Juzar Ali, MD, FRCP(C) FCCP has the following disclosures to make:

Interferon Gamma Release Assays (IGRA) Juzar Ali, MD, FRCP(C) FCCP June 27, 2018 TB Intensive June 26-29, 2018 San Antonio, TX EXCELLENCE EXPERTISE INNOVATION Juzar Ali, MD, FRCP(C) FCCP has the following disclosures to make: No conflict of interests No relevant financial relationships with any commercial companies pertaining to this educational activity 1

INTERFERON GAMMA RELEASE ASSAY AMIDST THE CHANGING MYCOBACTERIAL /TB LANDSCAPE Juzar Ali, MD, FRCP(C) FCCP Klein Professor of Medicine Section of Pulmonary/Critical Care & Allergy/Immunology LSU SOM & Health Sciences Center, New Orleans LSU Health & Network Clinics Director, LSUHSC-Wetmore Office of Public Health TB Clinics & Clinic for Mycobacterial Diseases, University Medical Center, New Orleans Faculty, School of Nursing, LSUHSC and School of PH & TM, Tulane University Disclosures, Introduction, Background Juzar Ali, MD, FRCP(C) FCCP Klein Professor of Clinical Medicine Section of Pulmonary/Critical Care & Allergy/Immunology LOUISIANA STATE UNIVERSITY Health Sciences Center, New Orleans LSU Health & Network Clinics ***** Director, LSUHSC-Wetmore Office of Public Health TB Clinics & Clinic for Mycobacterial Diseases,***** University Medical Center, New Orleans Faculty, School of Nursing LSUHSC and School of PH & TM TULANE UNIVERSITY Consultant to Oxford Immunotec ***** \ Consultant to Oxford Immunotec 2

Objectives TB: Brief overview Review latest guidelines Share with participants updated data on TB screening/igra test results in various subsets of population Experience with IGRA: The pros and cons Overcoming challenges Jeopardy 1 Must check for active TB Do not forget to look for Extrapulmonary TB 3

What do you do before starting treatment for latent TB/TB Infection? Remember the one definitive contraindication** Positive TST/IGRA: Suggested Traffic Light Plan* Drivin g Path A: DATA B: EVALUATE C: SCAN D: TREAT LTBI Quantify; Assess Borderline Indeterminate Discordant Results Rule Out Active TB Disease Based on Symptoms and Risk Rule out Extra-Pulmonary Disease Dx; LTB Infection Should we Offer Rx? Assess Risk Benefit Ratio and patient engagement, understanding and adherence to treatment. Document Symptoms History / Physical Detailed Review of Systems: ROS 101 ; Repeat Physical exam specially LN Exam Be aware of risk of adverse drug reactions and potential side effects Check HIV Other Immune status Chest Radiograph CT Scan if needed Correlate with Chest and other imaging if applicable Pre-Rx start : Lab Check Risk Stratify Check Source Case Culture/ Sensitivity Sputum Smear/NAAT; Induce Sputum if needed May need Direct Observed Sputum Evaluation (DOSE) Assess Background History again? Treat for TB Infection based on Risk Benefit Ratio Conclude after Full Evaluation: Proceed to Steps B-E Assess Pre-Test Probability; Treat for active TB disease f index of suspicion is high Treat for active TB disease f index of suspicion is high Monitor Side Effects; Drug-Drug Interaction; Establish Care Coordination with Primary Care Team **Designed by JA 4

To Be or Not To Be Risk, Signs, Symptoms, Micro (Smear, NAAT, Culture, Probe, Final Culture) Images Tissue Dx BUT WHAT IS NOT MENTIONED? Looks like a duck, walks like a duck, must be...but IS IT? 1000 800 600 400 200 0 Public Health Image Library: ID#5789 Ziehl-Neelsen stain BCG Does that matter? NTM - Is that important? 19 20 20 20 20 20 20 20 20 20 20 20 99 00 01 02 03 04 05 06 07 08 09 10 Number of TB 930776764784711657648652554585529569 related deaths Number of NTM 219221219215240262274226270282284278 related deaths Based on 2003 2007 Medicare data Projected 8% annual increase in prevalence Estimated 86,244 cases in 2010 at an annual cost $815 million; 87% inpatient 70% of NTM disease cases occurred in oceanic coast line & gulf states Medication cost: 76% of all total cost Modified Public Health CLASSIFICATION 0 No Exposure; Not Infected 1 Exposure; Not Infected 2 Infected; No Active Disease 3 Active Disease Pulmonary and or Extra-Pulmonary 4 Old Disease or UDA 5 Under Evaluation for Active TB 6 TB wannabes /Atypical/NTM 7 Associate Involvement 8 Exposed Contacts 5

