A Summary of Clinical Evidence

A Summary of Clinical Evidence Supporting the use of the Alere Determine HIV-1/2 Ag/Ab Combo Rapid Test to assist in the diagnosis of Human Immunodeficiency Virus (HIV) TAP HERE TO SEE THE PRODUCTS

Table of Contents Overview of HIV...3 Natural History and Immunopathogenesis of HIV-1 Infection...4-5 Treatment of HIV...6 Screening recommendations...7 A History of HIV diagnostic tests...7 Alere Determine HIV 1/2 Ag/Ab Combo...8 Alere Determine HIV 1/2 Ag/Ab Combo: HIV-1 Clinical Data...9 Alere Determine HIV 1/2 Ag/Ab Combo: HIV-1 Preclinical Data...10 Published Studies... 11-12 Summary of device description and biological principle of the procedure...13 Product Information...14 References...15

Alere Determine HIV-1/2 Ag/Ab Combo 3 Overview of HIV (Human Immunodeficiency Virus) 1.2 Million According to the CDC, there were almost 1.2 million people (1 out of every 250) in U.S. that were HIV positive in 2011. 1 HIV, or human immunodeficiency virus, is the virus that causes AIDS. It attacks the immune system by destroying CD4 positive (CD4+) T cells that are vital to fighting off infection. HIV belongs to a class of viruses known as retroviruses. Within that class, HIV is in the lentivirus subgroup, the members of which are known for having a long time period between initial infection and the beginning of serious symptoms. This is why there are many people who are unaware of their HIV infection and can unknowingly spread the virus to others. Early detection of HIV infection is critical in helping to minimize further transmission of the disease. The four main routes of infection are unsafe sex, contaminated needles, breast milk from an infected mother, and birth to an infected mother. Transmission through blood and blood products has largely been eliminated as a result of screening of blood donations. According to the CDC, there were almost 1.2 million people (1 out of every 250) in U.S. that were HIV positive in 2009. 18% (nearly ¼ of million) are not aware of their positive status. 1 This group is responsible for an estimated 2/3 (54-70%) of all new HIV infections. 2

Alere Determine HIV-1/2 Ag/Ab Combo 4 Natural History and Immunopathogenesis of HIV-1 Infection 3 Laboratory Markers Infection Stage Viral RNA + Gag p24 antigen + ELISA + Western Blot ± Viral Load (virions/ml of plasma) Viral Load Days since infection Data taken from Cohen S.M. et al. The Detection of Acute HIV-1 Infection. Journal of Infectious Disease, 2010 202; s270-277 The progression of HIV-1 infection can be depicted as discrete stages based on the viral load and the sequential appearance of different markers at any given point in time. How HIV infection can be detected during each of these phases will differ. Only certain tests are able to detect the different markers present during these phases. The actual timing for each of these key stages of HIV infection will vary for any individual patient. Immediately following initial exposure, HIV-1 seeks out sanctuary in the mucosal tissues where it continues the process of replication. This period, after infection and prior to detection of viral RNA, is defined as the eclipse phase. During this phase (which varies from 7 to 21 days), the virus (as measured by the viral load) cannot be detected in plasma. Viral RNA is the first detectable marker to appear and is the most common marker used to follow progression (Phase I). Once HIV-1 RNA reaches a certain concentration in viral load plasma, the virus can be detected using nucleic acid amplification testing (NAAT). The presence of viral RNA, absent of antibodies to HIV, confirms an acute HIV infection.

Alere Determine HIV-1/2 Ag/Ab Combo 5 The appearance of gag p24 protein antigen marks the next stage (Phase II) of HIV infection. During this stage where the viral load is nearing its peak, an HIV-1 infection can be characterized as either acute or early as follows 4 : n Acute: appearance/detection of free (unbound) p24 antigen (no antibodies). n Acute/Early: both free p24 antigen AND HIV Ab and/or Ag/Ab complex detected. n Early: p24 antigen/hiv-antibodies complex detected (no free p24 Ag). During the next stage where the viral load has peaked and is now reaching its set-point (Phase III), the gag p24 protein antigen is only found as an antigen-antibody immune-complex present in conjunction with free HIV-1 protein antibodies detected using an enzyme-linked immunosorbent assay (ELISA). Infections in this stage can either be early/later-stage of more established HIV infection. In the next stage where the viral load plateaus (Phase IV), the gag p24 protein antigen is present only as an Ag/Ab complex. The HIV-1 protein antibodies may now be detectable. Infections in this stage can either be early/later-stage of more established HIV infection. In fact, a person with HIV infection during the acute stage, where viral load is peaking, is 43 times more infectious than someone with a more established infection. 5 This is why early screening is vital to preventing HIV.

