting in a relative sensitivity of 100.0% (95 % CI = 99.3% - 100.0%). There were no invalid results (0/1388) observed in this study.

Detection of HIV-1 Antibody in Fingerstick Whole Blood Specimens from HIV-1 Seropositive Individuals

Study Population Number of Subjects

INSTI Reactive

Approved Test

ReactiveTrue Positive

HIV status unknown 905 34 34 34

Known HIV-1 Positive 483 483 483 483

TOTAL 1,388 517 517 517

Reactivity with HIV-1: Seroconversion Panels

Thirty (30) HIV-1 seroconversion panels (Boston Biomedica Inc.) were tested with INSTI. Each panel consisted of sequential serum/plasma specimens obtained from a single individual during seroconversion. The results of this study are presented in the table below and summarizes the INSTI HIV-1/HIV-2 assay data compared to US licensed and European approved HIV antibody enzyme immunoassays (EIA). Overall the INSTI HIV-1/HIV-2 Antibody Test has similar performance to commercially available anti-HIV EIA in the detection of HIV antibodies in seroconversion samples.

INSTI HIV-1/HIV-2 TEST: Number of Panels

Detected the earliest bleed that was detected by an EIA 15Detected within 1 bleed of earliest EIA positive 10Detected within 2 bleeds of earliest EIA positive 3Unknown** 2

**The last bleed in the panel was reactive by at least 1 EIA, non-reactive by INSTI

Reactivity with HIV-1: Low Titer Panel

A single low titer HIV-1 antibody panel (#PRB-108; Boston Biomedica) was tested with the INSTI HIV-1/HIV-2 Antibody Test. This low titer panel consisted of 15 serum/plasma specimens. Results of this study are summarized in the table below. This study demonstrated that the INSTI HIV-1/HIV-2Antibody Test has the capability of detecting antibodies to HIV-1 similar to currently available FDA licensed EIAs.

Specimen Number

Test 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

INSTI HIV-1/HIV-2 P P P P P P P P P P P N P P P

Abbott EIA P P P P P P P P P P P N P P P

Abbott HIVAB HIV-1/HIV2 (rDNA)EIA P P P P P P P P P P P N P P P

Cambridge Biotech Recombigen HIV-1 EIA P P P P P P P P P P P N P P P

Syva EIA P P P P P P P P P P P N P P P

Organon Teknika Vironostika Anti-HIV Uni-Form II P P P P P P P P P P P N P P P

Murex HIV 1/2 EIA P P P P P P P P P P P N P P P

Ortho HIV-1/HIV-2 EIA P P P P P P P P P P P N P P P

Sorin ETI-Ab-HIV 1/2K EIA P P P P P P P P P P P N P P P

Syva Microtrak II EIA P P P P P P P P P P P N P P P

Behringwerke ENZ PLUS Anti HIV 1/2 EIA P P P P P P P P P P P N P P P

Biotest Anti-HIV-1/HIV-2 Recombinant EIA P P P P P P P P P P P N P P P

Boehringer Mannheim Anti HIV-1/HIV-2 EIA P P P P P P P P P P P N P P P

IAF Biochem Detect-HIV-EIA P P P P P P P P P P P N P P P

Diagnostic Pasteur Genelavia EIA P P P P P P P P P P P N P P P

bioMerieux VIDAS anti-HIV-1/2 EIA P P P P P P P P P P P N P P P

Murex Wellcozyme HIV-1/HIV-2 EIA P P P P P P P P P P N N P P P

Behringwerke Enzygnost Anti HIV 1+2 EIA N P N P P P P P P P P N P P P

Cellular Products HIV-1 EIA N P P P P P P P N P P N P P P

Genetic Systems LAV EIA N P P P P P P P N P P N P P P

Genetic Systems HIV-1/HIV-2 EIA N P N P P P P P P P P N P P P

*These samples were confirmed positive (P) by EIA and Western Blotting(Data obtained from Boston Biomedica package insert, May 1995 p.2)

Interfering Substances and Unrelated Medical Conditions

To assess the impact of unrelated medical conditions or interfering substances on the sensitivity of the INSTI HIV-1/HIV-2 Antibody Test, 195 serum/plasma specimens from a variety of medical conditionsunrelated to HIV-1 infection and 217 specimens with interfering substances were spiked with an HIV-1positive specimen; see table in the Specificity section for list of medical conditions and substances tested. All spiked specimens gave reactive results.

DETECTION OF ANTIBODIES TO HIV-2 IN SPECIMENS FROM INDIVIDUALS INFECTED WITH HIV-2

A total of 137 individual HIV-2 positive samples were obtained from European sources. 49 sera from individuals with chronic HIV-2 infection were reactive on the INSTI HIV-1/HIV-2 Antibody Test. An additional 88 HIV-2 positive serum and plasma samples were prepared as contrived whole blood; all 88 contrived samples were reactive on the INSTI HIV-1/HIV-2 Antibody test. Combining the results of

the two studies, the relative sensitivity of the INSTI HIV-1/HIV-2 Antibody test for the detection of HIV-2 antibodies in these studies was calculated to be 100% (137/137).

HIV-1 SUBTYPE TESTING

To assess the sensitivity of the INSTI HIV-1/HIV-2 Antibody test for HIV-1 variants from various geographic regions, a total of 118 individual confirmed HIV-1 antibody-positive non-B subtype serum/plasma specimens were tested; of these 118 samples, 109 were non-B subtypes including 23 sub-type O samples. All 118 of these specimens were reactive using INSTI, generating an overall sensitivity of the INSTI HIV-1/HIV-2 Antibody Test for HIV-1 non-B subtypes of 100%.

SPECIFICITY

A specificity study was performed using 1386 freshly obtained specimens collected from low or unknown risk and high risk individuals as part of a multicenter prospective clinical trial. Of the 1386 samples,1376 gave a Non-Reactive result with INSTI and 4 were invalid. INSTI HIV-1/HIV-2 Antibody Test results were compared to results from a composite reference method (comparator method) which consisted of an FDA approved EIA with supplemental Western blot and PCR as required. A total of 7 INSTI false Reactive results (1 from the high risk group, 6 from the low or unknown risk group) were obtained from the 1382 specimens from HIV-negative individuals that produced valid INSTI results. From this data, the overall specificity of the INSTI HIV-1/HIV-2 Antibody Test in fingerstick whole blood specimens from the combined high risk and low or unknown risk populations, minus the invalid results, was calculated to be 1375/1382 = 99.5% (95% CI = 99.0% - 99.8%).

Performance of the INSTI HIV-1/HIV-2 Antibody Test on Fingerstick Whole Blood Specimens from Individuals Presumed to be Negative for HIV Infection

Test Group

Total Specimens

INSTI Non-

Reactive3

Approved Test Non-Reactive2

True Negative2

Low Risk 626 620 626 626High Risk

782 7561 7602 760

TOTAL 1408 1376 1386 13861 4 invalid results were not included in the calculation of specificity. The 4 specimens which gave invalid results on INSTI were Non-Reactive on the approved test.2 22 Reactives were confirmed by licensed HIV-1 Western Blot and excluded from the calculation of specificity.3 Of the 22 INSTI Reactive specimens, one was Non-Reactive by the approved test, i.e. INSTI false Reactive.

Interfering Substances and Unrelated Medical Conditions

To asses the impact of unrelated medical conditions or interfering substances on the specificity of the INSTI HIV-1/HIV-2 Antibody Test, 195 serum/plasma specimens from a variety of medical conditions unrelated to HIV-1 infection and 217 specimens with interfering substances were analyzed. Five specimens from individuals with multiple myeloma gave invalid results. No false reactive results were obtained.

Medical Condition (n=195) No. of Specimens

INSTI Reactive

INSTI Nonreactive

Toxoplasmosis 20 0 20

Rheumatoid Factor 20 0 20

Multiple Myeloma 10 0 5

Syphilis 30 0 30

SLE 5 0 5

Rubella 20 0 20

Cytomegalovirus 20 0 20

Epstein Barr Virus 20 0 20

HTLV-I/II panel 15 0 15

Hepatitis B Virus 20 0 20

Hepatitis A Virus 15 0 15

Interfering Substances (n=217)

Icteric 20 0 20

Elevated Bilirubin (>8.0mg/dL) 19 0 19

Lipemic 20 0 20

Visual Hemolysis 5 0 5

Elevated Triglyceride (>292mg/dL) 19 0 19

Elevated Hemoglobin (>12g/100mL) 20 0 20

Elevated Albumin (11.5-13.0g/dL) 15 0 15

EDTA 13 0 13

Sodium Heparin 13 0 13

Sodium Citrate 13 0 13

Bacterially Contaminated 60 0 60

In addition, a total of 208 specimens from pregnant women in various trimesters of pregnancy confirmed to be HIV-1 negative by a 3rd Generation HIV EIA were tested. One sample (1/208) produced invalid result, all other INSTI results were non-reactive.

EQUIVALENCE STUDIES

The INSTI HIV-1/HIV-2 Antibody Test was evaluated using matched serum and plasma specimens, Testing was performed with 50 anti-HIV-1 negative specimens (25 serum and 25 plasma) and 50 anti-HIV-1-spiked positive specimens. All samples produced acceptable assay performance. These results indicate 100% relative sensitivity and 100% relative specificity with the matched serum and plasma panel provided, and that serum and plasma sample types are equivalent.

REPRODUCIBILITYThe reproducibility of the INSTI HIV-1/HIV-2 Antibody Test was tested at 3 laboratory sites using 3 lots of the INSTI device on 3 separate days. A panel of 9 blind-coded plasma samples, consisting of 4 antibody positive, 1 very low antibody level sample, and 4 antibody negative samples was tested at each site.

A total of 729 tests were conducted, 243 at each site.

For the 4 antibody positive and 4 antibody negative samples, the overall reproducibility was 99.7% (646/648, two antibody negative samples were read as weak positive at 1 site). For the 1 very low level antibody sample, 59% (48/81) of the results were reactive while 41% (33/81) were non-reactive.

BIBLIOGRAPHY1. Guyader, M., Emerman, M., Sonigo, P., et al. Genome organization and transactivation of the human immunodeficiency virus type 2. Nature 326:662-669, 1987.2. Blattner, W., Gallo, R.C., and Temin, H.M. HIV causes AIDS. Science 241:515, 1988.3. Curran, J.W., Morgan, W.M., Hardy, A.M., et al. The epidemiology of AIDS; Current status and future prospects. Science 229:1352-1357, 19854. Sarngadharan, M.G., Popovic, M., Bruch, L., Schüpback, J., and Gallo, R.C. Antibodies reactive with human T-lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 224:506-508,19845. Gallo, R.C., Salahuddin, S.Z., Popovic, M., et al. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224:500-503, 19846. Weber, J.N., Weiss, R.A., Roberts, C., et al. Human immunodeficiency virus in two cohorts of homosexual men; Neutralising sera and association of anti-gag antibody with prognosis. Lancet 1:119-124, 19877. Clavel, F., Guétard, D., Brun-Vézinet, F., et al. Isolation of a new human retrovirus from West African patient with AIDS. Science 233:343-346, 19868. Centers for Disease Control. Revision of the CDC surveillance case definition for acquired Immunodeficiency syndrome. MMWR 36 (suppl. no. 1S):1S-15S, 19879. World Health Organization/Global Programme on AIDS. Report of a WHO workshop on synthetic peptides in HIV diagnosis and AIDS-related research, Moscow 24-26 May 1989. WHO Report, AIDS1991, 5: WHO1-WHO910. Los Alamos National Laboratory. Human retroviruses and AIDS Database. A compilation of nucleic acid and amino acid sequences, 1993.11. World Health Organization/Global Programme on AIDS. Operational characteristics of commercially available assays to detect antibodies to HIV-1 and/or HIV-2 in human sera. Geneva, Switzerland: WHO documents GPA/BMR/89.4; GPA/BMR/90.1; GPA/RES/DIA90.1; GPA/RES/DIA/91.6; GPA/RES/DIA/ 92.8 and GPA/RES/DIA/93.412. World Health Organization/Global Programme on AIDS. Acquired immunodeficiency syndrome (AIDS proposed WHO criteria for interpreting results from Western blot assays for HIV-1, HIV-2 and HTLV-I/HTLVII). WHO Weekly Epidemiological Record 65(37):281-282, 199013. Malone, J.D., Smith, E.S., Sheffield, J., et al. Comparative evaluation of six rapid serological tests for HIV-1 antibody. Journal of Acquired Immune Deficiency Syndrome (JAIDS) 6:115-149, 199314. The Laboratory Biosafety Guidelines, 3rd Edition. Office of Laboratory Security, Health Canada. 3.1.2, 2004. 15. “Summary of Safety and Effectiveness Data, 50-1110.” bioLytical Laboratories, Inc. 28 January 2011, Table 3.

TECHNICAL INFORMATION

For further information or assistance, contact the Technical Services at 1-604-644-4677.

Reference herein to any specific third party by name, trade name, trade-mark, manufacturer or otherwise does not constitute or imply an endorsement or recommendation of this Kit by such third party, or of the products or services of such third party by bioLytical or that such products or services are necessarily best suited for the intended purpose.

M Manufactured by:

bioLytical Laboratories, Inc.13351 Commerce Parkway Suite 1108Richmond, BC, V6V 2X7CanadaPhone: 1-604-204-6784Fax: 1-604-244-8399www.biolytical.com

Authorized Representative: EMERGO EUROPEPrinsessegracht 20, 2514 AP, The Hague, The NetherlandsPhone: +31.70.345.8570 Fax: +31.70.346.7299

51-1037O 14-Feb-2018© Copyright 2018, All rights reserved.

0543

INVALID ► The test is invalid if any of the following occurs:A. There is no dot on the membraneB. The test dot appeared without the control dotC. Uniform tint across the membraneD. Only blue specks appear on the membrane

A B C D

NOTE: Invalid tests with fingerstick blood samples in POC settings should be repeated with a fresh sample using a new membrane unit, kit components and support materials. Invalid tests with EDTA whole blood, EDTA plasma or serum samples in laboratory settings should be repeated using a new membrane unit and kit components.

INDETERMINATE ► The test is indeterminate if a faint background ring appeared on the test area. Following an indeterminate INSTI test result, a venous blood sample must be drawn in a lavender-top EDTA collection tube (for whole blood or plasma) or red-top tube (for serum), and forwarded to a laboratory for HIV confirmatory testing.

Please note the following:

1. Following a reactive or indeterminate INSTI test result, a venous blood sample must be drawn in a lavender-top EDTA collection tube (for whole blood or plasma) or red-top tube (for serum), and forwarded to a laboratory for HIV confirmatory testing.

2. Depending on the antibody titer, a reactive specimen may be less intense in colour than the procedural control, or vice versa.

3. Only a blue spot of colour discernibly darker than the background colour should be interpreted as reactive or positive. In rare instances, a faint background ring may appear around the test spot; this should not be interpreted as a reactive result. Only tests exhibiting distinct fully formed blue test dot combined with a distinct fully formed blue control dot should be interpreted as reactive. Colour intensity may be variable within or between the dots.

4. An invalid result indicates that the test was performed incorrectly or there is a problem with the sample or device. The absence of a distinct control dot usually indicates that the sample volume was insufficient. An invalid test must be repeated.

5. A test resulting in a uniform blue tint across the entire membrane, thus obscuring the control and test spots, can occur when more than 60μL of whole blood is used and the flow through the assay membrane is obstructed.

6. An individual who has a non-reactive result but was involved in HIV-risk activity is likewise recommended to obtain additional testing over the next months.

7. To significantly reduce the risk of HIV transmission, it is advisable to refrain from high risk activities such as unprotected sex and needle sharing at all times.

LIMITATIONS OF THE TEST Flow Times

In some instances, samples may exhibit longer than normal flow times (from the time the Sample Diluent specimen mixture is poured in the membrane well to the time the Clarifying Solution has fully flowed through the membrane). This is due to variable factors such as cellular components, especially with whole blood. In instances of long flow times, a faint shadow in the form of a ring may appear at the test spot location, but this should not be interpreted as a reactive result. This should be considered as an indeterminate result. In these instances, a venous blood sample should be drawn in a lavender-top EDTA collection tube, and forwarded to a laboratory for HIV confirmatory testing.

The INSTI HIV-1/HIV-2 Antibody Test procedure and the interpretation of result must be followed closely when testing for the presence of antibodies to HIV in serum, plasma or whole blood.

Insufficient data are available to interpret tests performed on other body fluids, pooled blood or pooled serum and plasma, or products made from such pools; therefore, testing of these specimens is not recommended.

The INSTI HIV-1/HIV-2 Antibody Test has not been validated for detection of antibodies to HIV-1Group N subtypes.

The INSTI HIV-1/HIV-2 Antibody Test detects antibodies to HIV-1/HIV-2 and is useful in establishing infection with HIV. Because a variety of factors may cause non-specific reactions, a patient found to be positive using the INSTI HIV-1/HIV-2 assay should have an EDTA blood sample drawn for laboratory-based confirmatory testing. A person who has antibodies to HIV is presumed to be infected with the virus and appropriate counseling and medical evaluation should be offered. The presence of HIV antibodies indicates past exposure to HIV but is not a diagnosis of AIDS, which can only be made by a physician. However, a non-reactive test does not rule out past exposure to HIV. The risk of an asymptomatic person with repeated reactive serum developing AIDS is not known. The prevalence of HIV infection in various groups, as well as clinical and public health guidelines, are available in the CDC Morbidity and Mortality Report.8

PERFORMANCE CHARACTERISTICS

SENSITIVITY DETECTION OF ANTIBODIES TO HIV-1 IN SPECIMENS FROM INDIVIDUALS INFECTED WITH HIV-1

A multi-center prospective study was conducted to evaluate the clinical performance of the INSTI HIV Antibody Test. There were 483 subjects known to be HIV-1 positive, and 905 subjects with unknown HIV status. The subjects with unknown HIV status were tested with INSTI and by a composite reference method (comparator method) which consisted of an licensed/approved EIA with supplemental Western blot and PCR assays as required. The result of INSTI was compared to the known or determined HIV status of the subject.

In this study, all 517/517 true HIV antibody positive subjects were identified as reactive by the INSTI HIV-1/HIV-2 Antibody Test, resulting in a relative sensitivity of 100.0% (95 % CI = 99.3% - 100.0%). There were no invalid results (0/1388) observed in this study.

Detection of HIV-1 Antibody in Fingerstick Whole Blood Specimens from HIV-1 Seropositive Individuals

Study Population Number of Subjects

INSTI Reactive

Approved Test

ReactiveTrue Positive

HIV status unknown 905 34 34 34

Known HIV-1 Positive 483 483 483 483

TOTAL 1,388 517 517 517

Reactivity with HIV-1: Seroconversion Panels

Thirty (30) HIV-1 seroconversion panels (Boston Biomedica Inc.) were tested with INSTI. Each panel consisted of sequential serum/plasma specimens obtained from a single individual during seroconversion. The results of this study are presented in the table below and summarizes the INSTI HIV-1/HIV-2 assay data compared to US licensed and European approved HIV antibody enzyme immunoassays (EIA). Overall the INSTI HIV-1/HIV-2 Antibody Test has similar performance to commercially available anti-HIV EIA in the detection of HIV antibodies in seroconversion samples.

INSTI HIV-1/HIV-2 TEST: Number of Panels

Detected the earliest bleed that was detected by an EIA 15Detected within 1 bleed of earliest EIA positive 10Detected within 2 bleeds of earliest EIA positive 3Unknown** 2

**The last bleed in the panel was reactive by at least 1 EIA, non-reactive by INSTI

Reactivity with HIV-1: Low Titer Panel

A single low titer HIV-1 antibody panel (#PRB-108; Boston Biomedica) was tested with the INSTI HIV-1/HIV-2 Antibody Test. This low titer panel consisted of 15 serum/plasma specimens. Results of this study are summarized in the table below. This study demonstrated that the INSTI HIV-1/HIV-2Antibody Test has the capability of detecting antibodies to HIV-1 similar to currently available FDA licensed EIAs.