The Hidden Reservoir of TB Smear negative cases: 13-22% of cohort can acquire disease from smear negative contacts1 Thou do DOTS but Doth do DOSE? Incomplete or Erratic Contact tracing Undocumented immigrants with prolonged symptoms with poor access to health care2 Low index of suspicion when specialists and Healthcare Workers (HCWs)* in the hospital have decreased awareness, with resultant missed or delayed diagnosis and treatment3 *12 million HCWs in US4 10 million immunocompromised patients in US5 1. Tostmann A, Kik SV, Kalisvaart NA, et al. Tuberculosis Transmission by Patients with Smear-Negative Pulmonary Tuberculosis in a Large Cohort in The Netherlands. Clin Infect Dis. 2008;47(9):1135-1142. 2. Achkar JM, Sherpa T, Cohen HW, Holzman RS. Differences in Clinical Presentation among Persons with Pulmonary Tuberculosis: A Comparison of Documented and Undocumented Foreign-born to US-born Persons. Clin Infect Dis. 2008;47(10):1277-1283. 3. Chen T-C, Lu P-L, Lin W-R, et al. Diagnosis and Treatment of Pulmonary Tuberculosis in Hospitalized Patients Are Affected by Physician Specialty and Experience: The American Journal of the Medical Sciences. 2010;340(5):367372. 4. May 2014 Occupational Profiles. Retrieved May 3 2015, from http://www.bls.gov/oes/current/oes_stru.htm 5. Redelman-Sidi G, Sepkowitz KA. IFN-gamma release assays in the diagnosis of latent tuberculosis infection among immunocompromised adults. Am J Respir Crit Care Med. 2013;188(4):422-431. WHO 2018 Updated and Consolidated Guidelines Pooled estimates of risk for active TB among household contacts stratified by age and baseline LTBI status as compared with the general population LTBI-positive at baseline Age (years) General population Follow-up < 12 months 15 Follow-up < 12 months Follow-up < 24 months No. of studies No. of studies Risk ratio No. of studies No. of studies Risk ratio - 1.0 (reference) - 1.0 (reference) - 1.0 (reference) - 1.0 (reference) 24.3 (0.73-811.0) 3 22.9 (7.7 68.6) 3 25.9 (16.9 39.7) 5 14.8 (9.8 22.3) 2 27.1 (17.5 54.1) 3 8.2 (2.3 29.4) 3 24.1 (16.9 34.4) 5 6.3 (2.9 13.7) 1 30.7 (17.5 54.1) 2 13.4 (9.5 18.8) 1 24.7 (14.2 43.0) 3 11.7 (7.6 18.0) 0 4 5 14 Regardless of baseline LTBI status Follow-up < 24 months Risk ratio Risk ratio WHO. Latent tuberculosis infection Updated and consolidated guidelines for programmatic management. 2018. 6

US Immunocompromised Population Estimated # of US Persons Living with Condition 1.2 million 1.5 million 1.1 million 320,000 49,000 2.4 million 1.0 million 0.87 million 1.0 million 120,000 Condition HIV infection Rheumatoid arthritis Inflammatory bowel disease Systemic lupus erythematosus Systemic sclerosis Spondyloarthropathies Vasculitis End-stage renal disease Hematologic malignancies Solid organ transplant candidates Total 10 million Double Jeopardy (Disease and Rx) JA Note: something s missing!! Redelman-Sidi G, Sepkowitz KA. IFN-gamma release assays in the diagnosis of latent tuberculosis infection among immunocompromised adults. Am J Respir Crit Care Med. 2013;188(4):422-431. 21C-TB-US-V9 TB Infection Flow Chart (Diagram 1) PAUCIBACILLARY DORMANT STATE POSITIVE TST/IGRA TB INFECTION (LTBI) 10% Lifetime Risk 7-10% Annual Risk* Adapted from Core Curriculum on Tuberculosis: What the Clinician Should Know, 5th ed. Atlanta, GA: US Department of Health and Human Services, CDC; 2011. with addenda and modifications by authors 7

Pathway of Pathogenesis in Non-Immune Compromised Patients with a Connection and yet a disconnect between Infection, Immune response and Disease Inhalation Macrophage phagocytosis Shedding and macrophage turnover Alveolar Dendritic Cell migration to regional LN Release of MTB Antigens ESAT -6; CFP-10 with CD4 /CD8 interaction Differentiate into INF-G, TNF TH1 or cytotoxic Tc1 cells respectively IL 17 21, 22 DTH Reaction- positive TST with Intragranuloma necrosis called Ghon Complex Ghon Complex is not necessarily limited to lung Post Primary IL- 4 IL -13 TH 2 response with central caseation Thus Granulomas are Dynamic Lesions with Continuous Turnover and Variable Bacillary Activity Variable Bacillary Activity or Disease Activity or Dormancy ** NO CLINICAL ACTIVITY and SIGNS HENCE CALLED LATENT TB ** LTBI: LATENT TB INFECTION OR LASTING TB IMMUNITY Mack U, Migliori GB, Sester M, et al. LTBI: latent tuberculosis infection or lasting immune responses to M. tuberculosis? A TBNET consensus statement. Eur Respir J. 2009;33(5):956-973. doi:10.1183/09031936.00120908 Immune Response1 TB Skin Testing In vivo measurement of cell-mediated immunity to mycobacterium antigens in the form of a delayed-type hypersensitivity reaction Previous exposure to mycobacterium results in production of sensitized lymphocytes Sensitized lymphocytes secrete cytokines to attract neutrophils, memory CD4 T cells, CD8 T cells, which cause induration and erythema Induration measured 48 72 hours post implantation Sensitivity of the tuberculin skin test is limited in immunocompromised individuals Specificity is limited because of cross-reactivity due to prior infection with environmental mycobacteria or BCG vaccination Interferon-Gamma Release Assay In vitro measurement of INF-gamma released by effector T cells responding to specific TB antigen stimulation, such as ESAT-6 and CFP10 Previous exposure to M. tuberculosis results in production of sensitized T cells Sensitized T cells secrete IFN- (cytokine) when they reencounter specific M. tuberculosis antigens IFN- secreted by effector T cells measured ~20 hours poststimulation Antigens used in IGRAs (ESAT-6, CFP10, TB7.7) are not present M. bovis BCG and in most environmental mycobacteria 1. Mack U, Migliori GB, Sester M, et al. Eur Respir J. 2009;33(5):956-973. 8

