OnSite Dengue Rapid Test

Overview Dengue: Global impact and diagnostic needs CTK Biotech: The complete solution to dengue diagnosis CTK product line: Technical details Summary and suggested literature

WHO: The global burden of disease 2004 update Dengue is a global health concern endemic to tropical and sub-tropical areas including Africa, Asia and the Americas. 2.5 billion people (40% of the world population) are at risk of infection. Annual Statistics 50-100 million infections reported annually 22 thousand deaths (mainly children) 2012 marked the spread to Europe with the first sustained transmission of dengue fever since the 1920’s Brazil and Mexico make up 14% percent of cases WHO: The global burden of disease 2004 update

WHO: The global burden of disease 2004 update Southeast Asia bears a large portion of the global burden with Thailand, Indonesia and India already experiencing fatal outbreaks as peak season approaches. Estimated 70% of world’s serious dengue cases in 2012 were in Asia 2013 figures reported since January are on pace for a record setting Dengue season WHO: The global burden of disease 2004 update

Two of the main reasons for the increase in reported cases, global spread of the virus and rise in the severity of cases reported are: Environmental factors and urbanization of areas conducive to mosquito reproduction  increased risk of infection because of increased exposure to transmitting mosquitoes All 4 serotypes have been identified in recent outbreaks; historically only 1 or 2 subtypes were identified in a particular outbreak  increase in secondary infections which are more severe than primary infections

Four serotypes of dengue virus exist in the endemic areas of the world Four serotypes of dengue virus exist in the endemic areas of the world. The virus is arthropod-borne, transmitted by mosquitos. Human Aedes aegypti Gunzman et al. Nature review 2010; Center for Disease Control (CDC) Dengue is a member of the flavi virus family Four serotypes exist (DENV 1, 2, 3, 4) The virus is transmitted by tiger mosquitos (Aedes spp.) Screening and limiting the number of mosquito bites is critical to managing the spread of the disease.

Primary infection results in mild symptoms and confers immunity to subsequent infection by the same serotype. Secondary infections are more severe leading to Dengue hemorrhagic fever. recovery 1° infection 2° infection Immunity to 1 serotype DENV serotype DENV serotype Any of the 4 DENV serotypes DENV serotype different from 1° infection Disease Disease Symptoms: Mild (Dengue Fever DF) Similar to other infection (Flu or malaria) Confers immunity against 1 DENV serotype Symptoms: Life-threatening (Dengue hemorrhagic fever DHV and Dengue shock syndrome DSS) Antibody enhanced infection Secondary infection is the most significant individual risk factor for severity of disease. No vaccine is available & treatment is limited to replenishing fluids and electrolytes. Therefore, early diagnosis that can distinguish between primary and secondary infections is critical to disease management allowing faster treatment and limit mortality and morbidity rates.

Dengue infections can be detected at all stages of disease Dengue infections can be detected at all stages of disease. Different Ab responses during primary and secondary infections enable differentiation between the two types of infection. Primary dengue infection Secondary dengue infection Disease onset Disease onset virus virus IgG IgG IgM IgM 0 1 2 3 4 5 6 7 8 9 10 30 .. 60...>90 0 1 2 3 4 5 6 7 8 9 10 30 .. 60…>90 days days viral antigen (Day 1-5) viral antigen (Day 1-5) Anti-dengue IgM (day 3-30) Anti-dengue IgM low and varying Anti-dengue IgG (from day 7-11 on) Anti-dengue IgG (from day 3 on) Adapted from Sa-Ngasanget al. Epidemiol. Infect. (2006), Gunzman et al. Int. Jour. Inf. disease 2004

Diagnostic needs to manage dengue Detection of dengue as early as possible to initiate medical care Discrimination of dengue from other infections with similar symptoms such as Leptospira, rubella Detection of all 4 DENV serotypes (DENV 1, 2, 3, 4) Detection during all phases of a dengue infection Differentiation between primary and secondary dengue infections

Training requirements Application in the field OnSite Rapid Tests offer many advantages compared to other diagnostic methods: minimal resource requirements, faster time to results, use in unlimited test settings Virus isolation RT-PCR ELISA OnSite Rapid Test Sensitivity high High-medium Specificity Equipment required medium low Training requirements Application in the field no yes Time days 6-12h 60-75min 15-25min Direct and indirect methods 1. Direct: Detection of virus (virus isolation, detection of viral RNA by RT-PCR, detection of viral antigens) 2. Indirect: Serology to detect of anti dengue antibodies Opportunity Resources