Specimen Number

Test 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

INSTI HIV-1/HIV-2 P P P P P P P P P P P N P P P

Abbott EIA P P P P P P P P P P P N P P P

Abbott HIVAB HIV-1/HIV2 (rDNA)EIA P P P P P P P P P P P N P P P

Cambridge Biotech Recombigen HIV-1 EIA P P P P P P P P P P P N P P P

Syva EIA P P P P P P P P P P P N P P P

Organon Teknika Vironostika Anti-HIV Uni-Form II P P P P P P P P P P P N P P P

Murex HIV 1/2 EIA P P P P P P P P P P P N P P P

Ortho HIV-1/HIV-2 EIA P P P P P P P P P P P N P P P

Sorin ETI-Ab-HIV 1/2K EIA P P P P P P P P P P P N P P P

Syva Microtrak II EIA P P P P P P P P P P P N P P P

Behringwerke ENZ PLUS Anti HIV 1/2 EIA P P P P P P P P P P P N P P P

Biotest Anti-HIV-1/HIV-2 Recombinant EIA P P P P P P P P P P P N P P P

Boehringer Mannheim Anti HIV-1/HIV-2 EIA P P P P P P P P P P P N P P P

IAF Biochem Detect-HIV-EIA P P P P P P P P P P P N P P P

Diagnostic Pasteur Genelavia EIA P P P P P P P P P P P N P P P

bioMerieux VIDAS anti-HIV-1/2 EIA P P P P P P P P P P P N P P P

Murex Wellcozyme HIV-1/HIV-2 EIA P P P P P P P P P P N N P P P

Behringwerke Enzygnost Anti HIV 1+2 EIA N P N P P P P P P P P N P P P

Cellular Products HIV-1 EIA N P P P P P P P N P P N P P P

Genetic Systems LAV EIA N P P P P P P P N P P N P P P

Genetic Systems HIV-1/HIV-2 EIA N P N P P P P P P P P N P P P

*These samples were confirmed positive (P) by EIA and Western Blotting(Data obtained from Boston Biomedica package insert, May 1995 p.2)

Interfering Substances and Unrelated Medical Conditions

To assess the impact of unrelated medical conditions or interfering substances on the sensitivity of the INSTI HIV-1/HIV-2 Antibody Test, 195 serum/plasma specimens from a variety of medical conditionsunrelated to HIV-1 infection and 217 specimens with interfering substances were spiked with an HIV-1positive specimen; see table in the Specificity section for list of medical conditions and substances tested. All spiked specimens gave reactive results.

DETECTION OF ANTIBODIES TO HIV-2 IN SPECIMENS FROM INDIVIDUALS INFECTED WITH HIV-2

A total of 137 individual HIV-2 positive samples were obtained from European sources. 49 sera from individuals with chronic HIV-2 infection were reactive on the INSTI HIV-1/HIV-2 Antibody Test. An additional 88 HIV-2 positive serum and plasma samples were prepared as contrived whole blood; all 88 contrived samples were reactive on the INSTI HIV-1/HIV-2 Antibody test. Combining the results of

the two studies, the relative sensitivity of the INSTI HIV-1/HIV-2 Antibody test for the detection of HIV-2 antibodies in these studies was calculated to be 100% (137/137).

HIV-1 SUBTYPE TESTING

To assess the sensitivity of the INSTI HIV-1/HIV-2 Antibody test for HIV-1 variants from various geographic regions, a total of 118 individual confirmed HIV-1 antibody-positive non-B subtype serum/plasma specimens were tested; of these 118 samples, 109 were non-B subtypes including 23 sub-type O samples. All 118 of these specimens were reactive using INSTI, generating an overall sensitivity of the INSTI HIV-1/HIV-2 Antibody Test for HIV-1 non-B subtypes of 100%.

SPECIFICITY

A specificity study was performed using 1386 freshly obtained specimens collected from low or unknown risk and high risk individuals as part of a multicenter prospective clinical trial. Of the 1386 samples,1376 gave a Non-Reactive result with INSTI and 4 were invalid. INSTI HIV-1/HIV-2 Antibody Test results were compared to results from a composite reference method (comparator method) which consisted of an FDA approved EIA with supplemental Western blot and PCR as required. A total of 7 INSTI false Reactive results (1 from the high risk group, 6 from the low or unknown risk group) were obtained from the 1382 specimens from HIV-negative individuals that produced valid INSTI results. From this data, the overall specificity of the INSTI HIV-1/HIV-2 Antibody Test in fingerstick whole blood specimens from the combined high risk and low or unknown risk populations, minus the invalid results, was calculated to be 1375/1382 = 99.5% (95% CI = 99.0% - 99.8%).

Performance of the INSTI HIV-1/HIV-2 Antibody Test on Fingerstick Whole Blood Specimens from Individuals Presumed to be Negative for HIV Infection

Test Group

Total Specimens

INSTI Non-

Reactive3

Approved Test Non-Reactive2

True Negative2

Low Risk 626 620 626 626High Risk

782 7561 7602 760

TOTAL 1408 1376 1386 13861 4 invalid results were not included in the calculation of specificity. The 4 specimens which gave invalid results on INSTI were Non-Reactive on the approved test.2 22 Reactives were confirmed by licensed HIV-1 Western Blot and excluded from the calculation of specificity.3 Of the 22 INSTI Reactive specimens, one was Non-Reactive by the approved test, i.e. INSTI false Reactive.

Interfering Substances and Unrelated Medical Conditions

To asses the impact of unrelated medical conditions or interfering substances on the specificity of the INSTI HIV-1/HIV-2 Antibody Test, 195 serum/plasma specimens from a variety of medical conditions unrelated to HIV-1 infection and 217 specimens with interfering substances were analyzed. Five specimens from individuals with multiple myeloma gave invalid results. No false reactive results were obtained.

Medical Condition (n=195) No. of Specimens

INSTI Reactive

INSTI Nonreactive

Toxoplasmosis 20 0 20

Rheumatoid Factor 20 0 20

Multiple Myeloma 10 0 5

Syphilis 30 0 30

SLE 5 0 5

Rubella 20 0 20

Cytomegalovirus 20 0 20

Epstein Barr Virus 20 0 20

HTLV-I/II panel 15 0 15

Hepatitis B Virus 20 0 20

Hepatitis A Virus 15 0 15

Interfering Substances (n=217)

Icteric 20 0 20

Elevated Bilirubin (>8.0mg/dL) 19 0 19

Lipemic 20 0 20

Visual Hemolysis 5 0 5

Elevated Triglyceride (>292mg/dL) 19 0 19

Elevated Hemoglobin (>12g/100mL) 20 0 20

Elevated Albumin (11.5-13.0g/dL) 15 0 15

EDTA 13 0 13

Sodium Heparin 13 0 13

Sodium Citrate 13 0 13

Bacterially Contaminated 60 0 60

In addition, a total of 208 specimens from pregnant women in various trimesters of pregnancy confirmed to be HIV-1 negative by a 3rd Generation HIV EIA were tested. One sample (1/208) produced invalid result, all other INSTI results were non-reactive.

EQUIVALENCE STUDIES

The INSTI HIV-1/HIV-2 Antibody Test was evaluated using matched serum and plasma specimens, Testing was performed with 50 anti-HIV-1 negative specimens (25 serum and 25 plasma) and 50 anti-HIV-1-spiked positive specimens. All samples produced acceptable assay performance. These results indicate 100% relative sensitivity and 100% relative specificity with the matched serum and plasma panel provided, and that serum and plasma sample types are equivalent.

REPRODUCIBILITYThe reproducibility of the INSTI HIV-1/HIV-2 Antibody Test was tested at 3 laboratory sites using 3 lots of the INSTI device on 3 separate days. A panel of 9 blind-coded plasma samples, consisting of 4 antibody positive, 1 very low antibody level sample, and 4 antibody negative samples was tested at each site.

A total of 729 tests were conducted, 243 at each site.

For the 4 antibody positive and 4 antibody negative samples, the overall reproducibility was 99.7% (646/648, two antibody negative samples were read as weak positive at 1 site). For the 1 very low level antibody sample, 59% (48/81) of the results were reactive while 41% (33/81) were non-reactive.

BIBLIOGRAPHY1. Guyader, M., Emerman, M., Sonigo, P., et al. Genome organization and transactivation of the human immunodeficiency virus type 2. Nature 326:662-669, 1987.2. Blattner, W., Gallo, R.C., and Temin, H.M. HIV causes AIDS. Science 241:515, 1988.3. Curran, J.W., Morgan, W.M., Hardy, A.M., et al. The epidemiology of AIDS; Current status and future prospects. Science 229:1352-1357, 19854. Sarngadharan, M.G., Popovic, M., Bruch, L., Schüpback, J., and Gallo, R.C. Antibodies reactive with human T-lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 224:506-508,19845. Gallo, R.C., Salahuddin, S.Z., Popovic, M., et al. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224:500-503, 19846. Weber, J.N., Weiss, R.A., Roberts, C., et al. Human immunodeficiency virus in two cohorts of homosexual men; Neutralising sera and association of anti-gag antibody with prognosis. Lancet 1:119-124, 19877. Clavel, F., Guétard, D., Brun-Vézinet, F., et al. Isolation of a new human retrovirus from West African patient with AIDS. Science 233:343-346, 19868. Centers for Disease Control. Revision of the CDC surveillance case definition for acquired Immunodeficiency syndrome. MMWR 36 (suppl. no. 1S):1S-15S, 19879. World Health Organization/Global Programme on AIDS. Report of a WHO workshop on synthetic peptides in HIV diagnosis and AIDS-related research, Moscow 24-26 May 1989. WHO Report, AIDS1991, 5: WHO1-WHO910. Los Alamos National Laboratory. Human retroviruses and AIDS Database. A compilation of nucleic acid and amino acid sequences, 1993.11. World Health Organization/Global Programme on AIDS. Operational characteristics of commercially available assays to detect antibodies to HIV-1 and/or HIV-2 in human sera. Geneva, Switzerland: WHO documents GPA/BMR/89.4; GPA/BMR/90.1; GPA/RES/DIA90.1; GPA/RES/DIA/91.6; GPA/RES/DIA/ 92.8 and GPA/RES/DIA/93.412. World Health Organization/Global Programme on AIDS. Acquired immunodeficiency syndrome (AIDS proposed WHO criteria for interpreting results from Western blot assays for HIV-1, HIV-2 and HTLV-I/HTLVII). WHO Weekly Epidemiological Record 65(37):281-282, 199013. Malone, J.D., Smith, E.S., Sheffield, J., et al. Comparative evaluation of six rapid serological tests for HIV-1 antibody. Journal of Acquired Immune Deficiency Syndrome (JAIDS) 6:115-149, 199314. The Laboratory Biosafety Guidelines, 3rd Edition. Office of Laboratory Security, Health Canada. 3.1.2, 2004. 15. “Summary of Safety and Effectiveness Data, 50-1110.” bioLytical Laboratories, Inc. 28 January 2011, Table 3.

TECHNICAL INFORMATION

For further information or assistance, contact the Technical Services at 1-604-644-4677.

Reference herein to any specific third party by name, trade name, trade-mark, manufacturer or otherwise does not constitute or imply an endorsement or recommendation of this Kit by such third party, or of the products or services of such third party by bioLytical or that such products or services are necessarily best suited for the intended purpose.

M Manufactured by:

bioLytical Laboratories, Inc.13351 Commerce Parkway Suite 1108Richmond, BC, V6V 2X7CanadaPhone: 1-604-204-6784Fax: 1-604-244-8399www.biolytical.com

Authorized Representative: EMERGO EUROPEPrinsessegracht 20, 2514 AP, The Hague, The NetherlandsPhone: +31.70.345.8570 Fax: +31.70.346.7299

51-1037O 14-Feb-2018© Copyright 2018, All rights reserved.

0543

TRAINING PACKHIV

16 17

Test Controls IFU Bibliography

HIV-1/HIV-2 Test Controls Product Number: 90-1036

Store at -20°C to +8°C H Use by

Y Caution M Manufacturer

i Consult instructions for use C CE Mark

R22 – Harmful if swallowed

Read this Instructions for Use and the INSTI® HIV-1/HIV-2 Antibody Test Instructions for Use before using this product. Conformance with the test procedure is necessary to ensure accurate results. Before performing the test, all operators must become familiar with Universal Precautions for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B Virus, and other Blood-borne Pathogens in Health Care Settings1 NAME AND INTENDED USE The INSTI HIV-1/HIV-2 Test Controls are intended to be used only with the INSTI HIV-1/HIV-2 Antibody Test. SUMMARY INSTI HIV-1/HIV-2 Positive and Negative Controls should be used in conjunction with Good Laboratory Procedures. They should be run under the following circumstances: for new INSTI operator verification prior to performing testing on patient specimens when switching to a new lot number of INSTI test kits whenever a new shipment of kits is received when temperature during storage of the kit falls outside of 2°-30°C (35.6°-86°F) when the temperature of the test area falls outside of 15°-30°C (59°-86°F) at regular intervals as determined by the user facility. PRINCIPLES OF THE PROCEDURE The INSTI HIV-1/HIV-2 Test Controls have been designed for use with the INSTI HIV-1/HIV-2 Antibody Test to validate the correct performance of the test procedure in the hands of the operator. The INSTI HIV-1/HIV-2 Positive Controls are prepared from inactivated human plasma. It is negative for HBsAg and anti-HCV by U.S. FDA licensed test procedures. The Positive Controls have been designed to produce an easily visible but faint blue colour on the INSTI test spot and a darker blue colour on the control spot. The INSTI HIV-1/HIV-2 Negative Control is prepared from defibrinated human serum which is negative for Anti-HIV-1 and Anti-HIV-2, HBsAg, and Anti-HCV. The Negative Control will produce a blue colour on the procedure control spot, but no colour on the test spot, for a Non-Reactive INSTI test result. REAGENTS: HIV-1 POSITIVE CONTROL (90-1036) 1 vial containing 1.0 ml of inactivated human plasma. Each vial is sufficient for 20 INSTI tests. The source material has been heat inactivated at 60°C for 60 minutes. HIV-2 POSITIVE CONTROL (90-1036) 1 vial containing 1.0 ml of inactivated human plasma. Each vial is sufficient for 20 INSTI tests. The source material has been heat inactivated at 60°C for 60 minutes. NEGATIVE CONTROL (90-1036) 1 vial containing 1.0 ml of processed human serum substitute, non-reactive for antibodies to HIV and HCV and non-reactive for HBsAg. Each vial is sufficient for 20 INSTI tests. WARNINGS & PRECAUTIONS

For in vitro diagnostic use only.

Safety Precautions: 1. All specimens should be handled as if capable of transmitting infectious agents. 2. Thoroughly wash hands after handling or performing this test. 3. Do not smoke, eat, or drink in areas where specimens or reagents are being

handled. 4. Wear disposable gloves while handling reagents or specimens. Do not pipette by

mouth. 5. Avoid contact with skin and eyes. If contact occurs, wash affected areas with

water. 6. Avoid forming aerosols. 7. Dispose of all specimens and materials used to perform the test in a biohazard

waste container. The preferred method of disposal is sterilization by autoclaving for a minimum of one hour at 121°C. Disposable materials may be incinerated. Liquid waste may be mixed with sodium hypochlorite (bleach) in volumes such that the final mixture contains 1.0% sodium hypochlorite (using a freshly prepared solution containing 10% household bleach). Allow at least 60 minutes for decontamination to be completed. Do not autoclave solutions that contain bleach.

8. Spills should be cleaned up and decontaminated in accordance with the user facility’s established procedures for handling biohazardous spills.

9. For additional information on bio-safety refer to “Universal Precautions for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B Virus, and other Blood-borne Pathogens in Health Care Settings”1 and “Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposures to HBV, HCV, and HIV and Recommendations for Postexposure Prophylaxis”2.

Handling Precautions:

1. Do not use INSTI HIV-1/HIV-2 Test Controls beyond the expiration date. INDICATIONS OF REAGENT INSTABILITY OR DETERIORATION Positive or Negative Controls that are visibly turbid and/or contain particulate matter should not be used and should be discarded in accordance with safety precautions.

STORAGE INSTRUCTIONS 1. The INSTI HIV-1/HIV-2 Test Controls are shipped without temperature control.

Upon receipt, store frozen at -20°C (-4°F) for up to one year or refrigerate at 2°C (36°F) to 8°C (46°F) for up to one year.

2. The shelf life is dictated by the printed expiry date on the INSTI Test Controls. 3. It is recommended to store the vials in an upright position. 4. Once the controls are thawed continue storage at refrigeration temperatures (2-

8°C). They remain stable until expiry (up to 1 year). Do not re-freeze once the vials have been opened.

5. Fluctuations in temperature causing freeze-thaw cycles may affect the performance of the INSTI Test Controls.

PROCEDURE Materials Required but not Provided

Pipette capable of delivering 50μl of specimen. INSTI HIV-1/HIV-2 Antibody Test Instructions for Use.

Instructions for use

1. Read the INSTI HIV-1/HIV-2 Test Controls Instructions for Use prior to using the INSTI HIV-1/HIV-2 Test Controls.

2. Remove from storage at -20°C (-4°F) to 8°C (46°F) and allow the Controls to reach room temperature before testing with INSTI. Return Controls to refrigeration storage at 2-8°C after use.

3. Mix the Controls by swirling before use. 4. Uncap the HIV-1 Positive, HIV-2 Positive or Negative Control vial. Using a 50μl

pipette, collect 50μl of the Control. 5. Transfer the Control sample held in the pipette to the INSTI Sample Diluent vial

(Solution 1). Recap the vial and mix by inversion. 6. Follow the INSTI test procedure as described in the TEST PROCEDURE section of the

INSTI HIV-1/HIV-2 Antibody Test Instructions for Use. 7. All Controls should be tested in the same manner as patient samples. 8. The HIV-1 Positive Control, HIV-2 Positive Control and the Negative Control are

to be run on separate Membrane Units. INTERPRETATION OF RESULTS

Follow the interpretation guidelines provided in the INTERPRETATION OF RESULTS section of the INSTI HIV-1/HIV-2 Antibody Test Instructions for Use.

Reactive Result: Both the control spot and the test spot show blue colour development.

Non-Reactive Result: Only the control spot shows blue colour development. Invalid Result: The test is invalid if any of the following occurs:

-There is no blue colour on both the control spot and the test spot -There is blue colour on the test spot but not on the control spot -Uniform tint across the membrane -Only blue specks appear on the membrane

LIMITATIONS OF THE PROCEDURE The INSTI HIV-1/HIV-2 Test Controls are only validated for use with the INSTI HIV-1/HIV-2 Antibody Test.

1. The TEST PROCEDURE and INTERPRETATION OF RESULTS sections of the INSTI HIV-1/HIV-2 Antibody Test Instructions for Use must be adhered to when testing the INSTI HIV-1/HIV-2 Test Controls.

2. Deviations from the procedure outlined in the INSTI HIV-1/HIV-2 Antibody Test Instructions for Use may produce unreliable results.

3. Do not dilute the INSTI HIV-1/HIV-2 Test Controls. The INSTI HIV-1/HIV-2 Test Controls are intended for use in undiluted form.

4. Adverse shipping and storage conditions or use of expired reagents may produce erroneous results.

EXPECTED RESULTS The HIV-1 Positive Control and HIV-2 Positive Control must be Reactive with INSTI and the Negative Control must be Non-Reactive with INSTI. Controls that produce incorrect or invalid results must be re-tested with INSTI. Contact bioLytical Laboratories’ Technical Support if the INSTI HIV-1/HIV-2 Test Controls do not produce the expected results. REFERENCES

1. CDC. Universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health care settings. MMWR 1988; 37(24):377-388

2. CDC Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposures to HBV, HCV, and HIV and Recommendations for Postexposure Prophylaxis. MMWR 2001; 50(RR-11):1-42.

Manufactured by:

bioLytical Laboratories, Inc 13351 Commerce Parkway Suite 1108 Richmond, BC, V6V 2X7 Canada Phone: +1 604-204-6784 Fax: +1 604-244-8399 www.biolytical.com

Authorized Representative:

EMERGO EUROPE

Prinsessegracht 20, 2514 AP The Hague, The Netherlands Phone: +31.70.345.8570 Fax: +31.70.346.7299

51-1181D 02-JUL-2018 © Copyright 2018. All rights reserved

0543

Please contact the Business Development Manager in your area to request the QA controls.

I. Published and Submitted INSTI® Manuscripts, and Reports:Cook, D., Gilbert, M., DiFrancesco, L., Krajden, M. 2010. “Detection of early sero-conversion HIV infection using the INSTI® HIV-1 Antibody Point-Of-Care test.” The Open AIDS Journal 176-179.

This study compared INSTI® HIV-1 Antibody Point-of-Care (POC) Test to laboratory-based tests for detection of early sero-conversion (i.e. acute) HIV infections. Of 53 individuals with early HIV infection, the INSTI® test was reactive for 34/49 (sensitivity 69.4%; 95%, CI 54.6-81.8%) early-infected individuals whose laboratory-based 3rd generation HIV EIA test was reactive. In conclusion, the INSTI® POC test performs well compared with other POC tests for the detection of early sero-conversion HIV infection, but it may miss 20% to 30% of those detected by laboratory-based 3rd generation anti-HIV tests. Both POC and laboratory-based anti-HIV tests will fail to detect a proportion of infected individuals in the first weeks after infection.