Testing Methodologies for Immune Response Tuberculin skin testing (TST) Interferongamma release assay (IGRA) Mantoux test, Purified Protein Derivative (PPD) T-SPOT.TB test QuantiFERON -TB Gold/Plus Advantages & Disadvantages T-SPOT is a registered trademark of Oxford Immunotec, Ltd. QuantiFERON is a registered trademark of the QIAGEN Group. ATS/IDSA/CID/CDC Guidelines (2017) IGRA or TST General 5 years who are likely to be infected with Mtb, have a high risk of progression to disease and has been determined that testing for LTBI is warranted IGRA recommended 5 years old meeting the over TST following criteria: TST recommended over IGRA Likely to be infected with Mtb Have low or intermediate risk of disease progression It has been decided that testing for LTBI is warranted Either have a history of BCG vaccination or unlikely to return to have their TST read 5 years old who are likely to be infected with Mtb, have a low or intermediate risk of disease progression and it has been decided that testing for LTBI is warranted Periodic screening of persons who might have occupational exposure, such as surveillance programs for healthcare workers Healthy children <5 years old, where testing for LTBI is warranted *There is limited data regarding IGRA performance in children <5 years old maybe coming soon Lewinsohn DM, Leonard MK, LoBue PA, et al. Official American Thoracic Society/Infectious Diseases Society of America/Centers for Disease Control and Prevention Clinical Practice Guidelines: Diagnosis of Tuberculosis in Adults and Children. Clinical Infectious Diseases. 2017;64(2):e1-e33. 9

CDC Guidelines (2010) Testing with both IGRA and TST may be considered when initial test result (by IGRA or TST) is: Negative and risks for infection, progression, and poor outcome are increased and clinical suspicion exists for TB disease and confirmation of M. tuberculosis infection is desired Positive and additional evidence of infection is required to encourage compliance Indeterminate, Invalid, or Borderline** repeating IGRA or performing the TST might be useful in a healthy person who has a low risk for both infection and progression Mazurek GH, Jereb J, Vernon A, et al. Updated guidelines for using Interferon Gamma Release Assays to detect Mycobacterium tuberculosis infection - United States, 2010. MMWR Recomm Rep. 2010;59(RR-5):1-25. USA Centers for Disease Control and Prevention Guidelines: Diagnosis of Tuberculosis in Adults and Children Group Likely to be infected High Risk of Progression (TST 5mM Testing Strategy Children 5 years of age Preferred: TST Acceptable: IGRA or TST Consider dual testing where a positive result from either would be considered positive1 Likely to be infected Low to Intermediate Risk of Progression (TST 10mM) Unlikely to be infected (TST > 15mM) Considerations Adults Acceptable: IGRA OR TST Consider dual testing where a positive result from either result would be considered positive Preferred: IGRA where available Acceptable: IGRA or TST Testing for LTBI is not recommended If necessary: Preferred: IGRA where available Acceptable: Either IGRA or TST For serial testing: Acceptable: Either IGRA or TST Prevalence of BCG vaccination Expertise of staff and/or laboratory Test availability Patient perceptions Staff perceptions Programmatic concerns Consider repeat or dual testing where a negative result from either would be considered negative2 Lewinsohn DM, Leonard MK, LoBue PA, et al. Official American Thoracic Society/Infectious Diseases Society of America/Centers for Disease Control and Prevention Clinical Practice Guidelines: Diagnosis of Tuberculosis in Adults and Children. Clinical Infectious Diseases. 2017;64(2):e1-e33. ME-OI-0001 v1 10