CTK Biotech offers a complete line for the diagnosis of infection with dengue virus at various disease stages. OnSite Rapid Tests Dengue IgG/IgM Combo Rapid Test (R0061C) - detection and differentiation of anti-dengue IgG, IgM Duo Dengue Ag-IgG/IgM Rapid Test (R0062C) - detection and differentiation viral antigens and anti-dengue IgG, IgM Dengue Ag Rapid Test (R0063C) - detection of DENV antigens at early stage of infection (1-5 days) Positivia External Controls Dengue Ag Rapid Test Control Kit (C0063)

OnSite Dengue Ag Rapid Test R0063C

OnSite Dengue Ag Rapid Test (R0063C) Test Overview Intended use Qualitatively detects dengue Ag Specimen type Serum/Plasma/Whole Blood Reading time 25 minutes Performance Relative Sensitivity: 95.6%, Relative Specificity: 95.5% Shelf life 18 months Package size 30 tests

OnSite Dengue Ag Rapid Test (R0063C) Test procedure – as easy as 1, 2, 3 Step 1: Addition of specimen Step 2: Addition of sample diluent Step 3: Read Result Result 1 drop serum/plasma 1 drop sample diluent 25 minutes 2 drops whole blood 1 drop sample diluent

OnSite Dengue Ag Rapid Test (R0063C) Interpretation of Results Negative Positive Invalid A positive Dengue Ag test result indicates early stage, current infection. Treatment should be started immediately to minimize severity of symptoms.

OnSite Dengue Ag Rapid Test OnSite Dengue Ag Rapid Test (R0063C) Performance Data OnSite Dengue Ag Rapid Test Dengue Ag EIA Test Positive Negative Total 66 3 69 2 43 45 68 46 114 Relative Sensitivity: 95.6% Relative Specificity: 95.5% Overall Agreement: 95.6%

OnSite Dengue Ag Rapid Test (R0063C) External evaluations performed by independent reference labs in different geographic regions showed favorable results. Two leading reference labs in Ecuador and one in Sri Lanka recently performed independent evaluations of the OnSite Dengue Ag Rapid Test. Key findings in the reports: 100% agreement with the reference test used for evaluation No cross-reactivity with 7 other infectious disease samples Highly sensitive and specific

OnSite Dengue IgG/IgM Combo Rapid Test R0061C

OnSite Dengue IgG/IgM Combo Rapid Test (R0061C) Intended Use Qualitatively detects & differentiates IgG & IgM to DENV 1, 2, 3, 4 Antigen used Proprietary recombinant DENV 1, 2, 3 & 4 Ag Specimen type Serum/Plasma/Whole blood Reading time 25 minutes IgM Performance Relative Sensitivity: 96.9%; Relative Specificity: 98.9% IgG Performance Relative Sensitivity: 97.3% Relative Specificity: 99.3% Shelf life 18 months Package size 30 tests

OnSite Dengue IgG/IgM Combo Rapid Test (R0061C) Test procedure – as easy as 1, 2, 3 Step 1: Addition of specimen Step 2: Addition of sample diluent Step 3: Read Result Result 5 µL serum/plasma 25 minutes 3 drops sample diluent 5 µL whole blood

OnSite Dengue IgG/IgM Combo Rapid Test (R0061C) Interpretation of Results IgM Positive IgG Positive IgM, IgG Positive Negative Invalid Explain how each result relates back to the various stages of the disease

OnSite Dengue IgG/IgM Combo Rapid Test OnSite Dengue IgG/IgM Combo Rapid Test (R0061C) Performance Data for IgM test OnSite Dengue IgG/IgM Combo Rapid Test IgM EIA Test Positive Negative Total 31 1 32 3 279 282 34 280 314 Relative Sensitivity: 96.9% Relative Specificity: 98.9% Overall Agreement: 98.7%

OnSite Dengue IgG/IgM Combo Rapid Test OnSite Dengue IgG/IgM Combo Rapid Test (R0061C) Performance Data for IgG test OnSite Dengue IgG/IgM Combo Rapid Test IgG EIA Test Positive Negative Total 36 1 37 2 287 289 38 288 326 Relative Sensitivity: 97.3% Relative Specificity: 99.3% Overall Agreement: 99.1%

OnSite Dengue IgG/IgM Combo Rapid Test (R0061C) External evaluations performed by independent reference labs in different geographic regions showed favorable results Reference labs in Ecuador and Taiwan recently performed independent evaluations of the OnSite Dengue IgG/IgM Rapid Test. Key findings in the reports: 100% agreement with the reference test used for evaluation Report from Taiwan to be published

OnSite Duo Dengue Ag-IgG/IgM Rapid Test (R0062C) Combines the dengue Ag and IgG/IgM tests in a single cassette Intended use Qualitatively detects & differentiates IgG & IgM anti-dengue virus and dengue Ag Antigen used Proprietary recombinant DENV 1, 2, 3 & 4 Ag Specimen type Serum/Plasma/Whole blood Reading time 25 minutes Shelf life 18 months Package size 30 tests

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