QuantiFERON® Blood Test for the Detection of Latent Tuberculosis Infection

Active and Latent Tuberculosis Infection TB-related morbidity and mortality The TB pandemic – global emergency (1) Active TB disease in 2013 9 million people developed TB disease 1.5 million people died Latent TB infection (LTBI) in 2013 2 billion infected with M. tuberculosis 10% chance of developing active, contagious TB disease in their lifetimes Screening and treatment (2) 2014 WHO guidelines, part of Broad strategy to “End TB” by 2035: Identify and treat LTBI for upper-middle and high income countries with TB incidence <100/100k population Screen and treat for LTBI in most at-risk populations for progression to active TB Reduce TB deaths by 95%; cut active TB cases by 90% As active TB rates decrease, LTBI diagnosis & preventive treatment grows in importance 1. WHO. Global tuberculosis report 2014. http://apps.who.int/iris/bitstream/10665/137094/1/9789241564809_eng.pdf?ua=1, Dec 1, 2014. 2. WHO. Guidelines on the management of latent tuberculosis infection. 2014 http://apps.who.int/iris/bitstream/10665/136471/1/9789241548908_eng.pdf?ua=1, Dec 1, 2014. Internal Training Use Only - January 2017

Outcomes of Transmission of TB Infection TB infection and disease can re-occur, even after treatment. This can happen as a result of the patient contracting a different strain or because bacteria remain that the individual’s immune system can’t keep under control. If the individual assumes an unhealthy lifestyle or becomes immunocompromised, the bacteria can potentially progress to disease again. Outcomes depend on immune status and represent a spectrum of TB infection Internal Training Use Only - January 2017

Why is it important to detect LTBI? Internal Training Use Only - January 2017

Is it efficient enough to only consider active TB? Ultimate goal of WHO is to eradicate active TB by 2050 This will not be achievable if only the active TB cases are considered WHO Global Tuberculosis 2016 Report clearly states that treatment of LTBI is key for prevention of new infections Adapted from Abu-Raddad et al. Epidemiological benefits of more-effective tuberculosis vaccines, drugs, and diagnostics. PNAS. 2009; 106; 13980-5. Internal Training Use Only - January 2017

From Latent TB infection to Active TB: Risk Factors Risk factors to develop active TB form Latent TB infection Risk Factors Estimation of relative risk* AIDS 110 - 170 Well controlled HIV infection 50 - 110 Solid Organ Transplantation 20 - 74 Chronic Hemodialysis 10 - 25 Head and neck cancer 16 Recent tuberculosis infection (<2 years) 15 Systemic prolonged corticosteroids therapy 4.9 Anti-TNF  treatment 1.5 - 4 Diabetes 2 - 3.6 Malnutrition (body mass index < 20 kg/m2) 2 - 3 Passive smoking * Compared to a population without any risk factor HIV: Human Immunodeficiency Virus TNF: Tumor Necrosis Factor Leroy H. et al in La revue du praticien vol. 62, avril 2012 p 484, adapted from Landry J, Menzies D. Preventive chemotherapy. Where has it got us? Where to go next? Int J Tuberc Lung Dis 2008;12:1352-64. Internal Training Use Only - January 2017

How to diagnose LTBI? Internal Training Use Only - January 2017

Principle of immunological tests (IGRA and TST) Immune response: from latent to active Tuberculosis Active TB Latent TB Infection Cell Mediated Immunity Immune Response Mycobacterial Load Antibody Infection Active Disease TIME T0 (0 to 3 weeks) T3 Internal Training Use Only - January 2017

How to diagnose Latent TB Infection? Principle of the immune reaction Sources: Andersen P et al, Lancet 2000 Internal Training Use Only - January 2017

Specificity of QuantiFERON versus TST Mycobacterium tuberculosis complex ESAT-6 CFP-10 IDR Environmental Mycobacteria M. tuberculosis + M. Abcessus - M. africanum M. avium M. bovis M. branderi M. celatum BCG strains M. chelonae Gothenberg M. fortuitum Moreau M. gordonii Tice M. intracellulare Tokyo M. kansasii Danish M. malmoense Glaxo M. marinum Montréal M. oenavense Pasteur M. scrofulaceum M. smegmatis M. szulgai M. terra M. vaccae M. xenopi

Sampling tubes of QuantiFERON TB Gold Plus NIL Tube Negative control Allows adjustment for background noise. TB1 ANTIGEN Tube Includes Mycobacterium tuberculosis specific antigens ESAT-6 and CFP-10 Peptides recognized by MHC Class II to detect CD4 response. TB2 ANTIGEN Tube Peptides recognized by MHC Class I and II to detect CD4 and CD8 combined response. Mitogen Tube Positive control Includes PHA and allows to check the functionality of the immune system Objectives: To identify individuals with weakened immune system To validate specimen handling conditions Internal Training Use Only - January 2017

QuantiFERON-TB Gold Plus Protocol Step 1: Whole Blood incubation IFN- is stable at 2-8°C for at least 4 weeks Nil Control ESAT-6 CFP-10 Mitogen Control 2) 15 minutes Centrifugation 1) 1mL of whole blood (x4) and incubation at +37ºC for 16-24 h. Step 2: INF- ELISA testing 3) Add plasma and conjugate. Incubation 2H at room temperature 4) Wash then add Substrate. OD reading after 30 min. 5) Calculation and results printing Internal Training Use Only - January 2017

Results: Cut-off at 0.35 IU/mL Internal Training Use Only - January 2017

Results interpretation Interpretation of QuantiFERON results for LTBI screening Negative QFT Latent TB infection very unlikely A negative result allows to eliminate LTBI with a probability close to 100% (NPV of 99.7%*) Positive QFT Latent TB infection very likely In addition to clinical examination and anamnesis, a positive result allows to orient diagnosis toward a recent or old Latent TB infection (Specificity of 99.2%*) An indeterminate QFT result means that the patient’s immune system is weakened (immunocompromised). This kind of result is very informative and must orient toward a specific patient management. An indeterminate result can also be the consequence of sample mishandling which need to be verify before interpretation. Internal Training Use Only - January 2017

Benefits of QuantiFERON versus TST Advantages of QuantiFERON Antigens used in the test Tuberculin: more than 200 antigens ESAT6, CFP10 Specific to M. tuberculosis No cross-reactivity with BCG Patient Management Day 1: Injection Day 3: Reading Only one sampling Only one visit No loss of patients Type de test In vivo testing Operator dependent In vitro testing Single blood sampling Standardized reading Quality controls included No booster effect Reproducibility Accuracy Possible automation Controls None Internal negative and positive controls ( Nil, Mitogen) QFT Control Panel Avoid false negative results Certify the quality of the results Positive threshold Different thresholds Unique threshold defined at 0.35 IU/mL Better precision Specificity of 99.2% Sources: 1 – Diel et al, Performance of IGRAs and the TST: A new and up-to-date Meta-Analysis, Chest 2009 2 – Cheallaigh 2013 3 – Hsia 2012 Internal Training Use Only - January 2017

Which medical reasons remain for still using TST ? Internal Training Use Only - January 2017