Galli, R.A., Green, K.F., La Marca, A., Waldman, L.F. Powers. R.E., Daly, A.C., Shackleton, C.R. 2013. “Evaluation of the accuracy and ease of use of a rapid HIV-1 AntibodyTest performed by untrained operators at the point of care.” Journal of Clinical Virology 65-69.

This is a prospective study conducted to compare the results of the FDA-cleared, INSTI® HIV-1 Antibody Test (INSTI®) used by untrained operators on finger-stick whole blood to results obtained by trained laboratory professionals using FDA-cleared comparator methods (CM) on matching venous blood. Of the 517 HIV positive subjects (34 new positives and 483 known positives) the concordance between INSTI® performed by untrained operators and CM performed by trained laboratory professionals was 100% (95% CI = 99.3–100%). Concordance for HIV negative results (n = 871) was 99.8% (95%CI = 99.2–99.9%). In conclusion, untrained operators with no laboratory background were able to perform and interpret the results of INSTI® on finger-stick blood and limit of detection specimens with a high degree of accuracy by following only the manufacturer’s written instructions.

Lee B.E., Plitt S., Fenton J., Preiksaitis J.K., Singh A.E. 2011. “Rapid HIV tests in acute care settings in an area of low HIV prevalence in Canada.” Journal of Virological Methods 66-71.

In this study, rapid HIV testing was performed on serum samples in acute care settings in five hospitals from urban and rural regions in Alberta, Canada using the INSTI® HIV-1/HIV-2 Rapid Antibody Test. Parallel standard HIV antibody tests were performed at the provincial reference laboratory. The study found the sensitivity of the rapid HIV test compared to standard HIV testing was 100%, specificity was 99.9%, the positive predictive value was 96% and the negative predictive value was 100%.

Pavie, J., Rachline, A., Loze, B., Niedbalski, L., Delaugerre, C., Laforgerie, E., Plantier, J.C., Rozenbaum, W., Molina, J.M., Simon, F. 2010. “Sensitivity of five rapid HIV test on oral fluid or finger-stick whole blood: a real-time comparison in a healthcare setting.” Plos One e11581.

In this study, 200 adults with documented HIV-1 (n = 194) or HIV-2 infection (n = 6) were prospectively screened with five HIV rapid tests using either oral fluid (OF) or finger-stick whole blood (FSB). 20 randomly selected HIV-seronegative subjects served as controls, and the results were read blindly. Most patients had HIV-1 subtype B infection (63.3%) and most were on antiretroviral therapy (68.5%). Sensitivity was 86.5%, 94.5%, 98.5%, 94.9%, 95.8% and 99% respectively, with OraQuick OF, OraQuick FSB, Vikia, Determine, Determine Ag/Ab Combo and INSTI® (p<0.0001). In conclusion, rapid HIV tests were less sensitive on oral fluid than on finger-stick whole blood and less sensitive on finger-stick whole blood than on serum

Singh, A.E., Lee, B., Fenton, J., Preiksaitis, J. 2013. “The INSTI® HIV-1/HIV-2 antibody test: a review.” Expert Opinion Medical Diagnostics 299-308.

This article reviews available data on kit characteristics and current performance data on the INSTI® HIV-1/HIV-2 antibody test are presented together with six other rapid point-of-care tests (RPOCTs) for HIV antibody. The authors conclude the good performance of INSTI® HIV-1/HIV-2 antibody test, its ease of use, the rapid availability of results (< 5 min), and the lack of specialized equipment required to use the kit make this kit a useful addition to the global market. The unique antigen and flow through technology contained in the kit make it a strong

TRAINING PACKHIV

18 19

Bibliography Clinician’s Briefing Notes

addition to HIV RPOCTs and to rapid/rapid algorithms used in many resource-limited settings.WHO Prequalification of Diagnostics Programme. 2013. Product: INSITI HIV-1/HIV-2 Antibody Test. Number: PQDx 0002-002-00, Geneva: WHO PQDx PR.(Public Report)

This report is a summary of prequalification status for INSTI® HIV-1/HIV-2 Antibody Test. The test was accepted for the WHO list of prequalified diagnostics and was listed on 29 August 2013. The WHO outlines the manufac-turing, productions, quality and labeling reviewed and approved for the prequalification. (Add brief statement on INSTI® lab evaluation results, which were very good – “In a limited evaluation on a panel of 1079 clinical-ly-derived specimens, we found an initial sensitivity (95% CI) of 100% (99.1% - 100%) and an initial specificity (95% CI) of 99.7% (98.9% - 100%) compared to the reference assays. The final sensitivity (95% CI) was 100% (99.1% - 100%) and the final specificity (95% CI) was 99.7% (98.9% - 100%) compared to the reference assays. In this study, 0% of the results were recorded as indeterminate. Results were interpreted independently by three technicians; the inter-reader variability was 0%. The invalid rate was 0%.

User testimonialsSamedaydoctor is a busy sexual health clinic in London, England. We have been using INSTI® instant HIV testing for over six months and in that time have never had a false positive or false negative result. We always confirm positives by DUO testing and the INSTI® test is always correct. The test is easy to use, fast and extremely reliable.Dr Laurence Gerlis, Samedaydoctor

I’m very happy to say that we’ve found the test easy to use; to give reliable results which have on occasion been verified by independent testing; and to be an effective clear way of eliminating patients HIV concerns.Dr Séan Cummings MBBS DRCOG DFFP LLM MRCGP, Freedomhealth Ltd.

The INSTI® HIV rapid POCT kit has revolutionised our private practice. Prior to the test, we were able to offer a same day HIV testing service for our client group, who are mainly gay men and people who have had some risk and are now anxious. However, this service was dependent on the client coming into clinic, and during regular office hours, and involved providing results by telephone, some four hours after the blood was taken, making managing a reactive or positive result difficult. With the introduction of the INSTI® test, we have been able to offer a seven day a week testing service, with appointments into the evening and outside normal office hours. In addition we are also able to offer a visiting service, due to the portability of the INSTI® kit, allowing us to visit patients in their home, place of work or hotel. Having used other rapid HIV POCT tests in my NHS practice, I have been very impressed with the easy of use and rapid result time offered by the INSTI® HIV, whilst being reassured by the true human IgG control, which ensures a gold standard of test validity, which when explained to patients reassures them of the reliability of the test. I have recommended a number of colleagues to use the test and recently based a presentation at a national clinical meeting on the practical applications of the test in expanding innovative testing practice.Justin Gaffney RN, Consultant Nurse for STI Control/Managing Director Metrosexual Health Limited

At TPA we currently use two kinds of POCT for HIV, the INSTI® and the Determine 1/2. These tests have enabled people to come forward for testing and receive a full pre and post- test consultation along with their results all within the hour. This has encouraged those who would otherwise not have tested due to clinic waiting times and the length of time it takes to receive a result, to come forward for a check up. The INSTI® test has a lot of great benefits as it all packaged together and with using a plastic pipette as opposed to a glass capillary tube, they have proved to be a safe resource for staff when testing within prison settings and when testing high risk groups. The reliability of the tests are also a great benefit when reassuring clients of their results. TPA also took part in a project in Ghana establishing two rapid testing services and when training staff on both the Determine and the INSTI®, a high percentage of staff favoured the INSTI® for its simplicity and speed at providing a result. I would highly recommend any clinic considering POCT tests to use the test.Michael Kay, Manager, Teeside Positive Action

TRAINING PACKHIV

20

Four Simple Steps...

The INSTI® Multiplex HIV-1/2 Syphilis Ab Test is a single-use, rapid, flow-through, in vitro qualitative immunoassay for the detection of antibodies to Human Immunodeficiency Virus (HIV) Type 1/ Type 2 and Treponema pallidum in human EDTA-whole blood, fingerstick blood, serum or EDTA-plasma. The test is intended for use by trained personnel as an in vitro diagnostic device capable of providing results in less than one minute.

Developed by the same company that brought you the INSTI® Point of Care HIV test, The INSTI® Multiplex Test is an advancement in the testing process that allows the individual to be checked for both HIV and Syphilis. Just as the original, the test takes as little as 60 seconds for a result. Further advice, help and support can be given to anyone who tests positive without the need for a lengthy wait.

MULTIPLEX

• Built-in true human IgG Control• Use 50μL of EDTA-whole

blood, fingerstick blood, serum or plasma

• Rapid results at a glance (60 seconds)• Proven early antibody detection• Large scale trial data

available• Easy to understand

instructions• No timing required• No refrigeration required

Add 50μLof EDTA-whole blood, fingerstick blood, serum or EDTA-plasma with pipette to the sam-ple diluent (bottle #1). Close lid and invert the bottle (do not shake).

Pour the diluted sample into the Membrane Unit Well. HIV-1, HIV-2 and Syphilis antibodies, if present, are captured at the test spots.

Add the Colour Developer (bottle #2) to the center of the Membrane Unit well to generate a blue control spot and up to two additional test spots if HIV-1 and/or HIV-2 and/or Syphilis antibodies are present.

Add the Clarifying Solution (bottle #3) to the Membrane Unit well to reduce background colour and produce a more distinct test and/or control spot.

21

Interpretation of Results

TRAINING PACK

HIV SPOT INTERPRETATION

SYPHILIS SPOT INTERPRETATION

HIV & SYPHILIS SPOT INTERPRETATION

Strong Reactive

Always readresults withINSTI® logofacing you

Strong Reactive

Strong Reactive Weak Reactive

Weak Reactive

Weak Reactive

Indeterminate

Indeterminate

Negative

Negative

Control

Syphilis

Control

HIV

Always readresults withINSTI® logofacing you

Always readresults withINSTI® logofacing you

Control

HIV

Syphilis

22

Instructions for Use

MULTIPLEX

MULTIPLEX

HIV-1/2 Syphilis* Ab Test*C mark for Syphilis by self-declaration

Single-use rapid assay for the detection of antibodies toHuman Immunodeficiency Virus Type 1 (HIV-1), Type 2 (HIV-2), and T. pallidum90-1028 - One INSTI® Multiplex HIV-1/2 Syphilis Ab Test with support materials (for POC use)

Store at 15°C to 30°C K Sterilization using irradiation

Y CautionHarmful if swallowed g Lot number

V In Vitro diagnostic medical device h Catalogue Number

i Consult instructions for use M Manufacturer

D Do not reuse C CE Mark

H Use by

Store at 15°C – 30°C. For in vitro diagnostic use only.

i It is recommended that the entire Instructions for Use be read prior to beginning the test procedure. Although the assay is designed to be simple to use, conformance with the test procedure is necessary to ensure accurate results.

INTENDED USE - Not for donor screeningThe INSTI MULTIPLEX HIV-1/2 Syphilis Ab Test is a single use, rapid, flow-through in vitro qualitative immunoassay for the detection of antibodies to Human Immunodeficiency Virus Type 1/ Type 2 and Treponema pallidum in human EDTA-whole blood, fingerstick blood, serum or EDTA-plasma. The test is intended for use by trained personnel in medical facilities, clinical laboratories, emergency care situations, and physicians’ offices as an in vitro diagnostic device capable of providing results in less than one minute. It is suitable for near-patient or point-of-care (POC) testing, and is not currently approved for self-testing. All required pre and post-test counseling guidelines must be followed in each setting in which the INSTI Multiplex antibody test is used. The INSTI MULTIPLEX HIV-1/2 Syphilis Ab Test will be referred to as INSTI Multiplex Test in the remainder of this Instructions for Use. SUMMARYAcquired Immunodeficiency Syndrome (AIDS) is caused by at least two retroviruses, HIV-1 and HIV-2. HIV-1 and HIV-2 are similar in genomic structure, morphology and ability to cause AIDS.1 HIV is transmitted mainly by sexual contact, exposure to blood or blood products, or from an infected mother to her fetus. People with increased risk of HIV infection include haemophiliacs, intravenous drug-users and men having sex with men (MSM). HIV has been isolated from patients with AIDS, AIDS-related complex (ARC), and from persons at high risk of contracting AIDS.2-5 Antibodies specific for HIV envelope proteins are prevalent in sera from persons at high risk of contracting AIDS as well as in people with AIDS, or ARC.5-7 The presence of antibodies to HIV indicated previous exposure to the virus, but does not necessarily constitute a diagnosis of AIDS. The prevalence of antibodies to HIV in people not known to be at risk of acquiring HIV infection is unknown, but significantly less.5 Absence of antibodies to HIV does not indicate that an individual is free of HIV-1 or HIV-2; HIV has been isolated from seronegative individuals prior to seroconversion. Test specificity and sensitivity depend, amongst other factors, on: a) the selection of HIV antigens used for antibody detection, b) the classes of antibodies recognized by the detection conjugate, and c) complexity of the protocol used to perform the test.8 Non-specific reactions may be observed in some specimens. A reactive INSTI test result should be considered a preliminary result, with appropriate counseling provided in POC settings. Following a reactive HIV rapid test result, a venous blood sample must be drawn in an EDTA collection tube (for whole blood or plasma), and forwarded to a laboratory for HIV confirmatory test.

Treponema pallidum is the causative agent of syphilis. Some of the proteins of this organism are highly immunoreactive and infected persons develop antibodies soon after infection. These antibodies are unaffected by treatment and once induced they remain detectable for years. It is possible for a person to be antibody positive for T. pallidum, but have been cured of the infection. Following a reactive result for T. pallidum antibodies, a venous blood sample must be drawn in an EDTA collection tube (for whole blood or plasma) or red-top tube (for serum), and forwarded to a laboratory for syphilis confirmatory testing. A confirmatory test is required to determine active syphilis or past infection in the patient.

PRINCIPLES OF THE TESTThe INSTI Multiplex Test is a manual, visually read, flow through immunoassay for the qualitative detection of HIV-/HIV-2 and syphilis IgG and/or IgM17 antibodies in human blood, serum or plasma. The test consists of a synthetic filtration membrane positioned atop an absorbent material within a plastic cartridge, referred to as the INSTI Membrane Unit. The membrane has been specifically treated with HIV-1 and HIV-2 recombinant proteins, and syphilis antigens which react with HIV-1/HIV-2 and syphilis IgG and/or IgM antibodies in the specimen to produce distinct visual signals on the membrane. The membrane also includes a procedural control. The procedural control consists of a protein-A treated spot capable of capturing IgG or IgM antibodies normally present in blood and blood components. IgG or IgMantibodies react with a proprietary chromatic agent to produce a visual signal on the membrane.

Since IgG and/or IgM antibodies can be present in blood from normal or HIV or syphilis positive human specimens, the control dot provides a visual signal when the test is run, indicating that the test was performed correctly. If the control dot does not appear, the test is considered invalid. In the case of the test dots, recombinant HIV-1, HIV-2 and syphilis proteins, embedded in the membrane, capture specific antibodies, if present in the specimen. Antibodies captured in the test dots react with a proprietary chromatic agent to produce visible signals on the membrane. The membrane unit is designed to filter, absorb, and retain the test specimen and all the test reagents in such a manner as to limit leakage and exposure of personnel to potentially infectious materials.

Reagents required to conduct a test include Sample Diluent, Colour Developer and a Clarifying Solution. The test is performed by adding the blood, serum or plasma specimen to the vial of Sample Diluent, which lyses the red blood cells and dilutes the specimens. This specimen/diluent solution is then poured onto the well of the membrane unit. HIV-1/HIV-2 and syphilis antibodies, if present in the specimen, are captured by proteins on the filtration membrane. Colour developer is then added to the Membrane Unit. The Colour Developer reacts with the captured antibodies to generate a distinct blue dot at the location of the control spot and, in the case that HIV-1/HIV-2 and/or syphilis antibodies are present in the specimen, a blue dot also appears at the location of one or both of the test spots on the membrane. In the final step, the Clarifying Solution is then added to the membrane to decrease background colour in order to make the control and test dots more distinct.

Antigen Selection: The INSTI HIV-1/HIV-2 assay portion utilizes a combination of recombinant transmembrane proteins from HIV-1 (gp41) and HIV-2 (gp36). Use of these proteins overcomes sensitivity and specificity problems associated with tests based on viral lysates or a combination of core antigen and other viral proteins.9-13 The syphilis antigens bound to the membrane consist of a recombinant fusion protein derived from p17 and p47 domains of Treponema pallidum.

Antibody Detection: The INSTI Multiplex assay uses a unique reagent to detect antibodies to HIV-1/HIV-2 and syphilis. Although primarily designed to detect the IgG class of specific antibodies, the INSTI HIV-1/HIV-2 assay portion has been shown to detect IgM antibodies in samples obtained early in HIV infection during seroconversion, and low titer anti-HIV-1 samples obtained later in infection17

Test Complexity: The INSTI Multiplex Test was designed to reduce protocol complexity. The INSTI Multiplex assay does not require sample preparation, accurate timing, or several steps, which include multiple washes and reagents. These requirements increase the complexity of an assay and lead to procedural errors which may adversely affect sensitivity and specificity. Total test time may vary slightly depending on specimen type but results of valid tests are usually clearly readable within one minute.

SPECIMEN COLLECTION AND STORAGE1. For EDTA-whole blood, EDTA-plasma or serum specimens, follow venipuncture blood collection

procedures using lavender-top EDTA anticoagulant tubes (for whole blood and plasma) or red-top (noanticoagulant) tubes for serum.

2. If plasma or serum is to be used, separate from the blood cells by centrifugation.3. Serum or EDTA-plasma may be stored at 2-8°C for up to 5 days, stored frozen at ≤ -20°C for 3 months,

or stored frozen at ≤ -70°C for one year.4. Whole blood specimens collected in EDTA anticoagulant may be stored at 2-8°C and should be tested

within 48 hours. Do not heat or freeze whole blood specimens.5. Do not dilute prior to testing.

KIT COMPONENTS AND STORAGEINSTI components should be stored at 15-30°C. All kit components are individually packaged for single use only. Each test requires the following materials:

1. Membrane Unit, individually packaged, prepared with control (IgG and/or IgM capture), HIV test(gp41 and gp36 antigen) and T. pallidum (p17-p47 antigen) reaction spots. For single use only in the INSTI procedure.

2. Sample Diluent, Y Solution 1 vial, containing 1.5 mL of tris-glycine buffered solution containing celllysis reagents, with adequate space for addition of blood, serum or plasma samples being tested withINSTI. Ready to use, invert 2-3X immediately before use.

3. Colour Developer, Y Solution 2 vial, containing 1.5 mL of a blue-coloured borate bufferedproprietary indicator solution designed to detect IgG and IgM in the control spot and specific HIV andT. pallidum antibodies in the test spots.. Ready to use, invert 2-3X immediately before use.

4. Clarifying Solution, Y Solution 3 vial, containing 1.5 mL of a proprietary tris-glycine buffered clarifying solution designed to remove background staining from the membrane unit prior to reading the INSTI test results. Ready to use, no mixing or preparation required

All solutions contain 0.1% Sodium Azide as a preservative and are harmful if swallowed. All solutions are for single use only and are stable to date and under storage conditions indicated on labels.

SUPPORT MATERIALSThe following materials are required when testing fingerstick whole blood:1. Single-use Alcohol Swab

2. Single-use Lancet K 0344

3. Single-use Pipette 50μL

MATERIALS REQUIRED BUT NOT PROVIDED▪ Personal protective equipment ▪ Appropriate biohazard waste containers and disinfectants▪ Absorbent cotton balls for fingerstick or venipuncture wound closure

For venipuncture blood collection and testing:▪ Venipuncture apparatus if collecting blood samples▪ Appropriate blood collection tubes▪ Appropriate shipping containers▪ Precision pipette capable of delivering 50μL of sample

MATERIALS AVAILABLE AS AN ACCESSORY TO THE KIT

INSTI T. pallidum Antibody Positive Control: Separate vials of anti-T. pallidum positive de-fibrinatedhuman plasma control sample, product no. 90-1032 are available from bioLytical Laboratories. INSTI HIV-1/HIV-2 Test Controls: Separate HIV-negative human serum substitute and HIV-1/HIV-2positive de-fibrinated human plasma control samples product no. 90-1036 are available from bioLytical Laboratories, for use in quality control procedures. Please refer to the section on Quality Control, following the Assay Procedure, the INSTI Multiplex Test Controls Instructions for Use and the INSTI HIV-1/HIV-2 Test Controls Instructions for Use.

C0543for HIV-1/HIV-2 only

23

Instructions for Use

WARNINGS

For in vitro diagnostic use only VIt is recommended that the entire Instructions for Use be read prior to beginning the test procedure. Although the assay is designed to be simple to use, conformance with the test procedure is necessary to ensure accurate results.