Screening for Latent Tuberculosis Infection in Adults US Preventive Services Task Force Recommendation Statement (2016) US Preventive Services Task Force (USPSTF) recommends screening for LTBI in populations at increased risk screening for LTBI in persons at increased risk for infection provides moderate net benefit no direct evidence on the harms of screening for LTBI treatment of LTBI provides moderate health benefit in preventing progression to active disease harms of treatment of LTBI with CDC-recommended regimens is small The testing with IGRAs may be preferable for persons who have received a BCG vaccination or persons who may be unlikely to return for TST interpretation primary harm of treatment is hepatotoxicity The USPSTF found no evidence on the optimal frequency of screening for LTBI screening tests include TSTs and IGRAs; both are moderately sensitive and highly specific for the detection of LTBI Test Sensitivity T-SPOT.TB 90% (n = Specificity QFT -GIT 80% (n = 2321) 97% (n = 2053) TST 79% (n = 988) 97% (n = 9651) 984) 95% (n = 1810) Meta-analysis conducted by USPSTF Nahid P, Dorman SE, Alipanah N, et al. Official American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America Clinical Practice Guidelines: Treatment of Drug-Susceptible Tuberculosis. Clinical Infectious Diseases. 2016;63(7):e147-e195. US Preventive Services Task Force, Bibbins-Domingo K, Grossman DC, et al. Screening for Latent Tuberculosis Infection in Adults: US Preventive Services Task Force Recommendation Statement. JAMA. 2016;316(9):962. QFT is a registered trademark of the Qiagen Group. ATS/IDSA/CDC Guidelines for HIV (2017) Individuals who have a prior latent infection with Mtb (not treated) and then acquire HIV infection will develop TB disease at an approximate rate of 5% 10% per year (in the absence of effective HIV treatment) False-negative reactions from TST occur more frequently in persons with clinical conditions associated with immunosuppression such as HIV Treatment of LTBI has been demonstrated to reduce the incidence of TB disease include persons with HIV infection Individuals at high risk of progression to TB include those with HIV infection Sensitivity of IGRAs (QFT-IT and T-SPOT.TB) for detecting LTBI in individuals with HIV infection has been estimated to be from 65% to 100%, while the sensitivity of TST is only 43% Lewinsohn DM, Leonard MK, LoBue PA, et al. Official American Thoracic Society/Infectious Diseases Society of America/Centers for Disease Control and Prevention Clinical Practice Guidelines: Diagnosis of Tuberculosis in Adults and Children. Clinical Infectious Diseases. 2017;64(2):e1-e33. 11

American College of Rheumatology Recommendations (2015) Recommendations for TB screening in patients being considered for or receiving biologics or tofacitinib All patients being considered for biologics or tofacitinib, regardless of the presence of risk factors, should be screened with an IGRA or the TST JA: Consider prevalence of NTM in cohort) IGRA recommended over the TST in patients who have previously received a BCG vaccination, due to the high false-positive test rates for TST Repeat TST or IGRA could be considered after an initial negative test result in immunosuppressed RA patients with risk factors for LTBI Singh JA, Saag KG, Bridges SL, et al. 2015 American College of Rheumatology Guideline for the Treatment of Rheumatoid Arthritis: ACR RA Treatment Recommendations. Arthritis Care & Research. 2016;68(1):1-25. Issues Affecting the Use of TST Limitations Need for trained personnel to administer the intradermal injection and also interpret the test Inter- and intra-reader variability in interpretation Need for a return visit to have the test read False-positive results due to cross-reactivity of antigens within the PPD to both BCG and nontuberculous mycobacteria False-negative results due to infections and other factors, rare adverse effects, and complicated interpretation False-Positive Results Repeat TST can restore reactivity in persons whose TST reactivity has decreased over time False-Negative Results In persons with clinical conditions associated with Cross-reactivity with BCG vaccine immunosuppression or overwhelming illness Cross-reactivity for non-tuberculous mycobacteria After recent viral and bacterial infections (NTM) is increased for persons living in areas In association with treatment with immunosuppressive where nontuberculous mycobacteria is common drugs Errors in TST placement or reading Lewinsohn DM, Leonard MK, LoBue PA, et al. Official American Thoracic Society/Infectious Diseases Society of America/Centers for Disease Control and Prevention Clinical Practice Guidelines: Diagnosis of Tuberculosis in Adults and Children. Clin Infect Dis. December 2016:ciw694. 12

Diagnosis Acceptance in TB Contacts 641 tested with IGRA and 650 tested with TST 100% Contacts tested with IGRA were more likely to complete evaluation (64% vs 56%) 80% Infected contacts started (89% vs 72%) and completed (70% vs 53%) LTBI treatment more often in group tested with IGRA 20% 60% 89% 72% 70% 53% 40% 0% Started LTBI Positive IGRA results, but not positive TST results, correlated with the intensity, proximity, and duration of TB exposure TST Completed LTBI IGRA Grinsdale JA, Ho CS, Banouvong H, Kawamura LM. Programmatic impact of using QuantiFERON(R)-TB Gold in routine contact investigation activities. Int J Tuberc Lung Dis. 2011;15(12):1614-1620. TB-Specific Antigens Used in IGRAs Produce measureable immunologic responses in TB-infected persons In vitro measurement Are present in of INF-gamma released by effector T cells responding to specific TB antigen stimulation, such as ESAT-6 and CFP10 Mycobacterium tuberculosis complex organisms, including M. tuberculosis, M. bovis, M. africanum, M. microti, and M. canetti M. kansasii, M. szulgai, and M. marinum (nontuberculous mycobacteria) Are absent in and do not cross-react with M. bovis BCG sub strains M. avium and most other nontuberculous mycobacteria JA note: except KMSFG T-SPOT.TB Package Insert PI-TB-US-V5. Oxford Immunotec Ltd. Abingdon, UK. March 2015. QuantiFERON-TB Gold (QFT) ELISA Package Insert, 1075116 Rev. 02. Cellestis, Inc. Valencia, CA., April 2015. 13