Alere Determine HIV-1/2 Ag/Ab Combo 6 Treatment of HIV 6,7 Recent studies have shown that providing early antiretroviral drug treatment for the recently infected can be beneficial for HIV patients and their uninfected sexual partners. Starting antiretroviral treatment sooner, rather than delaying therapy, may result in patients staying healthy longer. A recent study showed an increase in survival rate and life-expectancy in those treated versus untreated. Anti-retroviral initiation within the first 6 months after infection has also been associated with lower levels of T-cell activation and smaller HIV DNA and RNA reservoir size during long-term therapy. Another benefit of early therapy is in preventing transmission of HIV to others. By reducing the viral load during the highly infectious early-stages of infection, transmission of HIV to noninfected can be reduced. Long-term viral load reduction can also help in reducing onset of HIV/AIDS-related co-morbidities leading to improved long-term health outcomes and financial benefits. There can also be a significant financial impact of treating patients earlier. Such benefits in HIV-infected patients are a result of fewer illnesses and deaths. It has been shown that the potential cost of treatment of HIV/AIDS-related infections that would have otherwise been prevented by early treatment of HIV patients outweighed the costs of antiretroviral therapy drugs. Studies have also suggested that the impact of early treatment on the reduction of HIV transmission would result in a lower burden on healthcare system and overall cost savings.

Alere Determine HIV-1/2 Ag/Ab Combo 7 8, 9,10,11,12 Screening recommendations USPSTF The United States Preventative Services Task Force has formed the basis of the clinical standards for many professional societies, health organizations, and medical quality review groups. They recommend the following for HIV testing: n Adolescents and adults ages 15-65. n All pregnant women. n Adolescents and adults who are at increased risk of exposure. Other Organizations n The Center for Disease Control recommends that diagnostic HIV testing and opt-out HIV screening be a part of routine clinical care in all health-care settings. n The Infectious Diseases Society of America recommends routine HIV screening for all sexually active adults. n The American Academy of Family Physicians recommends that clinicians screen adolescents and adults aged 18-65 years for HIV infection. 13, 14 A History of HIV diagnostic tests The first HIV diagnostic test was introduced in 1985 and was a first enzyme immunoassay (EIA) that detected HIV antibodies. These first generation assays lacked the sensitivity and specificity of current widely used tests. By using recombinant proteins or peptides to produce viral antigens, the second generation assays had increased specificity and were able to detect HIV antibodies an average of 28.5 days after infection, approximately 1 week earlier than the previous first generation assays. Third generation assays could detect both HIV-1/2 immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies and were a significant advancement in HIV diagnostic tests. They were considered to have higher sensitivity than previous generations (lab-based and rapid assays are able to detect HIV antibodies approximately 21.4 and 31.6 days after HIV infection respectively) and by 2007 they had replaced earlier assays. In 2010 advanced, fourth generation assays became available in the US, although similar assays had been utilized in other countries a decade earlier. They detected both p24 antigen and HIV-1/2 IgG and IgM antibodies simultaneously. Fourth generation assays have succeeded in significantly reducing the window of detection and the time to diagnosis. They have been shown to detect HIV as soon as 16.8 days after infection. Until very recently, with FDA approval of the first Ag/Ab combination rapid test for HIV, 4th generation Ag/Ab tests have not been able to distinguish free p24 Ag from Ag/Ab immunecomplexes. This is because the laboratory samples require a dissociation step which breaks-up any Ag/Ab complexes. The p24 measured will be from both free and dissociated p24 Ag/ Ab complexes. While this may increase the sensitivity to p24, the ability to identify acute HIV infection without use of a secondary NAAT test is lost.