1. Do not mix reagents from different lots.2. Do not use reagents or kits beyond the stated expiration date.3. Do not use the Membrane Unit if the foil pouch has been previously opened or if the packaging integrity

has been compromised. Once the Membrane Unit has been opened, it must be used immediately.4. Avoid microbial contamination of reagents.5. Y Sodium azide is present at 0.1% in all assay reagents. Sodium azide may react with lead or

copper plumbing to form highly explosive metal azides. If products containing sodium azide are discarded into a drain, flush with large amounts of water to prevent azide build-up. Check with local regulatory agencies to determine at what concentration sodium azide may cause a product to be regulated as hazardous waste.

6. The performance characteristics of the INSTI HIV-1/HIV-2 assay have not been established for bodyfluids other than EDTA whole blood, fingerstick blood, serum, and EDTA-plasma. The use of bloodcollected in anticoagulants other than EDTA has not been validated. Insufficient data are available tointerpret tests performed on other body fluids, pooled blood or pooled serum and EDTA-plasma, or products made from such pools.

7. Failure to use the recommended reagent and specimen volumes may result in leakage and/or overflow of liquids from the membrane unit.

8. If the test kit is exposed to temperatures outside of 15°–30°C, ensure it is brought to this temperature range before performing testing. Use the Syphilis INSTI Controls and validated HIV Controls to ensure proper kit performance

9. Y Patients that are on long term antiretroviral drug therapy may give a false negative HIV-1/HIV-2test result.

10.Samples from patients with severe hypogammaglobulinemia conditions such as multiple myeloma may result in false negative or invalid results for HIV with INSTI Multiplex.

11.Patients with elevated haemoglobin levels may test false negative for HIV with INSTI Multiplex.15

12.Because the INSTI Multiplex Test has a lower affinity to IgM antibody class compared to IgG, patients in the early primary stage of syphilis infection may test negative for T. pallidum antibodies with INSTI Multiplex.

PRECAUTIONS

1. Y All specimens should be handled as if capable of transmitting infectious diseases. It is recommended that Directive 2000/54/EC, or equivalent regulations, be observed.14

2. Thoroughly wash hands after handling or performing this test.3. Do not smoke, eat, or drink in areas where specimens or kit reagents are being handled.4. Wear a lab coat and disposable gloves while handling kit reagents or specimens. Do not pipette by

mouth.5. Avoid contact with skin and eyes. If contact occurs, wash affected areas with water.6. Avoid forming aerosols.7. Y Dispose of all specimens and materials used to perform the test as if they contained infectious

agents. The preferred method of disposal is sterilization by autoclaving for a minimum of one hour at 121°C followed by incineration. Liquid waste not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 0.5% sodium hypochlorite (a solution containing 10% household bleach). Allow at least 30 minutes for decontamination to be completed. Do not autoclave solutions that contain bleach.

8. Spills should be cleaned up and decontaminated in accordance with the user facility’s establishedprocedures for handling biohazardous spills.

ASSAY PROCEDURENOTE: All INSTI Membrane Units must be used immediately once opened. All reagents should be dispensed evenly in the center of the well.

Sampling Fingerstick Blood:1. Gather support materials (swab, lancet, pipette), one sealed test pouch containing INSTI Membrane

Unit, and one vial each of the Sample Diluent, Colour Developer, and Clarifying Solution for each test to be performed.

Y CAUTION! The amount of sample (fingerstick blood) is critical. To ensure that the proper amount of blood is achieved, follow these instructions carefully:

2. Massage the finger to allow the blood to move to the surface (fingertip will become pink). Use heatingpad if available to warm the hand. Hand must be positioned at waist level or lower.

3. Wipe the fingertip with the alcohol swab.4. As soon as the finger is dry, twist and remove the protective insert from the lancet. Press the finger

firmly at the point just below where the lancet will be applied. With the other hand, place the lancet on the side of the fingertip and press hard until it clicks. Immediately dispose the used lancet into a proper sharps container.

5. As the blood droplet forms, hold the pipette horizontally and touch the tip of the pipette to the blood sample. Capillary action automatically draws the sample to the fill line and stops. If very little blood trickles out of the puncture, gently apply intermittent pressure below the puncture site to obtain the required blood volume. If blood is inadequate, perform a second skin puncture using a new lancet.

CAUTION! Filling is automatic: Never squeeze the pipette bulb while sampling.

6. Transfer the blood held in the pipette to the Sample Diluent vial (Solution 1). Align the tip of the pipette with the Sample Diluent vial and squeeze the bulb to dispense the sample (See Figure A).NOTE: If the sample will not expel, hold the pipette vertically and slide a finger over (without pressing) the vent hole, then squeeze the bulb (See Figure B). Recap the vial and mix by inversion. Follow General Procedure after Sampling, below.

Sampling Whole Blood, serum, plasma and Test Controls:1. Bring specimens to room temperature and mix each specimen thoroughly prior to use. Do not heat

or repeatedly freeze/thaw specimens.2. Gather one sealed test pouch containing INSTI Membrane Unit, and one vial each of the Sample

Diluent, Colour Developer, and Clarifying Solution for each test to be performed.3. Using a pipette, add 50μL of whole blood, serum, plasma, or kit controls (see Note) to the Sample

Diluent vial. Recap the vial and mix by inversion 2-3 times.Y Adding an excessive amount of specimen may cause the device to overflow or leak.

NOTE: In POC settings, for INSTI kit controls, it is important to use a 50μL pipette device to add the control material to the Sample Diluent vial. Do not use the disposable single-use pipette provided for finger stick blood collection.

General Procedure after Sampling:1. Tear open the pouch and remove the INSTI Membrane Unit without touching the center well. Place

the unit on a level surface. For sample identification purposes the bottom tab of the Membrane Unit may be labeled with the patient’s name or number.NOTE: At this point, it is important that the following steps be performed immediately and in sequence.

2. Mix the Sample Diluent-specimen mixture by inverting several times and pour the entire contents to the center of the Membrane Unit well. (NOTE: Do this within 5 minutes after the specimen has been added to the Sample Diluent vial). The sample should be absorbed through the membrane in less than 30 seconds; however, absorption times will vary slightly depending upon sample type.

3. Re-suspend the Colour Developer by slowly inverting to mix the solution thoroughly until the reagent is evenly suspended. Open the Colour Developer and add the entire contents to the center of the Membrane Unit well. The coloured solution should flow through completely in about 20 seconds.

4. Open the Clarifying Solution and add the entire contents to the center of the Membrane Unit well. This will lighten the background colour and facilitatereading. Immediately read the result while the membrane is still wet. Do notread the results if more than 5 minutes have elapsed following the addition of Clarifying Solution.

NOTE: INSTI tests should be read and interpreted under adequate lighting.

QUALITY CONTROLKit Controls:The INSTI Multiplex Test has a built-in IgG and IgM capture procedural control that demonstrates assay validity and adequate sample addition. A blue colour on the control dot indicates that the proper specimen was added and that the assay procedure was performed correctly. The control dot will appear on all valid INSTI tests. (Refer to Interpretation of Results, below.)

Separate Syphilis Controls and HIV Controls are available for use with the INSTI Multiplex Test. The controls are used to verify Syphilis and HIV test performance and interpretation of results. Kit controlsshould be run under the following circumstances:▪ for new INSTI operator verification prior to performing testing on patient specimens▪ when switching to a new lot number of INSTI test kits▪ whenever a new shipment of INSTI kits is received▪ when temperature during storage of the kit falls outside of 15°-30°C ▪ when the temperature of the test area falls outside of 15°-30°C ▪ at regular intervals as determined by the user facility.

Refer to the INSTI T. pallidum Test Controls Instructions for Use and the INSTI HIV-1/HIV-2 Test Controls Instructions for Use for additional information on the use of these reagents. It is the responsibility of each user of the INSTI T. pallidum Test Controls to establish an adequate quality assurance program to ensure proper performance under their specific locations and conditions of use.

CAUTION! It is not recommended to use external controls that have not been validated for the INSTI Multiplex Test as these may not produce the expected results.

TRAINING PACK

24

Instructions for Use

MULTIPLEX

INTERPRETATION OF RESULTS▪ Do not read the results if more than 5 minutes have elapsed following the addition of Clarifying

Solution.▪ If using the syphilis control samples provided by bioLytical Laboratories, all syphilis Positive

Controls must be reactive with INSTI and all Negative Controls must be non-reactive with INSTI. Controls that produce incorrect or invalid results must be re-tested with INSTI. If results are still incorrect or invalid, inform bioLytical Laboratories immediately.

NON-REACTIVE ► One blue dot that is clearly discernable above anybackground tint should appear on the membrane. This is the procedural Control Dot and shows that the test has been performed correctly. The control dot is located towards the top of the read frame furthest from the plastic tab on the Membrane Unit. No reaction should be visible at either of the two test spots, located below the control. A non-reactive result indicates that antibodies to HIV-1/HIV-2 and syphilis were not detected in the specimen.

REACTIVE ► Two or three blue dots that are discernable above any background tint indicate that the specimen contains HIV-1 and/or HIV-2 and/or syphilis antibodies, depending on the position of the dots. One dot may be darker than the other. A sample giving these patterns is considered a preliminary reactive. Following a reactive rapid test result, a venous blood sample must be drawn in an EDTA collection tube (for whole blood or plasma) or red-top tube (for serum), and forwarded to a laboratory for HIV and/or syphilis confirmatory testing.

INVALID ► The test is invalid if any of the following occurs:A. There is no dot on the membraneB. The test dots appeared without the control dotC. Uniform tint across the membraneD. Only scattered blue specks appear on the membrane

NOTE: Invalid tests with fingerstick blood should be repeated with a fresh sample using a new membrane unit, kit components and support materials. Invalid tests with whole blood, plasma or serum samples should be repeated using a new membrane unit and kit components.

INDETERMINATE ► The test is indeterminate if a faint background ring appeared on the test areas.Following an indeterminate INSTI test result, a venous blood sample must be drawn in an EDTA collection tube (for whole blood or plasma) or red-top tube (for serum), and forwarded to a laboratory for HIV and/or syphilis confirmatory testing.

Please note the following:1. Following a reactive or indeterminate INSTI test result, a venous blood sample must be drawn in an EDTA collection tube (for whole blood or plasma) or red-top tube (for serum), and forwarded to a laboratory for HIV and/or syphilis confirmatory testing.2. Depending on the antibody titer, a reactive specimen may be less intense in colour than the procedural control, or vice versa.3. Only a solid blue spot of colour discernibly darker than the background colour should be interpreted as reactive or positive. In rare instances, a faint background ring may appear around the test spot; this should not be interpreted as a reactive result. Only tests exhibiting distinct fully formed blue test dot combined with a distinct fully formed blue control dot should be interpreted as reactive.

4. An invalid result indicates that the test was performed incorrectly or there is a problem with the sample or device. The absence of a distinct control dot usually indicates that the sample volume was insufficient. An invalid test must be repeated.5. A test resulting in a uniform blue tint across the entire membrane, thus obscuring the control and test spots, can occur when more than 60μL of whole blood is used and the flow through of the assay membrane is obstructed.6. An individual who has a non-reactive result but was involved in HIV-risk activity is recommended toobtain additional testing over the next months.7. To significantly reduce the risk of HIV or syphilis transmission, it is advisable to refrain from high risk activities such as unprotected sex and needle sharing at all times.

LIMITATIONS OF THE TEST▪ Flow Times: In some instances, samples may exhibit longer than normal flow times (from the time

the Sample Diluent specimen mixture is poured in the membrane well to the time the Clarifying Solution has fully flown through the membrane). This is due to variable factors such as cellular components, especially with whole blood. In instances of long flow times, a faint shadow in the form of a ring may appear at the test spot location, but this should not be interpreted as a reactive result. This should be considered as an indeterminate result.In these instances, a venous blood sample should be drawn in an appropriate collection tube, and forwarded to a laboratory for HIV and/or syphilis confirmatory testing.

▪ The INSTI Multiplex Test procedure and the interpretation of result must be followed closely whentesting for the presence of antibodies to HIV and/or syphilis in serum, plasma or whole blood.

▪ Insufficient data are available to interpret tests performed on other body fluids, pooled blood or pooled serum and plasma, or products made from such pools; therefore, testing of these specimens is notrecommended.

▪ The INSTI Multiplex Test has not been validated for detection of antibodies to HIV-1 Group N subtypes.▪ The INSTI Multiplex Test detects antibodies to HIV-1/HIV-2 and T. pallidum, and is useful in

establishing infection with HIV and/or syphilis. Because a variety of factors may cause non-specific reactions, a patient found to be positive for HIV or syphilis using the INSTI Multiplex assay should have a blood sample drawn for laboratory-based confirmatory testing. A person who has antibodies to HIV is presumed to be infected with the virus and appropriate counseling and medical evaluation should be offered. The presence of HIV antibodies indicates past exposure to HIV but is not a diagnosis of AIDS, which can only be made by a physician. However, a non-reactive test does not rule out past exposure to HIV. The risk of an asymptomatic person with repeated reactive results developing AIDS is not known. The prevalence of HIV infection in various groups, as well as clinical and public health guidelines, are available in the CDC Morbidity and Mortality Report.8 The presence of antibodies to T. pallidum may indicate current or past syphilis infection, and a blood sample should be collected and sent to a laboratory for confirmation of infection status. Antibodies to the syphilis antigens used in this test may persist for decades, even in spite of successful therapy. A positive syphilis test may not be an indication of an ongoing infection.

HIV PERFORMANCE CHARACTERISTICS (Note: The HIV-1/HIV-2 portion of the Multiplex assay is identical to CE Marked INSTI HIV-1/HIV-2 Antibody Test product 90-1015. All data presented in this section is based on the data presented in the INSTI HIV-1/HIV-2 Antibody Test Instructions for Use,document 51-1037)

SENSITIVITY DETECTION OF ANTIBODIES TO HIV-1 IN SPECIMENS FROM INDIVIDUALS INFECTED WITH HIV-1

A multi-center prospective study was conducted to evaluate the clinical performance of the INSTI HIV Antibody Test. There were 483 subjects known to be HIV-1 positive, and 905 subjects with unknown HIV status. The subjects with unknown HIV status were tested with INSTI and by a composite reference method (comparator method) which consisted of an licensed/approved EIA with supplemental Western blot and PCR assays as required. The result of INSTI was compared to the known or determined HIV status of the subject.

In this study, all 517/517 true HIV antibody positive subjects were identified as reactive by the INSTI HIV-1/HIV-2 Antibody Test, resulting in a relative sensitivity of 100.0% (95 % CI = 99.3% - 100.0%). There were no invalid results (0/1388) observed in this study.

Detection of HIV-1 Antibody in Fingerstick Whole Blood Specimens from HIV-1 Seropositive Individuals

Study PopulationNumber

of Subjects

INSTI Reactive

Approved Test

ReactiveTrue Positive

HIV status unknown 905 34 34 34

Known HIV-1Positive 483 483 483 483

TOTAL 1,388 517 517 517

Reactivity with HIV-1: Seroconversion PanelsThirty (30) HIV-1 seroconversion panels (Boston Biomedica Inc.) were tested with INSTI. Each panel consisted of sequential serum/plasma specimens obtained from a single individual during seroconversion. The results of this study are presented in the table below and summarizes the INSTI HIV-1/HIV-2 assay data compared to US licensed and European approved HIV antibody enzyme immunoassays (EIA). Overall the INSTI HIV-1/HIV-2 Antibody Test has similar performance to commercially available anti-HIV EIA in the detection of HIV antibodies in seroconversion samples.

INSTI HIV-1/HIV-2 TEST: Number of Panels

Detected the earliest bleed that was detected by an EIA 15

Detected within 1 bleed of earliest EIA positive 10

Detected within 2 bleeds of earliest EIA positive 3

Unknown** 2

**The last bleed in the panel was reactive by at least 1 EIA, non-reactive by INSTI

25

Instructions for Use

Reactivity with HIV-1: Low Titer Panel A single low titer HIV-1 antibody panel (#PRB-108; Boston Biomedica) was tested with the INSTI HIV-1/HIV-2 Antibody Test. This low titer panel consisted of 15 serum/plasma specimens. Results of this study are summarized in the table below. This study demonstrated that the INSTI HIV-1/HIV-2 Antibody Test has the capability of detecting antibodies to HIV-1 similar to currently available FDA licensed EIAs.

Specimen Number

Test 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

INSTI HIV-1/HIV-2 P P P P P P P P P P P N P P P

Abbott EIA P P P P P P P P P P P N P P P

Abbott HIVAB HIV-1/HIV-2 (rDNA)EIA P P P P P P P P P P P N P P P

Cambridge Biotech Recombigen HIV-1 EIA P P P P P P P P P P P N P P P

Syva EIA P P P P P P P P P P P N P P P

Organon Teknika Vironostika Anti-HIV Uni-Form II P P P P P P P P P P P N P P P

Murex HIV 1/2 EIA P P P P P P P P P P P N P P P

Ortho HIV-1/HIV-2 EIA P P P P P P P P P P P N P P P

Sorin ETI-Ab-HIV 1/2K EIA P P P P P P P P P P P N P P P

Syva Microtrak II EIA P P P P P P P P P P P N P P P

Behringwerke ENZ PLUS Anti HIV 1/2 EIA P P P P P P P P P P P N P P P

Biotest Anti-HIV-1/HIV-2 Recombinant EIA P P P P P P P P P P P N P P P

Boehringer Mannheim Anti HIV-1/HIV-2 EIA P P P P P P P P P P P N P P P

IAF Biochem Detect-HIV-EIA P P P P P P P P P P P N P P P

Diagnostic Pasteur Genelavia EIA P P P P P P P P P P P N P P P

bioMerieux VIDAS anti-HIV-1/2 EIA P P P P P P P P P P P N P P P

Murex Wellcozyme HIV-1/HIV-2 EIA P P P P P P P P P P N N P P P

Behringwerke Enzygnost Anti HIV 1+2 EIA N P N P P P P P P P P N P P P

Cellular Products HIV-1 EIA N P P P P P P P N P P N P P P

Genetic Systems LAV EIA N P P P P P P P N P P N P P P

Genetic Systems HIV-1/HIV-2 EIA N P N P P P P P P P P N P P P *These samples were confirmed positive (P) by EIA and Western Blotting (Data obtained from Boston Biomedica package insert, May 1995 p.2) Interfering Substances and Unrelated Medical Conditions To assess the impact of unrelated medical conditions or interfering substances on the sensitivity of the INSTI HIV-1/HIV-2 Antibody Test, 195 serum/plasma specimens from a variety of medical conditions unrelated to HIV-1 infection and 217 specimens with interfering substances were spiked with an HIV-1 positive specimen; see table in the Specificity section for list of medical conditions and substances tested. All spiked specimens gave reactive results.

DETECTION OF ANTIBODIES TO HIV-2 IN SPECIMENS FROM INDIVIDUALS INFECTED WITH HIV-2 A total of 137 individual HIV-2 positive samples were obtained from European sources. 49 sera from individuals with chronic HIV-2 infection were reactive on the INSTI HIV-1/HIV-2 Antibody Test. An additional 88 HIV-2 positive serum and plasma samples were prepared as contrived whole blood; all 88 contrived samples were reactive on the INSTI HIV-1/HIV-2 Antibody Test. Combining the results of the two studies, the relative sensitivity of the INSTI HIV-1/HIV-2 Antibody Test for the detection of HIV-2 antibodies in these studies was calculated to be 100% (137/137).