Commercially Available IGRAs Variables QuantiFERON-TB Gold QuantiFERON-TB Gold-Plus T-SPOT.TB Test Technology ELISA ELISA ELISPOT Test Substrate Whole blood Whole blood Peripheral blood mononuclear cells Sample Collection 3 specialized tubes 4 specialized tubes 1 standard tube Adjusted Cell Count No No Yes Cell Wash No No Yes Cell Targets CD4 CD4, CD8 CD4, CD8 Sample Stability 16 hours 16 hours 32 hours Diagnostic Performance Package Insert: sensitivity, 88.7%; specificity, 99.2% Package Insert: sensitivity and specificity cannot be practically evaluated Package Insert: sensitivity, 95.6%; specificity, 97.1% Readout Interferon-gamma concentration (international units per ml) Interferon-gamma concentration (international units per ml) Individual spots representing captured interferon-gamma Result Interpretation Positive, Negative, Indeterminate Positive, Negative, Indeterminate Positive, Borderline, Negative, Invalid Includes FDA-approved borderline category Accessibility In-house lab or available through reference laboratories In-house lab or available through reference laboratories In-house lab or available through Oxford Diagnostic Laboratories Oxford Immunotec. T-SPOT.TB Package Insert PI-TB-IVD-UK-V3. Abingdon, UK. March 2016. QuantiFERON-TB Gold (QFT) ELISA Package Insert, 1075116 Rev. 02. Cellestis, Inc. Valencia, CA., April 2015. Qiagen. QuantiFERON -TB Gold Plus (QFT -Plus) ELISA Package Insert. Published 2016. Accessed April 12, 2017. Oxford Diagnostic Laboratories is a registered trademark of Oxford Immunotec, Ltd. Head-to-head studies between QuantiFERON-TB Gold (QFT) and QuantiFERON-TB Gold Plus (QFT-Plus) Sensitivity % (n/n) Publication QFT QFT-Plus Comments Yi. Sci Rep, 2016. 93.6 (147/157) 91.1 (143/157) Active TB- 157 bacteriologically confirmed patients; 5 indeterminates excluded Hoffmann. Clin Microbiol Infect, 2016. 89.5 (51/57) 89.5 (51/57) Active TB- 24 bacteriologically confirmed, 33 without bacteriological confirmation Petruccioli. Journal of Infection, 2016. 89 (24/27) 85 (23/27) Active TB- 23 microbiologically diagnosed and 4 clinically diagnosed TB-US-DIA-MPN166-0001 V1 14

T-SPOT.TB Test Substrate Peripheral Blood Mononuclear Cells (PBMCs) PBMCs are a subset of white blood cells that includes lymphocyte and monocyte cell populations, with T cells being the major component PBMCs are isolated from whole blood, washed and counted to create a standardized cell suspension before stimulation with TB-specific antigens Why is washing important? enables removal of plasma and potentially interfering substances that may inhibit interferon-gamma secretion Why is counting important? allows for adjustment in cell concentration to correct for variations in patient cell counts and ensure a standard number of cells are used in the test Why is normalization important? helps to minimize the risk of false-negative or invalid results due to abnormal patient cell counts Oxford Immunotec. T-SPOT.TB Package Insert PI-TB-IVD-UK-V3. Abingdon, UK. March 2016. T-SPOT.TB Test Borderline Category Establishing test cut-off test result data from known active TB patients and low risk patients plotted to determine optimal test cut-off point for T-SPOT.TB test effect of different cut-off points on test sensitivity and specificity determined and a cut-off of 6 spots chosen to provide maximum specificity and optimal sensitivity Borderline (equivocal) results represented by ±1 spot from the cut-off of 6 spots (5-7 spots) are valid, clinically interpretable, and should be followed up indicate a clinical judgment should be made which includes patient history, and retesting of the patient is recommended, using a new sample A study of US healthcare workers 79.8% of borderline test results resolved as positive or negative upon retesting (n = 465 pairs) 23% retesting as positive, suggesting that the borderline is useful in maintaining test sensitivity and specificity King TC, Upfal M, Gottlieb A, et al. T-SPOT.TB Interferon-γ Release Assay Performance in Healthcare Worker Screening at Nineteen U.S. Hospitals. Am J Respir Crit Care Med. 2015;192(3):367-373. 15