Alere Determine HIV-1/2 Ag/Ab Combo 8 Alere Determine HIV 1/2 Ag/Ab Combo Alere Determine HIV 1/2 Ag/Ab Combo is the first FDA approved rapid point-of-care test that provides for the simultaneous and separate qualitative detection of free HIV-1 p24 antigen and antibodies to HIV-1 and HIV-2. This enables health care providers to diagnose HIV infection earlier allowing individuals to seek medical care sooner. Intended use 15 Alere Determine HIV 1/2 Ag/Ab Combo is an in vitro, visually read, qualitative immunoassay for the simultaneous detection of Human Immunodeficiency Virus Type 1 (HIV- 1) p24 antigen (Ag) and antibodies (Ab) to HIV Type 1 and Type 2 (HIV-1 and HIV-2) in human serum, plasma, capillary (fingerstick) whole blood or venipuncture (venous) whole blood. It is intended for use as a point-of-care test to aid in the diagnosis of infection with HIV-1 and HIV-2, including an acute HIV-1 infection, and may distinguish acute HIV-1 infection from established HIV-1 infection when the specimen is positive for HIV-1 p24 antigen and negative for anti-hiv-1 and anti-hiv-2 antibodies. The test is suitable for use in multi-test algorithms designed for the statistical validation of rapid HIV test results. When multiple rapid HIV test are available, this test can be used in appropriate multi-test algorithms. Alere Determine HIV-1/2 Ag/Ab Combo is not intended for newborn screening or for use with cord blood specimens or specimens from individuals less than 12 years of age. Alere Determine HIV-1/2 Ag/Ab Combo is not intended for use in screening blood, plasma, cell, or tissue donors. CLIA Complexity: Moderate

Alere Determine HIV-1/2 Ag/Ab Combo 9 Alere Determine HIV 1/2 Ag/Ab Combo: HIV-1 Clinical Data 15 Sample Type Sensitivity* Specificity Serum 99.9% 99.6% Plasma 99.9% 99.7% Venous Whole Blood 99.9% 99.7% Fingerstick Whole Blood 99.9% 99.8% HIV-1 Sensitivity n Serum: The estimated sensitivity of Alere Determine Combo for serum specimens in these studies was 925/926 = 99.9% (95% confidence interval 99.4 to 100.0%). n Plasma: The estimated sensitivity of Alere Determine Combo for plasma specimens in these studies was 924/925 = 99.9% (95% confidence interval 99.4 to 100.0%). n Venous Whole Blood: The estimated sensitivity of Alere Determine Combo for venous whole blood specimens in these studies was 924/925 = 99.9% (95% confidence interval 99.4 to 100.0%). n Fingerstick Whole Blood: The estimated sensitivity of Alere Determine Combo for fingerstick whole blood specimens in these studies was 929/930 = 99.9% (95% confidence interval 99.4 to 100.0%). HIV-2 Sensitivity n The sensitivity of the Alere Determine Combo for detection of antibodies to HIV-2 in these studies was estimated to be 250/250 = 100% (95 % C.I. 98.5 100). Specificity n Serum: The overall specificity of Alere Determine Combo using serum specimens in these studies was estimated to be 1680/1687 = 99.6% (95% confidence interval 99.2 to 99.8%). n Plasma: The overall specificity of Alere Determine Combo using plasma specimens in these studies was estimated to be 1678/1683 = 99.7% (95% confidence interval 99.2 to 99.8%). n Venous Whole Blood: The overall specificity of Alere Determine Combo using venous whole blood specimens in these studies was estimated to be 1681/1686 = 99.7% (95% confidence interval 99.3 to 99.9%). n Fingerstick Whole Blood: The overall specificity of Alere Determine Combo using fingerstick whole blood specimens in these studies was estimated to be 1335/1337 = 99.8% (95% confidence interval 99.5 to 99.9%). Sensitivity was determined by testing specimens from individuals at high risk for HIV infection and individuals known to be infected with HIV. Specificity was determined by testing specimens from individuals at high risk for HIV infection and from individuals at low risk for HIV infection. *(95% CI = 99.6% - 100%)

Alere Determine HIV-1/2 Ag/Ab Combo 10 Alere Determine HIV 1/2 Ag/Ab Combo: HIV-1 Preclinical Data 15 HIV-1 p24 Antigen Detection in Culture Supernatants 15 Forty-three (43) HIV-1 culture supernatants, representing HIV-1 group M subtypes A, C, D, CRF01_AE, F, G, CRF02_AG, G/H, H, J, K, and G/A, and HIV-1 group O and group N, were tested on Alere Determine HIV-1/2 Ag/Ab Combo. Culture supernatants were diluted in HIV negative human serum before testing. Of the 43 culture supernatants, 40 tested positive for HIV-1 p24 antigen at a 1:27 dilution. One HIV-1 group O, one HIV-1 subtype H, and one HIV-1 subtype F specimen tested negative for HIV-1 p24 antigen (see Table 1). Results from this study demonstrated that Alere Determine HIV-1/2 Ag/Ab Combo can detect HIV-1 p24 antigen from subtype B and from non-b subtypes, and from HIV-1 group O and group N. Table 1: Detection of HIV-1 p24 Antigen from Group M Subtype Viral Isolate Culture Supernatants using Alere Determine HIV-1/2 Ag/Ab Combo Type and Subtype* Positive / Specimens Tested HIV-1 A 4/4 HIV-1 B 4/4 HIV-1 C 4/4 HIV-1 D 4/4 HIV-1 CRF01_AE 5/5 HIV-1 F 2/3 HIV-1 G 2/2 HIV-1 CRF02_AG 4/4 HIV-1 G/H 1/1 HIV-1 H 2/3 HIV-1 J 2/2 HIV-1 K 1/1 HIV-1 G/A 1/1 HIV-1 group O 3/4 HIV-1 group N 1/1 Total 40/43 *Culture supernatant from various isolates was spiked into negative human serum for testing