HIV-1 SUBTYPE TESTING To assess the sensitivity of the INSTI HIV-1/HIV-2 Antibody Test for HIV-1 variants from various geographic regions, a total of 118 individual confirmed HIV-1 antibody-positive non-B subtype serum/plasma specimens were tested; of these 118 samples, 109 were non-B subtypes including 23 sub-type O samples. All 118 of these specimens were reactive using INSTI, generating an overall sensitivity of the INSTI HIV-1/HIV-2 Antibody Test for HIV-1 non-B subtypes of 100%. SPECIFICITY A specificity study was performed using 1386 freshly obtained specimens collected from low or unknown risk and high risk individuals as part of a multicenter prospective clinical trial. Of the 1386 samples, 1376 gave a Non-Reactive result with INSTI and 4 were invalid. INSTI HIV-1/HIV-2 Antibody Test results were compared to results from a composite reference method (comparator method) which consisted of an FDA approved EIA with supplemental Western blot and PCR as required. A total of 7 INSTI false Reactive results (1 from the high risk group, 6 from the low or unknown risk group) were obtained from the 1382 specimens from HIV-negative individuals that produced valid INSTI results. From this data, the overall specificity of the INSTI HIV-1/HIV-2 Antibody Test in fingerstick whole blood specimens from the combined high risk and low or unknown risk populations, minus the invalid results, was calculated to be 1375/1382 = 99.5% (95% CI = 99.0% - 99.8%). Performance of the INSTI HIV-1/HIV-2 Antibody Test on Fingerstick Whole Blood Specimens from Individuals Presumed to be Negative for HIV Infection

Test Group

Total Specimens

INSTI Non-Reactive3

Approved Test Non-Reactive2

True Negative2

Low Risk 626 620 626 626

High Risk 782 7561 7602 760

TOTAL 1408 1376 1386 1386

1 4 invalid results were not included in the calculation of specificity. The 4 specimens which gave invalid results on INSTI were Non-Reactive on the approved test. 2 22 Reactives were confirmed by licensed HIV-1 Western Blot and excluded from the calculation of specificity. 3 Of the 22 INSTI Reactive specimens, one was Non-Reactive by the approved test, i.e. INSTI false Reactive. Interfering Substances and Unrelated Medical Conditions To assess the impact of unrelated medical conditions or interfering substances on the specificity of the INSTI HIV-1/HIV-2 Antibody Test, 195 serum/plasma specimens from a variety of medical conditions unrelated to HIV-1 infection and 217 specimens with interfering substances were analyzed. Five specimens from individuals with multiple myeloma gave invalid results. No false reactive results were obtained.

Medical Condition (n=195) No. of Specimens

INSTI Reactive

INSTI Nonreactive

Toxoplasmosis 20 0 20

Rheumatoid Factor 20 0 20

Multiple Myeloma 10 0 5

Syphilis 30 0 30

SLE 5 0 5

Rubella 20 0 20

Cytomegalovirus 20 0 20

Epstein Barr Virus 20 0 20

HTLV-I/II panel 15 0 15

Hepatitis B Virus 20 0 20

Hepatitis A Virus 15 0 15

Interfering Substances (n=217)

Icteric 20 0 20

Elevated Bilirubin 19 0 19

Lipemic 20 0 20

Visual Hemolysis 5 0 5

Elevated Triglyceride 19 0 19

Elevated Hemoglobin 20 0 20

Elevated Albumin 15 0 15

EDTA 13 0 13

Sodium Heparin 13 0 13

Sodium Citrate 13 0 13

Bacterially Contaminated 60 0 60

In addition, a total of 208 specimens from pregnant women in various trimesters of pregnancy confirmed to be HIV-1 negative by a 3rd Generation HIV EIA were tested. One sample (1/208) produced invalid result, all other INSTI results were non-reactive.

EQUIVALENCE STUDIES The INSTI HIV-1/HIV-2 Antibody Test was evaluated using matched serum and plasma specimens, Testing was performed with 50 anti-HIV-1 negative specimens (25 serum and 25 plasma) and 50 anti-HIV-1-spiked positive specimens. All samples produced acceptable assay performance. These results indicate 100% relative sensitivity and 100% relative specificity with the matched serum and plasma panel provided, and that serum and plasma sample types are equivalent.

HIV REPRODUCIBILITY The reproducibility of the INSTI HIV-1/HIV-2 portion of the Multiplex Test was tested at 3 laboratory sites using 3 lots of the INSTI HIV-1/HIV-2 Antibody Test on 3 separate days. A panel of 9 blind-coded plasma samples, consisting of 4 antibody positive, 1 very low antibody level sample, and 4 antibody negative samples was tested at each site. A total of 729 tests were conducted, 243 at each site. For the 4 antibody positive and 4 antibody negative samples, the overall reproducibility was 99.7% (646/648, two antibody negative samples were read as weak positive at 1 site). For the 1 very low level antibody sample, 59% (48/81) of the results were positive while 41% (33/81) were negative. SYPHILIS (T. pallidum) PERFORMANCE CHARACTERISTICS In-house Studies Data from parallel INSTI Multiplex and T. pallidum Particle Agglutination (TP-PA) in-house testing of frozen, archived serum and plasma samples obtained from commercial sources is provided below. INSTI Multiplex T. pallidum antibody test results were compared to a CE marked T. pallidum Particle Agglutination (TP-PA) assay, n=524 serum/plasma specimens known to be TP-PA negative or positive.

INSTI Multiplex T. pallidum

Antibody Results

TP-PA Final Interpretation

Positive Negative Total

Reactive 138 5 143

Non-Reactive 7 374 381

Total 145 379 524

NOTE: In an independent study conducted at the Centre National de la Reference de la Syphilis, Paris, France16, the sensitivity of INSTI Multiplex on samples from patients with known secondary syphilis was 100% (41/41). Field Study In an independent prospective field study conducted in Bangalore India in 2012-2013 on a population at risk for HIV and STI (n=1010 plasma), the performance of the INSTI Multiplex Test was assessed against the syphilis and HIV status for each patient as determined by an algorithm of syphilis serology methods (RPR, TPHA) and HIV antibody tests (two rapid lateral flow HIV-1/HIV-2 assays plus one rapid HIV-1/HIV-2 ELISA test. The results are presented below.

TRAINING PACK

26

Instructions for Use

MULTIPLEX

Syphilis Performance of the INSTI Multiplex Test against the Rapid Plasma Reagin (RPR) test for the prospective field study population, n=1010.

Rapid Plasma Reagin (RPR)Test

INSTI Multiplex T. pallidum Antibody Positive

INSTI Multiplex T. pallidum Antibody Negative

Positive 121 52

Negative 23 991

Total 14 996

1. 11/12 were confirmed positive by T. pallidum Haemagglutination Assay (TPHA) and 1/12 is considered false positive by INSTI and RPR. 2. 3/5 were confirmed positive by TPHA, ie INSTI false negative and 2/5 were negative by TPHA, ie RPR biological false positive. 3. 2/2 were confirmed positive by TPHA, ie RPR false negative The number of true T. pallidum antibody positive patients (n=16) in this prospective study, as determined by TPHA is too low for calculation of relative sensitivity of the INSTI Multiplex test for detection of T. pallidum antibodies. Of the total 994 samples that were considered negative for active syphilis (by RPR and/or TPHA), INSTI Multiplex was negative for 993, for a negative percent agreement of 99.9%. (It is important to note that a negative RPR result does not rule out past syphilis infection, therefore this is not a measure of true T. pallidum antibody specificity.) HIV Performance of the INSTI Multiplex Test against the HIV test algorithm results for the prospective field study population, n=1010

Panel INSTI Multiplex HIV-1/HIV-2 Positive

INSTI Multiplex HIV-1/HIV-2 Negative

HIV known positive (n=136) 136 0

HIV negative (n=874) 0 874

Total 136 874

The positive and negative percent agreement for HIV antibody detection between the INSTI Multiplex Test and the patients’ HIV status determined by the three-test HIV algorithm was 100%. Mixed Titre Panel The INSTI Multiplex Test was tested against a commercial syphilis mixed titre performance panel to compare performance to other T. pallidum antibody tests for detection of high and low levels of T. pallidum antibodies. The results are presented below. Syphilis Mixed Titre Performance Panel 1111-272-00123 (ZeptoMetrix Corporation)

Panel Member ID Number

T. pallidum Test Method 1 2 3 4 5 6 7 8 9 10

US FDA Licensed Test

Pheonix Biotech Trep Sure P P P N N N N N P P

Trinity Captia IgG P P P N N N N N P P

Trinity Captia IgM N N N N N N N N P N

Serodia-TP.PA P P P N N N N N P P

INSTI Multiplex T. pallidum Antibody P P P N N N N N P P

N = negative P = positive Whole Blood Testing Performance of the INSTI Multiplex Test compared to TP-PA for EDTA Whole Blood Specimens, un-spiked (n=105) and spiked with T.pallidum (n=64) .

The positive percent and negative percent agreement was 96.9% and 100% for this subset of specimens, comparing favorably to the corresponding values obtained from in-house testing (95.2% and 98.7%) indicating that there is no performance difference in the detection of T. pallidum antibodies in whole blood, serum or plasma samples. INSTI Multiplex Test Result compared to TP-PA for specimens that tested positive for other diseases or medical conditions (n = 380)

1 all were TP-PA positive unless otherwise indicated. 2 all were TPPA negative unless otherwise indicated 3 2 specimens tested negative on TP-PA, ie INSTI false positive 4 2 specimens tested positive on TP-PA, ie INSTI false negative 5 1 specimen tested negative on TP-PA test, ie INSTI false positive 6 1 specimen tested positive on TP-PA, ie INSTI false negative 7 2 specimens tested negative on TP-PA, ie INSTI false positive. 8 3 specimens tested positive on TP-PA test, ie INSTI false negative.

HIV and Syphilis Reproducibility The reproducibility of the HIV-1/HIV-2 and syphilis portions of the Multiplex Test was tested using 3 distinct lots of the INSTI Multiplex Test components by 3 operators over 3 separate days. A panel of 5 blind-coded plasma samples, designed to produce HIV and syphilis results ranging from strongly reactive to weakly reactive to negative, was used for the study. Each panel member sample was tested 33 times, for a total of 165 INSTI Multiplex tests. For HIV. 165/165 results were in agreement with the expected results across all operators, component lots and days of testing for an overall reproducibility of 100%. For syphilis, 164/165 results were in agreement with the expected results across all operators, component lots and days of testing for an overall reproducibility of 99.4%. BIBLIOGRAPHY 1. Guyader, M., Emerman, M., Sonigo, P., et al. Genome organization and transactivation of the human

immunodeficiency virus type 2. Nature 326:662-669, 1987. 2. Blattner, W., Gallo, R.C., and Temin, H.M. HIV causes AIDS. Science 241:515, 1988. 3. Curran, J.W., Morgan, W.M., Hardy, A.M., et al. The epidemiology of AIDS; Current status and future

prospects. Science 229:1352-1357, 1985 4. Sarngadharan, M.G., Popovic, M., Bruch, L., Schüpback, J., and Gallo, R.C. Antibodies reactive with

human T-lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 224:506-508,1984

5. Gallo, R.C., Salahuddin, S.Z., Popovic, M., et al. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224:500-503, 1984

6. Weber, J.N., Weiss, R.A., Roberts, C., et al. Human immunodeficiency virus in two cohorts of homosexual men; Neutralising sera and association of anti-gag antibody with prognosis. Lancet 1:119-124, 1987

7. Clavel, F., Guétard, D., Brun-Vézinet, F., et al. Isolation of a new human retrovirus from West African patient with AIDS. Science 233:343-346, 1986

8. Centers for Disease Control. Revision of the CDC surveillance case definition for acquired Immunodeficiency syndrome. MMWR 36 (suppl. no. 1S):1S-15S, 1987

9. World Health Organization/Global Programme on AIDS. Report of a WHO workshop on synthetic peptides in HIV diagnosis and AIDS-related research, Moscow 24-26 May 1989. WHO Report, AIDS 1991, 5: WHO1-WHO9

10. Los Alamos National Laboratory. Human retroviruses and AIDS Database. A compilation of nucleic acid and amino acid sequences, 1993.

11. World Health Organization/Global Programme on AIDS. Operational characteristics of commercially available assays to detect antibodies to HIV-1 and/or HIV-2 in human sera. Geneva, Switzerland: WHO documents GPA/BMR/89.4; GPA/BMR/90.1; GPA/RES/DIA90.1; GPA/RES/DIA/91.6; GPA/RES/DIA/ 92.8 and GPA/RES/DIA/93.4

12. World Health Organization/Global Programme on AIDS. Acquired immunodeficiency syndrome (AIDS proposed WHO criteria for interpreting results from Western blot assays for HIV-1, HIV-2 and HTLV-I/HTLVII). WHO Weekly Epidemiological Record 65(37):281-282, 1990

13. Malone, J.D., Smith, E.S., Sheffield, J., et al. Comparative evaluation of six rapid serological tests for HIV-1 antibody. Journal of Acquired Immune Deficiency Syndrome (JAIDS) 6:115-149, 1993

14. Directive 2000/54/EC of the European Parliament and of the Council of 18 September 2000 on the Protection of Workers from Risks Related to Exposure to Biological Agents at Work.

15. “Summary of Safety and Effectiveness Data, 50-1110.” bioLytical Laboratories, Inc. 28 January 2011, Table 3.

16. Dr Anne Bianchi, CNR, Paris, France, study completed Nov. 2010 / Oct. 2011, personal communication.

17. N. Moshgabadi et al. Sensitivity of a rapid point of care assay for early HIV antibody detection is enhanced by its ability to detect HIV gp41 IgM antibodies. Journal of Clinical Virology 71 (2015) 67–72

INSTI Multiplex T. pallidum

Antibody Results

TP-PA Final Interpretation

Positive Negative Percent Agreement

Reactive 62 0 Positive percent agreement 96.9% (62/64)

Non-Reactive 2 105 Negative percent agreement 100% (105/105)

Total 64 105 169

Condition No. of Specimens

INSTI Multiplex Syphilis

Reactive1

INSTI Multiplex Syphilis

Nonreactive2

Cytomegalovirus (CMV) 10 1 9

Epstein Barr Virus (EBV) 9 0 9

Helicobactor pylori 10 0 10

Hepatitis A Virus (HAV) 40 63 344

Hepatitis B Virus (HBV) 40 15 396

Hepatitis C Virus (HCV) 121 67 1158

Human Immunodeficiency Virus (HIV) 25 1 24

Herpes Simplex Virus (HSV) 10 0 10

Lyme Disease 5 0 5

Myeloma 10 0 10

Pregnancy 50 0 50

Rheumatoid Factor 5 0 5

Rubella 10 0 10

Systemic Lupus Erythematosus (SLE) 5 1 4

Toxoplasmosis 20 0 20

Varicella Zoster Virus (VZV) 10 0 10

Syphilis Test Control IFU

27

Syphilis (T. pallidum) Positive Control

Product Number: 90-1032

Store at -20°C to +8°C H Use by

Y Caution M Manufacturer

i Consult instructions for use C CE Mark

R22 – Harmful if swallowed

Read this Instructions for Use and the INSTI® Multiplex HIV-1/HIV-2/Syphilis Antibody Test Instructions for Use before using this product. Conformance with the test procedure is necessary to ensure accurate results. Before performing the test, all operators must become familiar with Universal Precautions for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B Virus, and other Blood-borne Pathogens in Health Care Settings1. NAME AND INTENDED USE The INSTI Syphilis (T. pallidum) Positive Control is intended to be used only with the INSTI Multiplex HIV-1/HIV-2/Syphilis Antibody Test, product number 90-1028. SUMMARY INSTI Syphilis (T. pallidum) Positive Control should be used in conjunction with Good Laboratory Procedures. It should be run under the following circumstances: for new INSTI Multiplex operator verification prior to performing testing on patient

specimens when switching to a new lot number of INSTI Multiplex test kits whenever a new shipment of kits is received when temperature during storage of the kit falls outside of 15°- 30°C (59°- 86°F) when the temperature of the test area falls outside of 15°- 30°C (59°- 86°F) At regular intervals as determined by the user facility PRINCIPLES OF THE PROCEDURE The INSTI Syphilis (T. pallidum) Positive Control has been designed for use with the INSTI Multiplex HIV-1/HIV-2/Syphilis Antibody Test to validate the correct performance of the test procedure in the hands of the operator. The INSTI Syphilis (T. pallidum) Positive Control is prepared from inactivated human plasma. It is negative for HBsAg and anti-HCV by U.S. FDA licensed test procedures. The INSTI Syphilis (T. pallidum) Positive Control has been designed to produce an easily visible but faint blue colour on the INSTI test spot and a darker blue colour on the control spot. REAGENTS SYPHILIS (T. pallidum) POSITIVE CONTROL 1 vial containing 1.0 ml of inactivated human plasma. Each vial is sufficient for 20 INSTI Multiplex tests. The source material has been heat inactivated at 60°C for 60 minutes. WARNINGS & PRECAUTIONS

For in vitro diagnostic use only.

Safety Precautions: 1. All specimens should be handled as if capable of transmitting infectious agents. 2. Thoroughly wash hands after handling or performing this test. 3. Do not smoke, eat, or drink in areas where specimens or kit reagents are being

handled. 4. Wear disposable gloves while handling kit reagents or specimens. Do not pipette

by mouth. 5. Avoid contact with skin and eyes. If contact occurs, wash affected areas with

water. 6. Avoid forming aerosols. 7. Dispose of all specimens and materials used to perform the test in a biohazard

waste container. The preferred method of disposal is sterilization by autoclaving for a minimum of one hour at 121°C. Disposable materials may be incinerated. Liquid waste may be mixed with sodium hypochlorite (bleach) in volumes such that the final mixture contains 1.0% sodium hypochlorite (using a freshly prepared solution containing 10% household bleach). Allow at least 60 minutes for decontamination to be completed. Do not autoclave solutions that contain bleach.

8. Spills should be cleaned up and decontaminated in accordance with the user facility’s established procedures for handling biohazardous spills.

9. For additional information on bio-safety refer to “Universal Precautions for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B Virus, and other Blood-borne Pathogens in Health Care Settings”1 and “Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposures to HBV, HCV, and HIV and Recommendations for Postexposure Prophylaxis”2.

Handling Precautions: Do not use INSTI Syphilis (T. pallidum) Positive Control beyond the expiration date. INDICATIONS OF REAGENT INSTABILITY OR DETERIORATION INSTI Syphilis (T. pallidum) Positive Control that is visibly turbid and/or contains particulate matter should not be used and should be discarded in accordance with safety precautions.

STORAGE INSTRUCTIONS 1. The INSTI Syphilis (T. pallidum) Positive Control is shipped without temperature

control. Upon receipt, store at 2 - 8°C (36 – 46°F) for up to 28 days or at -20°C (-4°F) for up to 1 year.

2. It is recommended to store the vials in an upright position. 3. Once the controls are thawed following storage at -20°C (-4°F), they remain stable

for 28 days at 2 - 8°C (36 – 46°F). Do not re-freeze once the vials have been opened.

PROCEDURE Materials Required but not Provided

Precision pipette capable of delivering 50μl of specimen. INSTI Multiplex HIV-1/HIV-2/Syphilis Antibody Test Instructions for Use.

Instructions for use

1. Read the INSTI Syphilis (T. pallidum) Positive Control Instructions for Use prior to using the INSTI Syphilis (T. pallidum) Positive Control.

2. Remove from storage at 2 – 8°C (36 – 46°F) or -20 °C (-4°F) and allow the Controls to reach room temperature before testing with INSTI Multiplex. Return Controls to storage at 2-8°C after use.

3. Mix the Controls by swirling before use. 4. Uncap the INSTI Syphilis (T. pallidum) Positive Control vial. Using a 50μl pipette,

collect 50μl of the Control. 5. Transfer the Control sample held in the pipette to the INSTI Multiplex Sample

Diluent vial (Solution 1). Recap the vial and mix by inversion. 6. Follow the INSTI Multiplex test procedure as described in the TEST PROCEDURE

section of the INSTI Multiplex HIV-1/HIV-2/Syphilis Antibody Test Instructions for Use.

7. All Controls should be tested in the same manner as patient samples. 8. The Syphilis Positive Control, HIV-1 Positive Control, HIV-2 Positive Control and

the Negative Control are to be run on separate Membrane Units. INTERPRETATION OF RESULTS

Follow the interpretation guidelines provided in the INTERPRETATION OF RESULTS section of the INSTI Multiplex HIV-1/HIV-2/Syphilis Antibody Test Instructions for Use.

Reactive Result: Both the control spot and the syphilis test spot show blue colour development.

Non-Reactive Result: Only the control spot shows blue colour development. Invalid Result: The test is invalid if any of the following occurs:

-There is no blue colour on both the control spot and the test spot -There is blue colour on the test spot but not on the control spot -Uniform tint across the membrane -Only blue specks appear on the membrane

LIMITATIONS OF THE PROCEDURE The INSTI Syphilis (T. pallidum) Positive Control is only validated for use with the INSTI Multiplex HIV-1/HIV-2/Syphilis Antibody Test.

1. The TEST PROCEDURE and INTERPRETATION OF RESULTS sections of the INSTI Multiplex HIV-1/HIV-2/Syphilis Antibody Test instructions for use must be adhered to when testing the INSTI Syphilis (T. pallidum) Positive Control.

2. Deviations from the procedure outlined in the INSTI® Multiplex HIV-1/HIV-2/Syphilis Antibody Test Instructions for Use may produce unreliable results.

3. Do not dilute the INSTI Syphilis (T. pallidum) Positive Control. The INSTI Syphilis (T. pallidum) Positive Control is intended for use in undiluted form.