King Study conclusion 79.8% of borderline test results resolved as positive or negative upon retesting 23% retesting as positive, suggesting that the borderline is useful in maintaining test sensitivity and specificity King TC, Upfal M, Gottlieb A, et al. T-SPOT.TB Interferon-γ Release Assay Performance in Healthcare Worker Screening at Nineteen U.S. Hospitals. Am J Respir Crit Care Med. 2015;192(3):367-373. QFT-GIT and T-SPOT.TB Test Sensitivity Summary of Head-to-Head Studies in Confirmed TB T-SPOT.TB Test Demonstrates T-SPOT.TB test is more sensitive than QFT-GIT TB confirmed through direct detection of MTB via culture or PCR Updated 08/03/2015 Analysis based on peer-reviewed articles published between 01/01/2007 and update date Indeterminate [invalid] results excluded prior to sensitivity calculation, but not quantified within the article are listed as excluded NR indicates indeterminate [invalid] results not reported within the article Sensitivity for T-SPOT.TB higher in 9/14 publications (64.3%) Publication Sensitivity % (n/n) Invalid [Indeterminate] % (n) QFT-GIT Sensitivity % (n/n) Indeterminate % (n) Detjen Clin Infect Dis, 2007. 92.9 (26/28) 0 (0) 92.9 (26/28) 0 (0) Chee J Clin Microbiol, 2008. 92.7 (254/274) 1.5 (4) 81.8 (224/274) 10 (3.6) Domínguez Diagn Microbiol Infect Dis, 2009. 84.2 (32/38) 5.3 (2) 71.1 (27/38) 2.6 (1) Kampmann Eur Respir J, 2009. 58.3 (14/24) 0 (0) 80.0 (20/25) 8.0 (2) 85.0 (34/40) Latorre. Diagn Microbiol Infect Dis, 2009. 94.9 (37/39) NR Markova Biotechnol. & Biotechnol. Eq, 2009. 61.5 (8/13) 30.8 (4) 92.3 (12/13) 0 (0) Lai Eur J Clin Microbiol Infect Dis, 2011. 87.8 (86/98) 0 (0) 65.3 (64/98) 6.1 (6) 11.6 (16) Ling Eur Respir J, 2011. NR 84.1 (116/138) 0.7 (1) 76.1 (105/138) Kobashi Intern Med, 2012. 95.5 (21/22) 0 (0) 86.4 (19/22) 4.5 (1) Theron Eur Respir J, 2012. 85.0 (91/107) excluded 84.9 (90/106) excluded Chiappini Pediatr Infect Dis J, 2014. 75.0 (21/28) 0 (0) 89.3 (25/28) 0 (0) Kobashi OJRD, 2014. 91.7 (11/12) 0 (0.) 83.3 (10/12) 8.3 (1) Young Eur Respir J, 2014. 61.9 (13/21) 28.6 (6) 61.9 (13/21) 33.3 (7) Yu Medicine, 2015. 95.8 (46/48) NR 70.8 (34/48) NR 87.2% (776/890) 2.4% (17) 78.9% (703/891) 6.3% (44) Total 16

Meta-analysis of QFT and T-SPOT.TB Test Sensitivity in HIV Patients With Culture-Confirmed TB Study QFT n/n (%) T-SPOT.TB n/n (%) Chee et al 2008 4/7 (57) 7/7 (100) Dheda et al 2009 1/5 (20) 5/5 (100) Leidl et al 2010 13/19 (68) 17/19 (89) Ling et al 2011 29/43 (67) 35/43 (81) Markova et al 2009 12/13 (92) 8/13 (62)* Pooled 59/87 (68) 72/87 (83) *4/13 patients with invalid T-SPOT.TB results included in this calculation. Excluding invalid results, T-SPOT.TB sensitivity was 8/9 (88%). Santin M, Munoz L, Rigau D. Interferon-gamma release assays for the diagnosis of tuberculosis and tuberculosis infection in HIV-infected adults: a systematic review and meta-analysis. PLoS One. 2012;7(3):e32482. Comparison of the Sensitivity of T-SPOT.TB and QFT-GIT According to Patient Age Retrospective review of medical records of diagnosed TB patients diagnosed with active pulmonary or extrapulmonary TB in Seoul, Korea from February 2008 to December 2013 defined as active TB based on either a positive culture or a positive PCR Test Result of IGRA Sensitivity of IGRAs across age group 29 years 30-49 years 50-69 years 70 years Overall Positive Negative Indeterminate T-SPOT.TB (n = 212) 96.7% 94.7% 87.5% 85.7% 91.0% 193 (91.0) 15 (7.1) 4 (1.9) QFT-GIT (n = 192) 93.3% 86.5% 76.8% 68.3% 80.2% 154 (80.2) 23 (12) 15 (7.8) QFT-GIT, but not T-SPOT.TB, was significantly affected by patient age Bae W, Park KU, Song EY, et al. Comparison of the Sensitivity of QuantiFERON-TB Gold In-Tube and T-SPOT.TB According to Patient Age. Shams H, ed. PLOS ONE. 2016;11(6):e0156917. 17