Alere Determine HIV-1/2 Ag/Ab Combo 11 Published Studies Data taken from Masciotra, S. et al. Performance of the Alere Determine HIV-1/2 Ag/Ab Combo Rapid Test with specimens from HIV-1 seroconverters from the US and HIV-2 infected individuals from Ivory Coast. J Clin Virol 2013 A CDC study by Masciotra was the first to evaluate the Alere Determine HIV 1/2 Ag/Ab Combo with samples collected in early stage infection. 16 The key findings included: n Alere Determine Combo is reactive 15.5 days before Western Blot, which is between 3rd & 4th generation lab IAs. Results from this study clearly define the value of the Alere Determine Combo and differentiate it from other available HIV tests. This includes not only comparisons to other rapid tests, but also to current laboratory tests such as the 4th generation Ag/Ab lab assay. n The Ab component of Alere Determine Combo detects HIV 7 days before Western Blot, sooner than some lateral flow tests and similar to flow-through RTs. n The data shows that Alere Determine Combo Ab component performs as well or better than Ab-only rapid tests currently in use in the US market. n The Ag component confers an advantage over other rapid tests in detecting early HIV infection.

Alere Determine HIV-1/2 Ag/Ab Combo 12 Number of Early HIV Infection Samples Identified (confirmed by NAAT) Data taken from Patel et al. Rapid HIV screening: Missed opportunities for HIV diagnosis and prevention JCV May 2012 This population study from the CDC 17 evaluated the sensitivity of rapid and 4th-generation tests for the detection of recent HIV infection. They screened approximately 100K samples of which 62 met the criteria of early HIV infection (based on positive NAAT and indeterminate or negative Ab). Of the 62 cases there was sufficient quantity to test 33 samples. Five FDA approved rapid tests demonstrated 22-33% sensitivity. The third-generation lab based assay demonstrated 57% sensitivity. The Alere Determine Combo identified 25 specimens (76%) of which 17 specimens were Ag-only positive providing diagnosis of acute infection. These findings suggest that current FDA-approved rapid tests fall short in detecting early and acute HIV infections and therefore miss the opportunity to interrupt onward HIV transmission especially when using these tests in high-risk populations. Of the 62 specimens, 33 (53%) had sufficient quantity for further testing. Because testing was done sequentially, the amount of specimen available for testing was exhausted in a few instances. n Fourth generation testing with Architect HIV Ag/Ab Combo was positive in 29 of 33 (88%) specimens. n Alere Determine Combo identified antibody or antigen (Ag) in 25 of 33 (76%) specimens n 8 specimens tested Ab positive/ag negative. n 13 specimens tested Ab negative/ag positive. n 4 specimens tested Ab positive/ag positive. n Genetic Systems HIV 1/2 + O correctly detected antibody in 19 of 33 (58%) specimens n Multispot HIV-1/HIV-2 detected 11 of 33 specimens (33%) n Clearview Complete HIV1/2 detected 8 of 27 specimens (30%) n Unigold Recombigen detected 8 of 33 specimens (24%) n Clearview HIV 1/2 STAT-PAK detected 7 of 31 specimens (23%) n OraQuick Advance Rapid HIV-1/2 detected 7 of 32 specimens (22%)