4. Adverse shipping and storage conditions or use of expired reagents may produce erroneous results.

EXPECTED RESULTS The INSTI Syphilis (T. pallidum) Positive Control must be Reactive with INSTI Multiplex. Contact bioLytical Laboratories’ Technical Support if the INSTI Syphilis (T. pallidum) Positive Control does not produce the expected results. REFERENCES

1. CDC. Universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health care settings. MMWR 1988; 37(24):377-388

2. CDC Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposures to HBV, HCV, and HIV and Recommendations for Postexposure Prophylaxis. MMWR 2001; 50(RR-11):1-42. Manufactured by:

bioLytical Laboratories, Inc 13351 Commerce Parkway Suite 1108 Richmond, BC, V6V 2X7 Canada Phone: +1 604-204-6784 Fax: +1 604-244-8399 www.biolytical.com

Authorized Representative:

EMERGO EUROPE

Prinsessegracht 20, 2514 AP The Hague, The Netherlands Phone: +31.70.345.8570 Fax: +31.70.346.7299

51-1169F 26-Sep-2017 © Copyright 2017. All rights reserved

HIV-1/HIV-2 Test Controls Product Number: 90-1036

Store at -20°C to +8°C H Use by

Y Caution M Manufacturer

i Consult instructions for use C CE Mark

R22 – Harmful if swallowed

Read this Instructions for Use and the INSTI® HIV-1/HIV-2 Antibody Test Instructions for Use before using this product. Conformance with the test procedure is necessary to ensure accurate results. Before performing the test, all operators must become familiar with Universal Precautions for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B Virus, and other Blood-borne Pathogens in Health Care Settings1 NAME AND INTENDED USE The INSTI HIV-1/HIV-2 Test Controls are intended to be used only with the INSTI HIV-1/HIV-2 Antibody Test. SUMMARY INSTI HIV-1/HIV-2 Positive and Negative Controls should be used in conjunction with Good Laboratory Procedures. They should be run under the following circumstances: for new INSTI operator verification prior to performing testing on patient specimens when switching to a new lot number of INSTI test kits whenever a new shipment of kits is received when temperature during storage of the kit falls outside of 2°-30°C (35.6°-86°F) when the temperature of the test area falls outside of 15°-30°C (59°-86°F) at regular intervals as determined by the user facility. PRINCIPLES OF THE PROCEDURE The INSTI HIV-1/HIV-2 Test Controls have been designed for use with the INSTI HIV-1/HIV-2 Antibody Test to validate the correct performance of the test procedure in the hands of the operator. The INSTI HIV-1/HIV-2 Positive Controls are prepared from inactivated human plasma. It is negative for HBsAg and anti-HCV by U.S. FDA licensed test procedures. The Positive Controls have been designed to produce an easily visible but faint blue colour on the INSTI test spot and a darker blue colour on the control spot. The INSTI HIV-1/HIV-2 Negative Control is prepared from defibrinated human serum which is negative for Anti-HIV-1 and Anti-HIV-2, HBsAg, and Anti-HCV. The Negative Control will produce a blue colour on the procedure control spot, but no colour on the test spot, for a Non-Reactive INSTI test result. REAGENTS: HIV-1 POSITIVE CONTROL (90-1036) 1 vial containing 1.0 ml of inactivated human plasma. Each vial is sufficient for 20 INSTI tests. The source material has been heat inactivated at 60°C for 60 minutes. HIV-2 POSITIVE CONTROL (90-1036) 1 vial containing 1.0 ml of inactivated human plasma. Each vial is sufficient for 20 INSTI tests. The source material has been heat inactivated at 60°C for 60 minutes. NEGATIVE CONTROL (90-1036) 1 vial containing 1.0 ml of processed human serum substitute, non-reactive for antibodies to HIV and HCV and non-reactive for HBsAg. Each vial is sufficient for 20 INSTI tests. WARNINGS & PRECAUTIONS

For in vitro diagnostic use only.

Safety Precautions: 1. All specimens should be handled as if capable of transmitting infectious agents. 2. Thoroughly wash hands after handling or performing this test. 3. Do not smoke, eat, or drink in areas where specimens or reagents are being

handled. 4. Wear disposable gloves while handling reagents or specimens. Do not pipette by

mouth. 5. Avoid contact with skin and eyes. If contact occurs, wash affected areas with

water. 6. Avoid forming aerosols. 7. Dispose of all specimens and materials used to perform the test in a biohazard

waste container. The preferred method of disposal is sterilization by autoclaving for a minimum of one hour at 121°C. Disposable materials may be incinerated. Liquid waste may be mixed with sodium hypochlorite (bleach) in volumes such that the final mixture contains 1.0% sodium hypochlorite (using a freshly prepared solution containing 10% household bleach). Allow at least 60 minutes for decontamination to be completed. Do not autoclave solutions that contain bleach.

8. Spills should be cleaned up and decontaminated in accordance with the user facility’s established procedures for handling biohazardous spills.

9. For additional information on bio-safety refer to “Universal Precautions for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B Virus, and other Blood-borne Pathogens in Health Care Settings”1 and “Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposures to HBV, HCV, and HIV and Recommendations for Postexposure Prophylaxis”2.

Handling Precautions:

1. Do not use INSTI HIV-1/HIV-2 Test Controls beyond the expiration date. INDICATIONS OF REAGENT INSTABILITY OR DETERIORATION Positive or Negative Controls that are visibly turbid and/or contain particulate matter should not be used and should be discarded in accordance with safety precautions.

STORAGE INSTRUCTIONS 1. The INSTI HIV-1/HIV-2 Test Controls are shipped without temperature control.

Upon receipt, store frozen at -20°C (-4°F) for up to one year or refrigerate at 2°C (36°F) to 8°C (46°F) for up to one year.

2. The shelf life is dictated by the printed expiry date on the INSTI Test Controls. 3. It is recommended to store the vials in an upright position. 4. Once the controls are thawed continue storage at refrigeration temperatures (2-

8°C). They remain stable until expiry (up to 1 year). Do not re-freeze once the vials have been opened.

5. Fluctuations in temperature causing freeze-thaw cycles may affect the performance of the INSTI Test Controls.

PROCEDURE Materials Required but not Provided

Pipette capable of delivering 50μl of specimen. INSTI HIV-1/HIV-2 Antibody Test Instructions for Use.

Instructions for use

1. Read the INSTI HIV-1/HIV-2 Test Controls Instructions for Use prior to using the INSTI HIV-1/HIV-2 Test Controls.

2. Remove from storage at -20°C (-4°F) to 8°C (46°F) and allow the Controls to reach room temperature before testing with INSTI. Return Controls to refrigeration storage at 2-8°C after use.

3. Mix the Controls by swirling before use. 4. Uncap the HIV-1 Positive, HIV-2 Positive or Negative Control vial. Using a 50μl

pipette, collect 50μl of the Control. 5. Transfer the Control sample held in the pipette to the INSTI Sample Diluent vial

(Solution 1). Recap the vial and mix by inversion. 6. Follow the INSTI test procedure as described in the TEST PROCEDURE section of the

INSTI HIV-1/HIV-2 Antibody Test Instructions for Use. 7. All Controls should be tested in the same manner as patient samples. 8. The HIV-1 Positive Control, HIV-2 Positive Control and the Negative Control are

to be run on separate Membrane Units. INTERPRETATION OF RESULTS

Follow the interpretation guidelines provided in the INTERPRETATION OF RESULTS section of the INSTI HIV-1/HIV-2 Antibody Test Instructions for Use.

Reactive Result: Both the control spot and the test spot show blue colour development.

Non-Reactive Result: Only the control spot shows blue colour development. Invalid Result: The test is invalid if any of the following occurs:

-There is no blue colour on both the control spot and the test spot -There is blue colour on the test spot but not on the control spot -Uniform tint across the membrane -Only blue specks appear on the membrane

LIMITATIONS OF THE PROCEDURE The INSTI HIV-1/HIV-2 Test Controls are only validated for use with the INSTI HIV-1/HIV-2 Antibody Test.

1. The TEST PROCEDURE and INTERPRETATION OF RESULTS sections of the INSTI HIV-1/HIV-2 Antibody Test Instructions for Use must be adhered to when testing the INSTI HIV-1/HIV-2 Test Controls.

2. Deviations from the procedure outlined in the INSTI HIV-1/HIV-2 Antibody Test Instructions for Use may produce unreliable results.

3. Do not dilute the INSTI HIV-1/HIV-2 Test Controls. The INSTI HIV-1/HIV-2 Test Controls are intended for use in undiluted form.

4. Adverse shipping and storage conditions or use of expired reagents may produce erroneous results.

EXPECTED RESULTS The HIV-1 Positive Control and HIV-2 Positive Control must be Reactive with INSTI and the Negative Control must be Non-Reactive with INSTI. Controls that produce incorrect or invalid results must be re-tested with INSTI. Contact bioLytical Laboratories’ Technical Support if the INSTI HIV-1/HIV-2 Test Controls do not produce the expected results. REFERENCES

1. CDC. Universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health care settings. MMWR 1988; 37(24):377-388

2. CDC Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposures to HBV, HCV, and HIV and Recommendations for Postexposure Prophylaxis. MMWR 2001; 50(RR-11):1-42.

Manufactured by:

bioLytical Laboratories, Inc 13351 Commerce Parkway Suite 1108 Richmond, BC, V6V 2X7 Canada Phone: +1 604-204-6784 Fax: +1 604-244-8399 www.biolytical.com

Authorized Representative:

EMERGO EUROPE

Prinsessegracht 20, 2514 AP The Hague, The Netherlands Phone: +31.70.345.8570 Fax: +31.70.346.7299

51-1181D 02-JUL-2018 © Copyright 2018. All rights reserved

0543

Syphilis (T. pallidum) Positive Control

Product Number: 90-1032

Store at -20°C to +8°C H Use by

Y Caution M Manufacturer

i Consult instructions for use C CE Mark

R22 – Harmful if swallowed

Read this Instructions for Use and the INSTI® Multiplex HIV-1/HIV-2/Syphilis Antibody Test Instructions for Use before using this product. Conformance with the test procedure is necessary to ensure accurate results. Before performing the test, all operators must become familiar with Universal Precautions for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B Virus, and other Blood-borne Pathogens in Health Care Settings1. NAME AND INTENDED USE The INSTI Syphilis (T. pallidum) Positive Control is intended to be used only with the INSTI Multiplex HIV-1/HIV-2/Syphilis Antibody Test, product number 90-1028. SUMMARY INSTI Syphilis (T. pallidum) Positive Control should be used in conjunction with Good Laboratory Procedures. It should be run under the following circumstances: for new INSTI Multiplex operator verification prior to performing testing on patient

specimens when switching to a new lot number of INSTI Multiplex test kits whenever a new shipment of kits is received when temperature during storage of the kit falls outside of 15°- 30°C (59°- 86°F) when the temperature of the test area falls outside of 15°- 30°C (59°- 86°F) At regular intervals as determined by the user facility PRINCIPLES OF THE PROCEDURE The INSTI Syphilis (T. pallidum) Positive Control has been designed for use with the INSTI Multiplex HIV-1/HIV-2/Syphilis Antibody Test to validate the correct performance of the test procedure in the hands of the operator. The INSTI Syphilis (T. pallidum) Positive Control is prepared from inactivated human plasma. It is negative for HBsAg and anti-HCV by U.S. FDA licensed test procedures. The INSTI Syphilis (T. pallidum) Positive Control has been designed to produce an easily visible but faint blue colour on the INSTI test spot and a darker blue colour on the control spot. REAGENTS SYPHILIS (T. pallidum) POSITIVE CONTROL 1 vial containing 1.0 ml of inactivated human plasma. Each vial is sufficient for 20 INSTI Multiplex tests. The source material has been heat inactivated at 60°C for 60 minutes. WARNINGS & PRECAUTIONS

For in vitro diagnostic use only.

Safety Precautions: 1. All specimens should be handled as if capable of transmitting infectious agents. 2. Thoroughly wash hands after handling or performing this test. 3. Do not smoke, eat, or drink in areas where specimens or kit reagents are being

handled. 4. Wear disposable gloves while handling kit reagents or specimens. Do not pipette

by mouth. 5. Avoid contact with skin and eyes. If contact occurs, wash affected areas with

water. 6. Avoid forming aerosols. 7. Dispose of all specimens and materials used to perform the test in a biohazard

waste container. The preferred method of disposal is sterilization by autoclaving for a minimum of one hour at 121°C. Disposable materials may be incinerated. Liquid waste may be mixed with sodium hypochlorite (bleach) in volumes such that the final mixture contains 1.0% sodium hypochlorite (using a freshly prepared solution containing 10% household bleach). Allow at least 60 minutes for decontamination to be completed. Do not autoclave solutions that contain bleach.

8. Spills should be cleaned up and decontaminated in accordance with the user facility’s established procedures for handling biohazardous spills.

9. For additional information on bio-safety refer to “Universal Precautions for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B Virus, and other Blood-borne Pathogens in Health Care Settings”1 and “Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposures to HBV, HCV, and HIV and Recommendations for Postexposure Prophylaxis”2.

Handling Precautions: Do not use INSTI Syphilis (T. pallidum) Positive Control beyond the expiration date. INDICATIONS OF REAGENT INSTABILITY OR DETERIORATION INSTI Syphilis (T. pallidum) Positive Control that is visibly turbid and/or contains particulate matter should not be used and should be discarded in accordance with safety precautions.

STORAGE INSTRUCTIONS 1. The INSTI Syphilis (T. pallidum) Positive Control is shipped without temperature

control. Upon receipt, store at 2 - 8°C (36 – 46°F) for up to 28 days or at -20°C (-4°F) for up to 1 year.

2. It is recommended to store the vials in an upright position. 3. Once the controls are thawed following storage at -20°C (-4°F), they remain stable

for 28 days at 2 - 8°C (36 – 46°F). Do not re-freeze once the vials have been opened.

PROCEDURE Materials Required but not Provided

Precision pipette capable of delivering 50μl of specimen. INSTI Multiplex HIV-1/HIV-2/Syphilis Antibody Test Instructions for Use.

Instructions for use

1. Read the INSTI Syphilis (T. pallidum) Positive Control Instructions for Use prior to using the INSTI Syphilis (T. pallidum) Positive Control.

2. Remove from storage at 2 – 8°C (36 – 46°F) or -20 °C (-4°F) and allow the Controls to reach room temperature before testing with INSTI Multiplex. Return Controls to storage at 2-8°C after use.

3. Mix the Controls by swirling before use. 4. Uncap the INSTI Syphilis (T. pallidum) Positive Control vial. Using a 50μl pipette,

collect 50μl of the Control. 5. Transfer the Control sample held in the pipette to the INSTI Multiplex Sample

Diluent vial (Solution 1). Recap the vial and mix by inversion. 6. Follow the INSTI Multiplex test procedure as described in the TEST PROCEDURE

section of the INSTI Multiplex HIV-1/HIV-2/Syphilis Antibody Test Instructions for Use.

7. All Controls should be tested in the same manner as patient samples. 8. The Syphilis Positive Control, HIV-1 Positive Control, HIV-2 Positive Control and

the Negative Control are to be run on separate Membrane Units. INTERPRETATION OF RESULTS

Follow the interpretation guidelines provided in the INTERPRETATION OF RESULTS section of the INSTI Multiplex HIV-1/HIV-2/Syphilis Antibody Test Instructions for Use.

Reactive Result: Both the control spot and the syphilis test spot show blue colour development.

Non-Reactive Result: Only the control spot shows blue colour development. Invalid Result: The test is invalid if any of the following occurs:

-There is no blue colour on both the control spot and the test spot -There is blue colour on the test spot but not on the control spot -Uniform tint across the membrane -Only blue specks appear on the membrane

LIMITATIONS OF THE PROCEDURE The INSTI Syphilis (T. pallidum) Positive Control is only validated for use with the INSTI Multiplex HIV-1/HIV-2/Syphilis Antibody Test.

1. The TEST PROCEDURE and INTERPRETATION OF RESULTS sections of the INSTI Multiplex HIV-1/HIV-2/Syphilis Antibody Test instructions for use must be adhered to when testing the INSTI Syphilis (T. pallidum) Positive Control.

2. Deviations from the procedure outlined in the INSTI® Multiplex HIV-1/HIV-2/Syphilis Antibody Test Instructions for Use may produce unreliable results.

3. Do not dilute the INSTI Syphilis (T. pallidum) Positive Control. The INSTI Syphilis (T. pallidum) Positive Control is intended for use in undiluted form.

4. Adverse shipping and storage conditions or use of expired reagents may produce erroneous results.

EXPECTED RESULTS The INSTI Syphilis (T. pallidum) Positive Control must be Reactive with INSTI Multiplex. Contact bioLytical Laboratories’ Technical Support if the INSTI Syphilis (T. pallidum) Positive Control does not produce the expected results. REFERENCES

1. CDC. Universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health care settings. MMWR 1988; 37(24):377-388

2. CDC Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposures to HBV, HCV, and HIV and Recommendations for Postexposure Prophylaxis. MMWR 2001; 50(RR-11):1-42. Manufactured by:

bioLytical Laboratories, Inc 13351 Commerce Parkway Suite 1108 Richmond, BC, V6V 2X7 Canada Phone: +1 604-204-6784 Fax: +1 604-244-8399 www.biolytical.com

Authorized Representative:

EMERGO EUROPE

Prinsessegracht 20, 2514 AP The Hague, The Netherlands Phone: +31.70.345.8570 Fax: +31.70.346.7299

51-1169F 26-Sep-2017 © Copyright 2017. All rights reserved

Please contact the Business Development Manager in your area to request the QA controls.

TRAINING PACK

TECHNICAL INFORMATION NOTE: The HIV assay portion of the INSTI Multiplex Test is CE Marked through CE0543 and the syphilis assay portion is CE Marked through bioLytical Laboratories by self-declaration. For further information, assistance, or problem reporting, contact Customer Service at 1-604-644-4677. Reference herein to any specific third party by name, trade name, trade-mark, manufacturer or otherwise does not constitute or imply an endorsement or recommendation of this Kit by such third party, or of the products or services of such third party by bioLytical or that such products or services are necessarily best suited for the intended purpose.

M Manufactured by:

C0543 for HIV-1/HIV-2 only

bioLytical Laboratories, Inc. 13351 Commerce Parkway, Suite 1108 Richmond, BC, V6V 2X7 Canada Phone: +1-604-204-6784 Fax: +1-604-244-8399 www.biolytical.com 51-1143K 25-Feb-2019 ©Copyright 2018, All rights reserved.

Authorized Representative:

P EMERGO EUROPE Prinsessegracht 20, 2514 AP The Hague, The Netherlands Phone: +31.70.345.8570 Fax: +31.70.346.7299

TECHNICAL INFORMATION NOTE: The HIV assay portion of the INSTI Multiplex Test is CE Marked through CE0543 and the syphilis assay portion is CE Marked through bioLytical Laboratories by self-declaration. For further information, assistance, or problem reporting, contact Customer Service at 1-604-644-4677. Reference herein to any specific third party by name, trade name, trade-mark, manufacturer or otherwise does not constitute or imply an endorsement or recommendation of this Kit by such third party, or of the products or services of such third party by bioLytical or that such products or services are necessarily best suited for the intended purpose.

M Manufactured by:

C0543 for HIV-1/HIV-2 only

bioLytical Laboratories, Inc. 13351 Commerce Parkway, Suite 1108 Richmond, BC, V6V 2X7 Canada Phone: +1-604-204-6784 Fax: +1-604-244-8399 www.biolytical.com 51-1143K 25-Feb-2019 ©Copyright 2018, All rights reserved.

Authorized Representative:

P EMERGO EUROPE Prinsessegracht 20, 2514 AP The Hague, The Netherlands Phone: +31.70.345.8570 Fax: +31.70.346.7299

Introduction

SELF TEST

28 29

TRAINING PACK

Instructions For Use

The World’s Fastest HIV TestGet highly accurate results instantly.

Early DetectionDetects HIV infection up to two weeks sooner than other leading brands.

Easy to UseSimple design. No timers, no fuss.

Convenient and DiscreetGet results from the privacy of your own home.

What is an INSTI® HIV Self TestINSTI® HIV Self Test is designed as a single-use HIV test kit that allows you to get results in the comfort of your home and at a time that works best for you. Sample, pour and read your results immediately. It’s that easy.

INSTI is designed to be a simple and straightforward home HIV test. Read the step-by-step instructions inside the box to learn how to use your test and get the results you need to take control of your health. You can visit www.hivhometest.com to watch a training video.