Negative Predictive Value of the T-SPOT.TB Test in HIV-infected Patients 608 HIV-infected patients tested with T-SPOT.TB T-SPOT.TB ( ) n = 534 (87.8%) Indeterminate [invalid] n = 10 (1.6%) No patients developed TB disease No patients developed TB disease T-SPOT.TB (+) n = 64 (10.5%) 1 patient developed TB disease Mean follow-up 2.57 years Negative predictive value of 100% Low indeterminate/invalid rate of 1.6% Sun HY, Hsueh PR, Liu WC, et al. Risk of Active Tuberculosis in HIV-Infected Patients in Taiwan with Free Access to HIV Care and a Positive T-Spot.TB Test. PLoS One. 2015;10(5):e0125260. Comparison of T-SPOT.TB and TST Test Sensitivity and Specificity in Rheumatic Disease Patients 311 subjects with rheumatic disease or probable rheumatic disease 83.9% BCG-vaccinated 256 patients (82.3%) on glucocorticoid or immunosuppressant therapy 28 patients (9.0%) clinically diagnosed with TB disease overall positivity rates TST test 37.8% (42/111) T-SPOT.TB test 14.2% (44/311) T-SPOT.TB test n/n (%) TST n/n (%) Sensitivity 92.9% (26/28) 81.8% (9/11) Specificity 93.6% (265/283) 67.0% (67/100) As a new immunoassay for TB diagnosis, the sensitivity and specificity of [T-SPOT.TB] is higher than TST. It is of great importance in the diagnosis of active or latent TB infection in rheumatic disease patients. Jiang B, Ding H, Zhou L, Chen X, Chen S, Bao C. Evaluation of interferon-gamma release assay (T-SPOT. TBTM) for diagnosis of tuberculosis infection in rheumatic disease patients. International Journal of Rheumatic Diseases. 2015. http://onlinelibrary.wiley.com/doi/10.1111/1756-185x.12772/full. Accessed March 1, 2016. 18

Issues and debate about IGRAs Practical Cost/front end load vs down stream cost Collection / Technical / Logistical issues/institutional and Health system Invalid T-SPOT.TB test meaning Indeterminate QFT meaning Borderline T-SPOT.TB test significance Variations in serial testing related to conversions and reversion Inter-observer variation 6-15 % variation on spot counting Academic/Research Quantitative Assessment? Antigen Specificity? Spot / PHA Ratio? Analysis of T-SPOT.TB Test Costs and Completion Rates Screening Health Care Workers with Interferon-γ Release Assay Versus Tuberculin Skin Test Prior TST Positives 75/113 (66.4%) T-SPOT.TB negative 69/75 (92.0%) BCG vaccinated Compliance 99% test completion rate without EH staff follow-up with the T-SPOT.TB test 71% test completion rate The SWITCH without EH staff follow-up Studywith TST TST Costs New hire = $81.38 Annual without follow-up = $54.09 Annual with follow-up = $88.15 T-SPOT.TB Costs Cost savings realized when material cost is $54.83 Wrighton-Smith P, Sneed L, Humphrey F, Tao X, Bernacki E. Screening health care workers with interferon-gamma release assay versus tuberculin skin test: impact on costs and adherence to testing (the SWITCH study). J Occup Environ Med. 2012;54(7):806-815. 19

King et al., Am J Respir Crit Care Med 2015 T-SPOT.TB HCW Serial Screening Study Summary of Publications Reporting Conversion and Reversion Rates in North American HCWs Study Setting IGRA Used This study 19 U.S. hospitals T-SPOT.TB Dorman et al. (3) 4 large urban U.S. hospitals T-SPOT.TB Dorman et al. (3) 4 large urban U.S. hospitals Fong et al. (9) Cleveland Clinic, OH Zwerling et al. (10) McGill University Health Centre, Montreal, Canada Central Arkansas Veterans Healthcare System Stanford University Medical Center, Palo Alto, CA Joshi et al. (4) Slater et al. (6) Number of Subjects Tested Conversion Rate (%) Reversion 19,630 0.8 17.6 2,418 8.3 63.9 QFN 2,418 6.1 56.8 QFN 1,857 2.8 80.0 QFN 258 5.3 61.5 QFN 2,303 3.2 45.0 QFN 9,153 4.4 38.7 Rate (%) Definition of abbreviations: HCW = healthcare workers; IGRA = interferon-gamma release assay; QFN = QuantiFERON. King TC, Upfal M, Gottlieb A, et al. T-SPOT.TB Interferon-γ Release Assay Performance in Healthcare Worker Screening at Nineteen U.S. Hospitals. Am J Respir Crit Care Med. 2015;192(3):367-373. T-SPOT.TB Test Performance in Serial Screening of Healthcare Workers Largest IGRA serial testing study to date First to represent routine use of the T-SPOT.TB test in US healthcare worker TB screening programs 16,076 HCWs from 19 hospitals assessing 19,630 serial pair test results As expected, higher baseline positivity and conversion rates in sites testing only higher-risk HCWs than those testing all HCWs (1.4% and 0.6% vs. 8.4% and 2.3%, respectively) Analysis Baseline positivity Concordance Invalid rate Conversion rate Reversion rate Specificity (minimum) T-SPOT.TB Test 2.3% 98.9% 0.4% 0.8% 17.6% of baseline positives or 0.4% of all pairs 98.6% King et al., American Thoracic Society 2015 T-SPOT.TB Interferon-Gamma Release Assay (IGRA) Performance in Healthcare Worker Screening at 19 US Hospitals 20