Alere Determine HIV-1/2 Ag/Ab Combo 13 Summary of device description and biological principle of the procedure Alere Determine HIV 1/2 Ag/Ab Combo is an immunochromatographic test for the simultaneous and separate qualitative detection of free HIV-1 p24 antigen and antibodies to HIV-1 and HIV-2. The test device is a laminated strip that consists of a Sample Pad containing monoclonal biotinylated anti-hiv-1 p24 antibody, a Conjugate Pad containing monoclonal anti-hiv-1 p24 antibody-colloidal selenium and HIV-1 and HIV-2 recombinant antigen-colloidal selenium, and a nitrocellulose membrane with an immobilized mixture of recombinant and synthetic peptide HIV-1 and HIV-2 antigens in the Lower Test Area, immobilized streptavidin in the Upper Test Area, and an immobilized mixture of anti-hiv-1 antibodies, HIV-1/2 antigens, and HIV-1 p24 recombinant antigen and anti-hiv-1 p24 monoclonal antibody in the Control Area. Lot Number Patient Identification Area Product Name Control Line Area Antigen Results Area Antibody Results Area Sampling Area

Alere Determine HIV-1/2 Ag/Ab Combo 14 Product Information Information Type Product Detail FOR ORDERING PLEASE CONTACT: Ordering: 1.877.441.7440 Method Time to results Storage conditions Lateral Flow 20 minutes 2-30 C (36-86 F) Test shelf life 15 months Sample type Fingerstick or Venous Whole blood/serum/plasma FOR TECHNICAL ASSISTANCE PLEASE CONTACT: Technical Service: 1.877.866.9335 Technical Service e-mail: ts.scr@alere.com Ordering Information Product Alere Determine HIV-1/2 Ag/Ab Combo (25 Tests) Order Number 7D2648 Alere Determine HIV-1/2 Ag/Ab Combo (100 Tests) Alere Determine HIV-1/2 Ag/Ab Combo External Controls Fingerstick Sample Collection Kit 7D2649* 7D2628 2604US199 * from date of manufacture *Not available until 2014

References 1. CDC. Monitoring selected national HIV prevention and care objectives by using HIV surveillance data United States and 6 U.S. dependent areas 2010. HIV Surveillance Supplemental Report 2012; 17(No. 3, part A). 2. http://www.arnothealth.org/news/general/new-hiv-testing-law-4152011 3. Cohen S.M. et al. The Detection of Acute HIV-1 Infection. Journal of Infectious Disease, 2010 202; s270-277 4. Fiebig E.W. et al. Dynamics of HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection. AIDS 2003, 17:1871 1879 5. Pinkerton, S. D, Probability of HIV Transmission During Acute Infection in Rakai, Uganda, AIDS Behav. 2008 September; 12(5): 677 684. doi:10.1007/s10461-007-9329-1. 6. http://www.webmd.com/hiv-aids/news/20131030/early-hiv-treatment-awin-win-researchersreport 7. Jain V. et. al. Antiretroviral Therapy Initiated Within 6 Months of HIV Infection Is Associated With Lower T-Cell Activation and Smaller HIV Reservoir Size, 1202 Journal of Infectious Disease, 2013 208:1-10 8. http://www.uspreventiveservicestaskforce.org/uspstf/uspshivi.htm 10. https://www.bcnepa.com/providers/qualitymanagement/practice_ guidelines/adult_hlth_main.pdf 11. Lubinski, C et al. HIV Policy: The Path Forward A Joint Position Paper of the HIV Medicine Association of the Infectious Diseases Society of America and the American College of Physicians, Clinical Infectious Diseases 2009; 48:1335 44 12. http://www.aafp.org/patient-care/clinical-recommendations/all/hiv.html 13. Cornett J. Laboratory Diagnosis of HIV in Adults: A Review of Current Methods. CID 2013: 57 14. http://www.cdc.gov/hiv/pdf/testing_advantages&disadvantages.pdf 15. Alere Determine HIV-1/2 Ag/Ab Combo Package Insert. 027332530 Rev: 04 2013/09 16. Masciotra, S. et al. Performance of the Alere Determine HIV-1/2 Ag/ Ab Combo Rapid Test with specimens from HIV-1 seroconverters from the US and HIV-2 infected individuals from Ivory Coast. J Clin Virol 2013: Published online 05 August 2013. DOI: 10.1016/j.jcv.2013.07.002 17. Patel P. et al. Rapid HIV screening: Missed opportunities for HIV diagnosis and prevention Journal of Clinical Virology May 2012 9. http://www.cdc.gov/mmwr/preview/mmwrhtml/rr5514a1.htm Alere Determine HIV 1/2 Ag/Ab Combo Summary of Clinical Evidence For more information, please visit alerehiv.com/us 2014 Alere. All rights reserved. The Alere Logo, Alere and Determine are trademarks of the Alere groups of companies. All other trademarks referenced are trademarks of their respective owners. 12000964E-01 04/14 TAP HERE TO RETURN TO THE COVER