Easy to UseA quick and easy blood test with three simple steps

SensitiveCan detect HIV up to 2 weeks sooner thanother brands

UniqueNovel flow- through platform

ClearSimple dots for easy interpretation

AccurateOver 99% accurate

In-Store Stockist Support: New Stockist Pack including Posters, Shelf Wobblers & Leaflets to help promote

the product Online portal of resources

Online Stockist Support: Online portal of resources to promote the product on your online platform How to use video which can be hosted on your website or linked to the product website

Bottle 1, 2, 3 Test Device

Lancet Bandage

®

Membrane Unit51-1061D

For

in vitro diagnostic use only.Store at 15-30°C

15°C

30°C

HI V- 1/HI V- 2 Antibody T es t

0050

STERILE R

PreparationTraining video available at: www.INSTI.com

Step 2: Test

This Test Contains:

2. Place test device on a flat surface. 3. Remove cap of Bottle 1. Place on flat surface. WARNING: Bottle 1 contains liquid. Handle with care.

1. Open test device pouch.IMPORTANT: Clean and dry hands.

Step 1: Collect BloodIf you have trouble collecting blood, see Frequently Asked Questions on reverse side.

2. Rub finger and hand to increase blood flow. 3. Place lancet on the side of finger tip.1. Twist and pull out lancet tip. Discard tip.

4. Rub finger to create a LARGE drop of blood.

5. Let 1 drop FALL into Bottle 1. Twist on cap of Bottle 1.

6. Apply adhesive bandage.

SHAKE 4 TIMES

SHAKE 4 TIMES

SHAKE 4 TIMES

POUR ALL POUR ALL POUR ALL

1. Shake and pour all liquid.Wait until liquid disappears.

2. Shake and pour all liquid.Wait until liquid disappears. TIP: You may need to gently tap Bottle 2 to get all the liquid out.

3. Shake and pour all liquid.Wait until liquid disappears.

Negative Positive InvalidYour test result is negative. Your test did not work.

Control dot must appear to indicate that the test has

been performed correctly.

CONTROLTEST

TIP: One dot may be lighter than the other. In rare instances, a faint ring may appear at the test dot; this is a positive result.

HIV Self TestInstructions For Use

READ BEFORE USE

Step 3: Read ResultRead result right away to within 1 HOUR.

A Negative ResultAs with many tests, there is a chance for false results. To reduce

the chance of false results, be sure to follow the instructions and use the test correctly. If you have a negative result but

you were involved in an HIV-risk activity in the past 3 months, you could be in what is called the “window period” and it is

recommended to repeat testing at a later date.

A Positive ResultConsult a doctor as soon as possible and inform him/her that

you have performed a self test for HIV. All positive results must be confirmed by a laboratory test.What Next After A Positive Result?

Having HIV does not mean you have AIDS. With early diagnosis and treatment, it is unlikely that you will develop AIDS.

51-1200C 27-Mar-2018

PRESS HARD

Two dots means your test result is positive. You are probably HIV

positive. Positive results MUST be confirmed by a doctor.

DisposalDispose in accordance with local regulations. Put all items back into the outer packaging. Throw away into waste bin.

Instructions For Use

SELF TEST

30 31

TRAINING PACK

Instructions For Use

Bottle 1, 2, 3 Test Device

Lancet Bandage

®

Membrane Unit51-1061D

For

in vitro diagnostic use only.Store at 15-30°C

15°C

30°C

HI V- 1/HI V- 2 Antibody T es t

0050

STERILE R

PreparationTraining video available at: www.INSTI.com

Step 2: Test

This Test Contains:

2. Place test device on a flat surface. 3. Remove cap of Bottle 1. Place on flat surface. WARNING: Bottle 1 contains liquid. Handle with care.

1. Open test device pouch.IMPORTANT: Clean and dry hands.

Step 1: Collect BloodIf you have trouble collecting blood, see Frequently Asked Questions on reverse side.

2. Rub finger and hand to increase blood flow. 3. Place lancet on the side of finger tip.1. Twist and pull out lancet tip. Discard tip.

4. Rub finger to create a LARGE drop of blood.

5. Let 1 drop FALL into Bottle 1. Twist on cap of Bottle 1.

6. Apply adhesive bandage.

SHAKE 4 TIMES

SHAKE 4 TIMES

SHAKE 4 TIMES

POUR ALL POUR ALL POUR ALL

1. Shake and pour all liquid.Wait until liquid disappears.

2. Shake and pour all liquid.Wait until liquid disappears. TIP: You may need to gently tap Bottle 2 to get all the liquid out.

3. Shake and pour all liquid.Wait until liquid disappears.

Negative Positive InvalidYour test result is negative. Your test did not work.

Control dot must appear to indicate that the test has

been performed correctly.

CONTROLTEST

TIP: One dot may be lighter than the other. In rare instances, a faint ring may appear at the test dot; this is a positive result.

HIV Self TestInstructions For Use

READ BEFORE USE

Step 3: Read ResultRead result right away to within 1 HOUR.

A Negative ResultAs with many tests, there is a chance for false results. To reduce

the chance of false results, be sure to follow the instructions and use the test correctly. If you have a negative result but

you were involved in an HIV-risk activity in the past 3 months, you could be in what is called the “window period” and it is

recommended to repeat testing at a later date.

A Positive ResultConsult a doctor as soon as possible and inform him/her that

you have performed a self test for HIV. All positive results must be confirmed by a laboratory test.What Next After A Positive Result?

Having HIV does not mean you have AIDS. With early diagnosis and treatment, it is unlikely that you will develop AIDS.

51-1200C 27-Mar-2018

PRESS HARD

Two dots means your test result is positive. You are probably HIV

positive. Positive results MUST be confirmed by a doctor.

DisposalDispose in accordance with local regulations. Put all items back into the outer packaging. Throw away into waste bin.

P.2

Instructions for Use

90-1041 INSTI® HIV Self Test, Pouch Format 90-1050 INSTI® HIV Self Test, Box Format

Store at 2 to 30°C K Sterilization using irradiation

Y Caution g Lot number

V In Vitro diagnostic medical device h Catalogue Number

i Consult Instructions for Use M Manufacturer

D Do not reuse C CE Mark

H Use by R22 – Harmful if swallowed.

Authorized Representative in the European Community

For in vitro diagnostic use only. V i Read this Instructions for Use prior to beginning the test procedure. Although the INSTI HIV Self Test is designed to be simple to use, conformance with the test procedure is necessary to ensure accurate results. BACKGROUND HIV stands for Human Immunodeficiency Virus. HIV is the virus that causes AIDS (Acquired Immunodeficiency Syndrome) if left untreated. It is estimated that there are over 30 million people living with HIV in the world today, and up to half of those people have not been diagnosed and are unaware of their infection. This undiagnosed population accounts for most of the HIV transmissions worldwide. Treatment for HIV is highly effective. It is important to start treatment as early as possible following infection, to reduce the risk of serious illness or death. INTENDED USE The INSTI HIV Self Test is a single use, rapid, flow-through in vitro qualitative immunoassay for the detection of antibodies to Human Immunodeficiency Virus Type 1 (HIV-1) and Type 2 (HIV-2) in human fingerstick whole blood. The test is intended for use by untrained lay users as a self test to aid in the diagnosis of HIV-1 and HIV-2 infection using a small drop (50µL) of blood obtained through fingerstick collection procedures. BIOLOGICAL PRINCIPLES OF THE TEST The INSTI HIV Self Test is an immunoassay for detecting HIV antibodies. The test device consists of a synthetic membrane positioned atop an absorbent pad within a plastic cartridge. One section of the membrane has been treated with non-hazardous HIV-1 and HIV-2 recombinant proteins, which capture HIV antibodies (test dot). The membrane also includes a control dot treated with protein-A that captures other non-specific antibodies normally present in human blood. The test is performed by adding a fingerstick blood sample to Bottle 1. The diluted blood in Bottle 1 is poured into the well of the test device. Any HIV antibodies in the sample are captured by the test dot and non-specific antibodies are captured by the control dot. Bottle 2 is then added to the test device. Bottle 2 solution reacts with captured antibodies to produce a blue dot at control dot and, if HIV antibodies are present, a blue dot also appears at test dot. In the final step, Bottle 3 is added to the membrane to make the control and test dots more visible. MATERIALS PROVIDED 1 Instructions for Use 1 Pouch with test device (labelled Membrane Unit) 1 Sample Diluent (Bottle 1, red top) 1 Colour Developer (Bottle 2, blue top) 1 Clarifying Solution (Bottle 3, clear top) 1 Sterile single-use lancet ( 0050) 1 Adhesive bandage 1 Resources Card Test device (packaged inside the pouch labelled Membrane Unit) The control and/or test dot will appear on the test device once the test is performed. The test device is prepared with control (IgM/IgG capture) and test (gp41 and gp36 antigen) reaction dots. It is individually packaged and is to be used only once to complete a single INSTI test. Sample Diluent (Bottle 1, red cap) A solution designed to dilute the blood sample and break down red blood cells. It contains 1.5 mL of colorless Tris-Glycine buffered solution containing cell lysis reagents. Color Developer (Bottle 2, blue cap) A blue solution that detects human antibodies. It contains 1.5 mL of a blue-coloured Borate buffered proprietary indicator solution designed to detect IgM/IgG in the control dot and HIV-specific antibodies in the test dot. Clarifying Solution (Bottle 3, clear cap) A solution to remove background blue color. It contains 1.5 mL of a colorless Tris-Glycine buffered solution designed to remove background staining from the test device prior to reading the INSTI test results. All solutions contain 0.1% Sodium Azide as a preservative and are harmful if swallowed. All solutions are for single use only and are stable to date and under storage conditions indicated on labels. LIMITATIONS OF THE TEST • In some instances, samples may exhibit longer than normal flow times from the time the diluted

blood-Bottle 1 mixture is poured into the test device, to the time the contents of Bottle 3 have fully flowed through the test device. This is due to variable factors, such as cellular components within the whole blood sample.

• The INSTI HIV Self Test procedure and the interpretation of results must be followed closely when testing for the presence of antibodies to HIV.

• This test has not been validated for detection of antibodies to HIV-1 Group N subtypes. • Because a variety of factors may cause non-specific reactions, a patient found to be positive using

the INSTI HIV Self Test must have the results confirmed by a doctor. • The presence of HIV antibodies indicates past exposure to HIV but is not a diagnosis of AIDS,

which can only be made by a physician. • A negative test result does not rule out exposure to HIV.

WARNINGS AND PRECAUTIONS • Each device is for single use only and is designed for self testing by one person. V • Keep out of the reach of children. • The test is for use only with human whole blood. • Do not use the INSTI HIV Self Test beyond the expiration date stated on the outer packaging. • Do not use the test device if the foil pouch has been damaged. • Wash your hands with warm water and ensure they are clean and dry before beginning the test. • Do not read the result if more than 1 hour has passed after completing the test procedure. • Failure to follow the instructions may result in leakage and/or overflow of liquids from the test device. • If you have been on long term antiretroviral drug therapy your test may give a false negative result. • If you have a severe blood disorder such as multiple myeloma you may obtain a false negative or

invalid result. • If you have higher than normal haemoglobin, you may test false negative. • Y All blood samples should be handled as if capable of transmitting infectious diseases. • Spills should be cleaned up with household bleach or disinfecting wipes. • Solutions in Bottle 1, 2 and 3 are harmful if swallowed due to the presence of Sodium Azide. • Test procedure must be completed in the proper sequence without delays between steps. • Adequate lighting is required to read the test results. Restrictions on Use • Not suitable for users who are afraid of needles • May not be suitable for patients who have been infected within the last 3 months • Not suitable for users who have a bleeding disorder • Not suitable for users below the age of 18 • Not suitable for users who are taking anti-retroviral treatment (ART) • Not suitable for users who have participated in a HIV vaccine study Storage • Store in the original packaging in a cool, dry location between 2 to 30°C. DO NOT FREEZE. • Do not store near a heat source or in direct sunlight. • The test should be performed at room temperature (15 to 30°C). • Do not open the test device pouch until you are ready to perform the test. Note that although the

test device pouch states storage at 15-30°C, it can be stored refrigerated, if required. Disposal Place all components back into outer packaging and dispose into waste bin. Dispose with accordance to local regulations. PERFORMANCE CHARACTERISTICS DIAGNOSTIC SENSITIVITY Diagnostic sensitivity of a test like the INSTI HIV Self Test is a measure of how well the test detects the presence of HIV antibodies. Sensitivity is expressed as a percentage and is calculated from data from a clinical trial or performance evaluation. Sensitivity is calculated by dividing the number of INSTI positive test results by the number of truly HIV positive persons tested. The higher the sensitivity the better the test is at correctly identifying truly infected persons. In a study conducted by untrained lay users (Table 1), 517/517 true HIV antibody positive subjects were identified as positive by the INSTI test, resulting in a relative sensitivity of 100%. Table 1 – Relative Sensitivity and Specificity of the INSTI Self Test compared to the HIV Status of Individuals with Known and Unknown HIV Status by Untrained Lay Users

Study Population

Number of Subjects

Relative Sensitivity

95% Confidence Interval

Relative Specificity

95% Confidence

Interval

HIV status unknown 905 100%

(34/34) 89.9% - 100% 99.8% (869/871)

99.2% - 99.9%

Known HIV-1 Positive 483 100%

(483/483) 99.2% -100% N.A. N.A.

Total 1,388 100% (517/517) 99.3% - 100% 99.8%

(869/871) 99.2% - 99.9%

Percent of invalid results was 0% (0/1388) Studies to calculate the HIV-2 sensitivity of the INSTI HIV Self Test The sensitivity of INSTI HIV-1/HIV-2 Antibody Test evaluated in an independent European study with 49 sera from Western Blot confirmed HIV-2 infected patients at the chronic stage of the infection was 100%. Additional studies conducted in-house with 88 different HIV-2 positive serum and plasma samples obtained from European sources and 24 different plasma samples obtained from Nigeria added into individual whole blood (to simulate HIV-2 positive blood) also showed 100% sensitivity of INSTI for HIV-2 antibody detection. Table 2: INSTI HIV-1/HIV-2 Antibody Test’s Sensitivity in HIV-2 Positive Specimens

Sample Source France1 France2 Nigeria3 Total

Positive Samples 49 88 24 161

INSTI Positives 49 88 24 161

Sensitivity 100% 100% 100% 100%

1. Tests performed in France using serum samples 2. Tests performed in-house using whole blood spiked with plasma (13 samples) and serum (75 samples) 3. Tests performed in-house using plasma samples In addition, 12 out of 500 prospectively collected plasma specimens from an HIV-2 endemic region (Ivory Coast) were tested and confirmed as HIV-2 true positive by an FDA-approved differentiation assay or an HIV-2 RNA quantitative assay. INSTI was reactive in all 12 of these specimens for a sensitivity of 100%. Results are summarized in Table 3. Table 3 - Detection of Antibody to HIV-2 in Specimens from HIV-2 Seropositive Individuals and Individuals from an HIV-2 Endemic Region

Specimen Group Total Specimens HIV-2 Positive INSTI HIV-1/HIV-2

Reactive

Endemic subjects 500 121 12 1Determined by an approved HIV-1/HIV-2 differentiation assay or HIV-2 RNA testing

DIAGNOSTIC SPECIFICITY Diagnostic specificity of a test like the INSTI HIV Self Test is a measure of how well the test detects healthy patients who do not have HIV. Specificity is expressed as a percentage and is calculated from data from a clinical trial or performance evaluation. Specificity is calculated by dividing the number of INSTI negative test results by the number of truly HIV negative persons that were tested. The higher the specificity the better the test is at correctly identifying truly non-infected persons. The INSTI HIV Self Test has a specificity of 99.8% in a performance evaluation conducted by untrained lay users. This means a positive result will be correct 998 out of every 1000 tests. A specificity study was performed on 1408 low or unknown risk and high risk individuals. Of the 1386 individuals identified as HIV negative using an approved comparator assay, 1376 were INSTI negative, and 4 were invalid. The overall specificity of the INSTI HIV Self Test in fingerstick whole blood specimens from the combined high risk and low or unknown risk populations, minus the invalid results, was calculated to be 1376/1382 = 99.5% which means a positive result will be correct 998 out of every 1000 tests. (see Table 4) Table 4 - Performance of the INSTI HIV-1/HIV-2 Antibody Test on Fingerstick Whole Blood Specimens from Individuals Presumed to be HIV Negative and Individuals at High Risk of HIV Infection

Test Group Total specimens

INSTI Non-

Reactive INSTI

Reactive INSTI

Invalid1 Approved Test Non- Reactive

Approved Test

Reactive True

Negative2

Low or Unknown Risk

626 620 6 0 626 0 626

High Risk 782 756 223 4 760 22 760 Total 1408 1376 28 4 1386 22 1386

1 Invalid results were not included in the calculation of specificity. The 4 specimens which gave invalid results on INSTI were Non-Reactive on the approved test. 2 Reactives were confirmed by licensed HIV-1 Western Blot and excluded from the calculation of specificity. 3 Of the 22 INSTI Reactive specimens, one was Non-Reactive by the approved test, i.e. INSTI false Reactive. Untrained Lay User Performance Evaluation The performance of INSTI was evaluated in a prospective study conducted over 4 months at 3 different sites. At each site, testing was conducted by non-professional lay users who had no laboratory experience. The 11 people running the tests had no training on how to use the test. Fingerstick blood from a total of 905 subjects with unknown HIV status and 483 subjects known to be HIV positive were tested with INSTI and results compared to those determined by FDA approved reference methods. The sensitivity of INSTI was 100% (517/517) and the specificity was 99.8% (869/871) There were no invalid results reported (see Table 1). Unrelated Medical Conditions and Potentially Interfering Substances To assess the impact of unrelated medical conditions or potentially interfering substances on the sensitivity and specificity of INSTI, 195 serum/plasma specimens from a cross section of medical conditions unrelated to HIV-1 infection and 178 specimens with potentially interfering substances were tested “unspiked” (HIV Nonreactive) and “spiked” with an HIV-1 positive specimen to give a low level of reactivity in the INSTI HIV-1 Antibody Test. No cross-reactivity or interference with INSTI test performance was observed with the following two exceptions:

1. Up to 5 specimens from individuals with multiple myeloma produced invalid INSTI results depending on the INSTI kit lot tested.

2. Of the 20 specimens from individuals with elevated hemoglobin, one tested false Non-Reactive in 2 out of 3 INSTI kit lots.

Reproducibility Studies The reproducibility of the INSTI test and ability of operators to consistently correctly interpret test results was evaluated at 3 laboratory sites using 3 lots of INSTI on 3 separate days with 9 operators (3 per site). A panel of 5 specimens, consisting of 4 HIV-1 antibody positive (one strong positive and three low positives) and 1 HIV-1 antibody negative specimen was tested at each site. A total of 405 tests were conducted, 135 at each site, with a total of 81 tests per panel specimen. Overall all operators interpreted the test results for each sample correctly, generating a reproducibility of the INSTI HIV Test of 100% (405/405 samples tested). FREQUENTLY ASKED QUESTIONS What is HIV and AIDS? HIV stands for Human Immunodeficiency Virus. HIV is the virus that causes AIDS (Acquired Immunodeficiency Syndrome) if left untreated. When a person becomes infected with HIV, the virus begins to attack his or her immune system, which is the body’s defense against illness. As a result, that person becomes more susceptible to disease and infection. When his or her body loses the ability to fight diseases, that person is diagnosed with AIDS. There is no cure for HIV infection. However, treatment for HIV is highly effective. How does someone get infected with HIV? HIV spreads through contact with blood, semen, pre-seminal fluid, rectal fluid, vaginal fluids, or breast milk of an infected person. Transmission can occur from unsafe sex. It can also result from exposure to blood through the sharing of used syringes or needles. Women living with HIV can pass the virus to their babies during pregnancy, childbirth, and breastfeeding. It is also possible to become infected with HIV through a blood transfusion, although this is now very rare. HIV cannot be passed on from one person to another through casual contact. There is no risk of infection when we share everyday items such as food, dishes, utensils, clothes, beds and toilets with a person living with HIV. The virus is not spread from contact with sweat, tears, saliva, or a casual kiss from an infected person. People do not become infected from eating food prepared by a person living with HIV. People have not become infected with HIV through insect bites. How do I make sure I get enough blood? Relax and have a drink of water about 20 minutes before you start the test. Warm your hands by washing them with warm water. Ensure your hands are dry. Place your hand below waist level to promote blood flow. Before using the lancet, look for a spot on the side of your finger tip that is smooth and not calloused and away from your fingernail. What is an antibody? Antibodies are proteins produced by your body’s immune system in response to harmful organisms like viruses and bacteria. Their purpose is to defend the body against infection by these organisms. How accurate is the test? Extensive research studies have shown that this test is extremely accurate when performed correctly. In a recent study performed by untrained users, the test sensitivity was 100%. It also has a proven specificity (a measure of reliability that the test will be negative for people who do not have HIV infection) of 99.5%. In the untrained user study, the specificity was 99.8%. *If you are unsure of your result you must go to a doctor to perform more testing.