Identification of False-positive QuantiFeron-TB Gold In-Tube Assays by Repeat Testing in HIV-Infected Patients at Low Risk of Tuberculosis N= 1364; with 94 (6.9%) with positive results Repeat testing in 49 of 94 (52 %) Of the 49 repeated QFT tests, 35 (71.4%) reverted to negative 12 (24.5%) remained positive, and 2 (4.1%) were indeterminate Gray J, Reves R, Johnson S, Belknap R. Identification of False-Positive QuantiFERON-TB Gold In-Tube Assays by Repeat Testing in HIV-Infected Patients at Low Risk for Tuberculosis. Clinical Infectious Diseases. 2012;54(3):e20-e23. Characteristics of HIV/TB Co-Infected Patients with Data of T-SPOT.TB Testing: Review of Practice Pattern in an HIV Outpatient Clinic in New Orleans, Louisiana Group 1 Reversion positive to negative 11/19 58% Remained positive 8/19 42% Borderline to negative 10/15 67% Borderline to positive 2/15 13% Remained Borderline 3/15 20% Group 2 Table 3. Results with Serial Repeat T.Spot TB Test During Study Period (n=34) of the 68 patients with either positive or borderline tests studied Group1 : 19 were repeated after initial positive Group 2: 15 were repeated after initial borderline Frontini M, Ige M, Ali J Characteristics of HIV/TB Co-Infected Patients with Data of T-Spot TB Testing: Review of Practice Pattern in an HIV Outpatient Clinic in New Orleans, Louisiana. J Tuberc Ther 2: 108. Published 2017. 21

Issues with Reproducibility Pre-analytical processing QFT ELISA Analytical testing EQUAL Assay manufacturing EQUAL Immune-modulation PPD BOOSTING (more than 3 days gap) / DRUGS / Pathogen Associated Molecular patterns (PAMP) Release of Interferon Gamma by other cells Sources of variability in the IGRA assay ( QFT* /T Spot** ) Phlebotomy, Tube Order, and Time of Blood Draw* Blood Volume* Tube Shaking* Incubation or Processing Delay* Incubation Duration* Analytical Error*/** Manufacturing Defects*/** Immunomodulation and Boosting*/** Pai M, Denkinger CM, Kik SV, et al. Gamma interferon release assays for detection of Mycobacterium tuberculosis infection. Clin Microbiol Rev. 2014;27(1):3-20. 22

**So how do we explain all of this and ***how to get around it as of now **Intra assay causes related to technical/transport/lab variability **Subject/timing variability with variable immune response **External subject variability related to stress/medication effect **Unknown ***Risk stratification of subject or cohort/population ***Incorporate TST data if applicable, timely and available ***Appropriate two step testing (TST IGRA) with acceptable interval between the two (See international and national guidelines) ***Using borderline category and quantification data of positive and borderline results and in short: Global Clinical Sense Having Said That Case Finding: The Real TST is Risk Stratification, Targeted Screening and Site and Focus of Team Efforts Identifying Active & Latent TB and the steps in diagnosis and Treatment Role of Primary Care and lack thereof** Care Coordination and Continuity of Care and Closing the Loop Coordination of Community, Academics, Clinical and Public Health Medicine with Case Management **Ref: JAMA Editorial Case Finding in TB 1941;116 **PICO based Case Management Paradigm & Strategy *ATS/CDC/IDSA Clinical Practice Guidelines for Drug Susceptible TB CID Advanced Access Aug 10.2016 **JAMA 2016 ;316(9):970-983 Kahwati et al Cochrane Analysis on Primary Care Screening & US Preventive Services Task Force 23

DOTS yes, but Do We Connect the Dots too? *GAPS IN INFORMATION *MIGRANTS *FOREIGN-BORN *DM *EXPOSED CONTACTS The missing ones TB *BARRIERS TO HEALTH CARE ACCESS *HIV *TB & NTM *COPD *OTHER CO MORBDITIES *OTHER INFECTIONS *NON-HIV IMMUNOCOMPROMISED *RESOURCE & LIFESTYLE CHALLENGES MODIFIED MODALITIES OUT OF THE BOX PGM V DOT etc. OI-US-PRES-0004-V1 Questions / Comments / Opinions/Feedback JALI@ LSUHSC.EDU Visit Us Thank you for your kind attention Juzar Ali 24