The contents of Bottle 1, Bottle 2 or Bottle 3 do not absorb into the test device. It is very rare for this to happen, but if it does, you will not be able to complete the test procedure and read the results. You will need to perform another test. What happens if I spill some of the contents of Bottle 1, Bottle 2 or Bottle 3 outside the test device? Keep going with the test procedure. As long as the control dot shows a visible dot after pouring Bottle 3 into the test device, the test results are valid. How early can this test detect HIV? Based on bioLytical's studies, INSTI demonstrates third generation performance and detects HIV antibodies of the IgM and IgG class. IgM antibodies are the earliest antibodies that the body produces after an HIV infection and are detectable within 21-22 days.1,2 Depending on how quickly a person’s immune system generates HIV antibodies after infection, it could still take up to 3 months to get a positive result. If you think you have been exposed to HIV within the last 3 months, and your results are negative, you will need to test again after at least 3 months have passed since your exposure. The time from HIV infection to when a test can correctly give a positive result is referred to as the ‘window period’. 1Moshgabadi N, Galli RA, Daly AC, Ko SM, Westgard TE, Bulpitt AF, Shackleton CR., 2015. “Sensitivity of a rapid point of care assay for early HIV antibody detection is enhanced by its ability to detect HIV gp41 IgM antibodies.” J Clin Virol. 2015 Oct; 71:67-72. 2M. Cohen, C. Gay, M. Busch, F. Hecht, The detection of acute HIV infection, J.Infect. Dis. 202 (2010) 270–277 I can’t see any dots Make sure you have adequate lighting. If no dots are visible, you may not have completed the test correctly, or collected enough blood. You will need to perform another test. How will I know if my test was done correctly? The INSTI HIV Self Test has a built-in control dot to show that the test has been performed correctly and that you have added the proper sample type and amount of blood sample. If the control dot does not appear (invalid test result), your test has not worked. It is not possible to draw conclusions from this result and you will need to perform another test. In the event of repeated invalid results, consult a doctor.

M Manufactured by: bioLytical Laboratories, Inc. 13351 Commerce Parkway, Suite 1108 Richmond, BC, Canada V6V 2X7 Phone: +1 604-204-6784 Fax: +1 604-244-8399 www.biolytical.com

Authorized Representative:

EMERGO EUROPE Prinsessegracht 20 2514 AP, The Hague The Netherlands Phone: +31.70.345.8570 Fax: +31.70.346.7299 51-1200C 27-Mar-2018 © Copyright 2018, All rights reserved.

0543

32

Instructions For Use

SELF TEST

P.2

Instructions for Use

90-1041 INSTI® HIV Self Test, Pouch Format 90-1050 INSTI® HIV Self Test, Box Format

Store at 2 to 30°C K Sterilization using irradiation

Y Caution g Lot number

V In Vitro diagnostic medical device h Catalogue Number

i Consult Instructions for Use M Manufacturer

D Do not reuse C CE Mark

H Use by R22 – Harmful if swallowed.

Authorized Representative in the European Community

For in vitro diagnostic use only. V i Read this Instructions for Use prior to beginning the test procedure. Although the INSTI HIV Self Test is designed to be simple to use, conformance with the test procedure is necessary to ensure accurate results. BACKGROUND HIV stands for Human Immunodeficiency Virus. HIV is the virus that causes AIDS (Acquired Immunodeficiency Syndrome) if left untreated. It is estimated that there are over 30 million people living with HIV in the world today, and up to half of those people have not been diagnosed and are unaware of their infection. This undiagnosed population accounts for most of the HIV transmissions worldwide. Treatment for HIV is highly effective. It is important to start treatment as early as possible following infection, to reduce the risk of serious illness or death. INTENDED USE The INSTI HIV Self Test is a single use, rapid, flow-through in vitro qualitative immunoassay for the detection of antibodies to Human Immunodeficiency Virus Type 1 (HIV-1) and Type 2 (HIV-2) in human fingerstick whole blood. The test is intended for use by untrained lay users as a self test to aid in the diagnosis of HIV-1 and HIV-2 infection using a small drop (50µL) of blood obtained through fingerstick collection procedures. BIOLOGICAL PRINCIPLES OF THE TEST The INSTI HIV Self Test is an immunoassay for detecting HIV antibodies. The test device consists of a synthetic membrane positioned atop an absorbent pad within a plastic cartridge. One section of the membrane has been treated with non-hazardous HIV-1 and HIV-2 recombinant proteins, which capture HIV antibodies (test dot). The membrane also includes a control dot treated with protein-A that captures other non-specific antibodies normally present in human blood. The test is performed by adding a fingerstick blood sample to Bottle 1. The diluted blood in Bottle 1 is poured into the well of the test device. Any HIV antibodies in the sample are captured by the test dot and non-specific antibodies are captured by the control dot. Bottle 2 is then added to the test device. Bottle 2 solution reacts with captured antibodies to produce a blue dot at control dot and, if HIV antibodies are present, a blue dot also appears at test dot. In the final step, Bottle 3 is added to the membrane to make the control and test dots more visible. MATERIALS PROVIDED 1 Instructions for Use 1 Pouch with test device (labelled Membrane Unit) 1 Sample Diluent (Bottle 1, red top) 1 Colour Developer (Bottle 2, blue top) 1 Clarifying Solution (Bottle 3, clear top) 1 Sterile single-use lancet ( 0050) 1 Adhesive bandage 1 Resources Card Test device (packaged inside the pouch labelled Membrane Unit) The control and/or test dot will appear on the test device once the test is performed. The test device is prepared with control (IgM/IgG capture) and test (gp41 and gp36 antigen) reaction dots. It is individually packaged and is to be used only once to complete a single INSTI test. Sample Diluent (Bottle 1, red cap) A solution designed to dilute the blood sample and break down red blood cells. It contains 1.5 mL of colorless Tris-Glycine buffered solution containing cell lysis reagents. Color Developer (Bottle 2, blue cap) A blue solution that detects human antibodies. It contains 1.5 mL of a blue-coloured Borate buffered proprietary indicator solution designed to detect IgM/IgG in the control dot and HIV-specific antibodies in the test dot. Clarifying Solution (Bottle 3, clear cap) A solution to remove background blue color. It contains 1.5 mL of a colorless Tris-Glycine buffered solution designed to remove background staining from the test device prior to reading the INSTI test results. All solutions contain 0.1% Sodium Azide as a preservative and are harmful if swallowed. All solutions are for single use only and are stable to date and under storage conditions indicated on labels. LIMITATIONS OF THE TEST • In some instances, samples may exhibit longer than normal flow times from the time the diluted

blood-Bottle 1 mixture is poured into the test device, to the time the contents of Bottle 3 have fully flowed through the test device. This is due to variable factors, such as cellular components within the whole blood sample.

• The INSTI HIV Self Test procedure and the interpretation of results must be followed closely when testing for the presence of antibodies to HIV.

• This test has not been validated for detection of antibodies to HIV-1 Group N subtypes. • Because a variety of factors may cause non-specific reactions, a patient found to be positive using

the INSTI HIV Self Test must have the results confirmed by a doctor. • The presence of HIV antibodies indicates past exposure to HIV but is not a diagnosis of AIDS,

which can only be made by a physician. • A negative test result does not rule out exposure to HIV.

WARNINGS AND PRECAUTIONS • Each device is for single use only and is designed for self testing by one person. V • Keep out of the reach of children. • The test is for use only with human whole blood. • Do not use the INSTI HIV Self Test beyond the expiration date stated on the outer packaging. • Do not use the test device if the foil pouch has been damaged. • Wash your hands with warm water and ensure they are clean and dry before beginning the test. • Do not read the result if more than 1 hour has passed after completing the test procedure. • Failure to follow the instructions may result in leakage and/or overflow of liquids from the test device. • If you have been on long term antiretroviral drug therapy your test may give a false negative result. • If you have a severe blood disorder such as multiple myeloma you may obtain a false negative or

invalid result. • If you have higher than normal haemoglobin, you may test false negative. • Y All blood samples should be handled as if capable of transmitting infectious diseases. • Spills should be cleaned up with household bleach or disinfecting wipes. • Solutions in Bottle 1, 2 and 3 are harmful if swallowed due to the presence of Sodium Azide. • Test procedure must be completed in the proper sequence without delays between steps. • Adequate lighting is required to read the test results. Restrictions on Use • Not suitable for users who are afraid of needles • May not be suitable for patients who have been infected within the last 3 months • Not suitable for users who have a bleeding disorder • Not suitable for users below the age of 18 • Not suitable for users who are taking anti-retroviral treatment (ART) • Not suitable for users who have participated in a HIV vaccine study Storage • Store in the original packaging in a cool, dry location between 2 to 30°C. DO NOT FREEZE. • Do not store near a heat source or in direct sunlight. • The test should be performed at room temperature (15 to 30°C). • Do not open the test device pouch until you are ready to perform the test. Note that although the

test device pouch states storage at 15-30°C, it can be stored refrigerated, if required. Disposal Place all components back into outer packaging and dispose into waste bin. Dispose with accordance to local regulations. PERFORMANCE CHARACTERISTICS DIAGNOSTIC SENSITIVITY Diagnostic sensitivity of a test like the INSTI HIV Self Test is a measure of how well the test detects the presence of HIV antibodies. Sensitivity is expressed as a percentage and is calculated from data from a clinical trial or performance evaluation. Sensitivity is calculated by dividing the number of INSTI positive test results by the number of truly HIV positive persons tested. The higher the sensitivity the better the test is at correctly identifying truly infected persons. In a study conducted by untrained lay users (Table 1), 517/517 true HIV antibody positive subjects were identified as positive by the INSTI test, resulting in a relative sensitivity of 100%. Table 1 – Relative Sensitivity and Specificity of the INSTI Self Test compared to the HIV Status of Individuals with Known and Unknown HIV Status by Untrained Lay Users

Study Population

Number of Subjects

Relative Sensitivity

95% Confidence Interval

Relative Specificity

95% Confidence

Interval

HIV status unknown 905 100%

(34/34) 89.9% - 100% 99.8% (869/871)

99.2% - 99.9%

Known HIV-1 Positive 483 100%

(483/483) 99.2% -100% N.A. N.A.

Total 1,388 100% (517/517) 99.3% - 100% 99.8%

(869/871) 99.2% - 99.9%

Percent of invalid results was 0% (0/1388) Studies to calculate the HIV-2 sensitivity of the INSTI HIV Self Test The sensitivity of INSTI HIV-1/HIV-2 Antibody Test evaluated in an independent European study with 49 sera from Western Blot confirmed HIV-2 infected patients at the chronic stage of the infection was 100%. Additional studies conducted in-house with 88 different HIV-2 positive serum and plasma samples obtained from European sources and 24 different plasma samples obtained from Nigeria added into individual whole blood (to simulate HIV-2 positive blood) also showed 100% sensitivity of INSTI for HIV-2 antibody detection. Table 2: INSTI HIV-1/HIV-2 Antibody Test’s Sensitivity in HIV-2 Positive Specimens

Sample Source France1 France2 Nigeria3 Total

Positive Samples 49 88 24 161

INSTI Positives 49 88 24 161

Sensitivity 100% 100% 100% 100%

1. Tests performed in France using serum samples 2. Tests performed in-house using whole blood spiked with plasma (13 samples) and serum (75 samples) 3. Tests performed in-house using plasma samples In addition, 12 out of 500 prospectively collected plasma specimens from an HIV-2 endemic region (Ivory Coast) were tested and confirmed as HIV-2 true positive by an FDA-approved differentiation assay or an HIV-2 RNA quantitative assay. INSTI was reactive in all 12 of these specimens for a sensitivity of 100%. Results are summarized in Table 3. Table 3 - Detection of Antibody to HIV-2 in Specimens from HIV-2 Seropositive Individuals and Individuals from an HIV-2 Endemic Region

Specimen Group Total Specimens HIV-2 Positive INSTI HIV-1/HIV-2

Reactive

Endemic subjects 500 121 12 1Determined by an approved HIV-1/HIV-2 differentiation assay or HIV-2 RNA testing

DIAGNOSTIC SPECIFICITY Diagnostic specificity of a test like the INSTI HIV Self Test is a measure of how well the test detects healthy patients who do not have HIV. Specificity is expressed as a percentage and is calculated from data from a clinical trial or performance evaluation. Specificity is calculated by dividing the number of INSTI negative test results by the number of truly HIV negative persons that were tested. The higher the specificity the better the test is at correctly identifying truly non-infected persons. The INSTI HIV Self Test has a specificity of 99.8% in a performance evaluation conducted by untrained lay users. This means a positive result will be correct 998 out of every 1000 tests. A specificity study was performed on 1408 low or unknown risk and high risk individuals. Of the 1386 individuals identified as HIV negative using an approved comparator assay, 1376 were INSTI negative, and 4 were invalid. The overall specificity of the INSTI HIV Self Test in fingerstick whole blood specimens from the combined high risk and low or unknown risk populations, minus the invalid results, was calculated to be 1376/1382 = 99.5% which means a positive result will be correct 998 out of every 1000 tests. (see Table 4) Table 4 - Performance of the INSTI HIV-1/HIV-2 Antibody Test on Fingerstick Whole Blood Specimens from Individuals Presumed to be HIV Negative and Individuals at High Risk of HIV Infection

Test Group Total specimens

INSTI Non-

Reactive INSTI

Reactive INSTI

Invalid1 Approved Test Non- Reactive

Approved Test

Reactive True

Negative2

Low or Unknown Risk

626 620 6 0 626 0 626

High Risk 782 756 223 4 760 22 760 Total 1408 1376 28 4 1386 22 1386

1 Invalid results were not included in the calculation of specificity. The 4 specimens which gave invalid results on INSTI were Non-Reactive on the approved test. 2 Reactives were confirmed by licensed HIV-1 Western Blot and excluded from the calculation of specificity. 3 Of the 22 INSTI Reactive specimens, one was Non-Reactive by the approved test, i.e. INSTI false Reactive. Untrained Lay User Performance Evaluation The performance of INSTI was evaluated in a prospective study conducted over 4 months at 3 different sites. At each site, testing was conducted by non-professional lay users who had no laboratory experience. The 11 people running the tests had no training on how to use the test. Fingerstick blood from a total of 905 subjects with unknown HIV status and 483 subjects known to be HIV positive were tested with INSTI and results compared to those determined by FDA approved reference methods. The sensitivity of INSTI was 100% (517/517) and the specificity was 99.8% (869/871) There were no invalid results reported (see Table 1). Unrelated Medical Conditions and Potentially Interfering Substances To assess the impact of unrelated medical conditions or potentially interfering substances on the sensitivity and specificity of INSTI, 195 serum/plasma specimens from a cross section of medical conditions unrelated to HIV-1 infection and 178 specimens with potentially interfering substances were tested “unspiked” (HIV Nonreactive) and “spiked” with an HIV-1 positive specimen to give a low level of reactivity in the INSTI HIV-1 Antibody Test. No cross-reactivity or interference with INSTI test performance was observed with the following two exceptions:

1. Up to 5 specimens from individuals with multiple myeloma produced invalid INSTI results depending on the INSTI kit lot tested.

2. Of the 20 specimens from individuals with elevated hemoglobin, one tested false Non-Reactive in 2 out of 3 INSTI kit lots.

Reproducibility Studies The reproducibility of the INSTI test and ability of operators to consistently correctly interpret test results was evaluated at 3 laboratory sites using 3 lots of INSTI on 3 separate days with 9 operators (3 per site). A panel of 5 specimens, consisting of 4 HIV-1 antibody positive (one strong positive and three low positives) and 1 HIV-1 antibody negative specimen was tested at each site. A total of 405 tests were conducted, 135 at each site, with a total of 81 tests per panel specimen. Overall all operators interpreted the test results for each sample correctly, generating a reproducibility of the INSTI HIV Test of 100% (405/405 samples tested). FREQUENTLY ASKED QUESTIONS What is HIV and AIDS? HIV stands for Human Immunodeficiency Virus. HIV is the virus that causes AIDS (Acquired Immunodeficiency Syndrome) if left untreated. When a person becomes infected with HIV, the virus begins to attack his or her immune system, which is the body’s defense against illness. As a result, that person becomes more susceptible to disease and infection. When his or her body loses the ability to fight diseases, that person is diagnosed with AIDS. There is no cure for HIV infection. However, treatment for HIV is highly effective. How does someone get infected with HIV? HIV spreads through contact with blood, semen, pre-seminal fluid, rectal fluid, vaginal fluids, or breast milk of an infected person. Transmission can occur from unsafe sex. It can also result from exposure to blood through the sharing of used syringes or needles. Women living with HIV can pass the virus to their babies during pregnancy, childbirth, and breastfeeding. It is also possible to become infected with HIV through a blood transfusion, although this is now very rare. HIV cannot be passed on from one person to another through casual contact. There is no risk of infection when we share everyday items such as food, dishes, utensils, clothes, beds and toilets with a person living with HIV. The virus is not spread from contact with sweat, tears, saliva, or a casual kiss from an infected person. People do not become infected from eating food prepared by a person living with HIV. People have not become infected with HIV through insect bites. How do I make sure I get enough blood? Relax and have a drink of water about 20 minutes before you start the test. Warm your hands by washing them with warm water. Ensure your hands are dry. Place your hand below waist level to promote blood flow. Before using the lancet, look for a spot on the side of your finger tip that is smooth and not calloused and away from your fingernail. What is an antibody? Antibodies are proteins produced by your body’s immune system in response to harmful organisms like viruses and bacteria. Their purpose is to defend the body against infection by these organisms. How accurate is the test? Extensive research studies have shown that this test is extremely accurate when performed correctly. In a recent study performed by untrained users, the test sensitivity was 100%. It also has a proven specificity (a measure of reliability that the test will be negative for people who do not have HIV infection) of 99.5%. In the untrained user study, the specificity was 99.8%. *If you are unsure of your result you must go to a doctor to perform more testing.

The contents of Bottle 1, Bottle 2 or Bottle 3 do not absorb into the test device. It is very rare for this to happen, but if it does, you will not be able to complete the test procedure and read the results. You will need to perform another test. What happens if I spill some of the contents of Bottle 1, Bottle 2 or Bottle 3 outside the test device? Keep going with the test procedure. As long as the control dot shows a visible dot after pouring Bottle 3 into the test device, the test results are valid. How early can this test detect HIV? Based on bioLytical's studies, INSTI demonstrates third generation performance and detects HIV antibodies of the IgM and IgG class. IgM antibodies are the earliest antibodies that the body produces after an HIV infection and are detectable within 21-22 days.1,2 Depending on how quickly a person’s immune system generates HIV antibodies after infection, it could still take up to 3 months to get a positive result. If you think you have been exposed to HIV within the last 3 months, and your results are negative, you will need to test again after at least 3 months have passed since your exposure. The time from HIV infection to when a test can correctly give a positive result is referred to as the ‘window period’. 1Moshgabadi N, Galli RA, Daly AC, Ko SM, Westgard TE, Bulpitt AF, Shackleton CR., 2015. “Sensitivity of a rapid point of care assay for early HIV antibody detection is enhanced by its ability to detect HIV gp41 IgM antibodies.” J Clin Virol. 2015 Oct; 71:67-72. 2M. Cohen, C. Gay, M. Busch, F. Hecht, The detection of acute HIV infection, J.Infect. Dis. 202 (2010) 270–277 I can’t see any dots Make sure you have adequate lighting. If no dots are visible, you may not have completed the test correctly, or collected enough blood. You will need to perform another test. How will I know if my test was done correctly? The INSTI HIV Self Test has a built-in control dot to show that the test has been performed correctly and that you have added the proper sample type and amount of blood sample. If the control dot does not appear (invalid test result), your test has not worked. It is not possible to draw conclusions from this result and you will need to perform another test. In the event of repeated invalid results, consult a doctor.

M Manufactured by: bioLytical Laboratories, Inc. 13351 Commerce Parkway, Suite 1108 Richmond, BC, Canada V6V 2X7 Phone: +1 604-204-6784 Fax: +1 604-244-8399 www.biolytical.com

Authorized Representative:

EMERGO EUROPE Prinsessegracht 20 2514 AP, The Hague The Netherlands Phone: +31.70.345.8570 Fax: +31.70.346.7299 51-1200C 27-Mar-2018 © Copyright 2018, All rights reserved.

0543