Sample & Assay Technologies For Internal Use Only Effectively screening for Latent TB HIV/STD/TB/Hepatitis Symposium North Dakota April Mary Shragal Area Director Sales, Northern Region USA Cellestis, Inc. a Qiagen Compan y QuantiFERON ® -TB Gold In-Tube

Sample & Assay Technologies For Internal Use Only A little history In the 1980’s the need for a better test for TB infection in cattle was addressed in Australia The tuberculin skin test in cattle had very similar problems to the TST in humans and thus a new test was needed I But also was a very messy test to perform

Sample & Assay Technologies For Internal Use Only History of QuantiFERON ® 1980’s Developed by Australian researchers at CSIRO for detecting TB in Australian cattle herds Early 1990’s CSL (Australia) acquired exclusive license to patents; and undertook commercialization of a cattle diagnostic test and development of a human diagnostic for TB 2000 Cellestis, founded by two of the inventors of the QuantiFERON ® technology, was chosen to commercialize the human TB diagnostic, known as QuantiFERON ® -TB

Sample & Assay Technologies For Internal Use Only Developed in Cattle – An Excellent Model for Human TB 4

Sample & Assay Technologies For Internal Use Only Skin Testing Cows Australia 1990’s Injecting tuberculin from M. bovis into the caudal fold (base of tail) of a cow.

Sample & Assay Technologies For Internal Use Only Tuberculosis (TB) Review Bacterial infection caused by Mycobacterium tuberculosis complex organisms  M. tuberculosis, M. bovis, M. africanum Infection may be either  Active (with all symptoms and highly contagious)  Latent (without any symptoms, not contagious) Latent TB infection (LTBI)  Needs treatment  Progression to active disease Treatment involves 6-9 months antibiotic therapy; new therapy once per week for 12 weeks.

Sample & Assay Technologies For Internal Use Only LTBI Active TB LTBI means infection, no active disease, no symptoms, not contagious If undetected and untreated  10% will progress to disease  50% do so within 2 years  Higher for immuno- compromised individuals

Sample & Assay Technologies For Internal Use Only Active tuberculosis: Signs and symptoms

Sample & Assay Technologies For Internal Use Only Sample & Assay Technologies World Facts on TB At least one person becomes infected every second Each year, more than 9 million people develop TB disease The WHO estimates that TB takes a life every 17 sec Almost 2 million TB-related deaths occur each year TB is the leading killer of people who are HIV- infected Global mobility, immigration, and inadequate control strategies make it a worldwide problem

Sample & Assay Technologies For Internal Use Only Global travel makes it worse Journal of the American Medical Association

Sample & Assay Technologies For Internal Use Only Transmission of TB Family, friends, workmates etc. exposed Active TB: Infectious Not infected Infected, but no symptoms “Latent TB infection” If not identified & treated ~10% develop TB disease during their lifetime If identified & treated they don't develop TB disease

Sample & Assay Technologies For Internal Use Only Conventional TB Diagnostics, desperately in need of an upgrade Purified Protein Derivative (PPD) is injected intradermally 48 – 72 hours later the size of the resultant reaction is measured

Sample & Assay Technologies For Internal Use Only Tuberculin Skin Test (TST) Limitations: TST responses are often not read within time window Poor compliance Cost implications (follow up and re-testing) Employee health implications False positives due to BCG & NTM Inaccuracy of measuring induration; Subjective interpretation 1 in 3 TST’s failed to be properly diagnosed (Kendig et.al. 1998) 2-step testing required for new hires Up to 4 consultations (usually 10 days)

Sample & Assay Technologies For Internal Use Only TB and the 21st Century QuantiFERON®-TB Gold ‘‘In-tube’

Sample & Assay Technologies For Internal Use Only And counting… 1,100,000 tests/year in the US Testing rate >3,000,000 per year and growing US rate >1,100,000 per year –Majority are serial screening of HCW’s –In Europe, mainly contacts, immunesuppressed, TB suspects –In Asia, contacts, TB suspects, HCWs Worldwide > 1000 labs running QFT –In the US >300 routinely using QFT

Sample & Assay Technologies For Internal Use Only for…HCW Screening by…Public Health Departments for… Clinical & Immune Suppressed Patients for… Contact Investigations Homeless Refugees Recent Immigrants TB/Chest Clinics by… Major University Hospitals & Medical Centers VA Hospitals Military Facilities Foreign Students at University Hospitals HIV & Other Infectious Disease Clinics TNF inhibitors (Rheumatologists) Principle use of QFT in the United States…

Sample & Assay Technologies For Internal Use Only Immunological Basis for QuantiFERON ® Testing In normal circumstances, there is no Interferon Gamma (IFN-  ) within the blood. In the presence of the TB specific antigens, T cells of infected persons are stimulated to produce IFN-  In the QFT test whole blood is exposed to 3 TB specific antigens T cells of infected persons are activated and secrete IFN-  Measurement of IFN-  using an ELISA assay is the basis for the QFT test T-cells activate and secrete IFN- γ.

Sample & Assay Technologies For Internal Use Only QFT Species Specificity vs. TST Tuberculosis Complex ESAT- 6 CFP- 10 TB-7.7TST Environmental Strains ESAT- 6 CFP- 10 TB- 7.7 TST M. tuberculosis ++++ M. abcessus ---+ M. africanum ++++ M. avium ---+ M. bovis ++++ M. branderi ---+ M. celatum ---+ BCG Substrain ESAT- 6 CFP- 10 TB-7.7TST 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 ---+

Sample & Assay Technologies For Internal Use Only QuantiFERON ® -TB Gold Procedures & Guidance Blood Collection Laboratory ELISA Data Analysis

Sample & Assay Technologies For Internal Use Only In the field: 3 tubes: TB specific antigen, Nil & Mitogen Blood collected directly into tubes (1mL each) In the lab: ELISA for detection of IFN-gamma

Sample & Assay Technologies For Internal Use Only Blood Collection Set of three collection tubes: Nil, TB-Antigen, Mitogen Draw 1 mL of blood into each of the 3 tubes Black side marking on the tube indicates the 1mL fill line

Sample & Assay Technologies For Internal Use Only Shaking of Tubes Tubes are mixed by shaking for 5 seconds (~10x) After shaking, the entire inner surface of each tube should be coated with blood Proper shaking will lead to some frothing of the blood

Sample & Assay Technologies For Internal Use Only After Blood Collection and Shaking Tubes can be held at Room Temperature for up to 16 hours Following incubation, tubes have up to 3 days for transfer to lab for QFT ELISA Within 16 hours of collection/shaking, tubes must be incubated at 37ºC for hours Option 2:

Sample & Assay Technologies For Internal Use Only Data Analysis and Results Results are reported as: Positive Negative Indeterminate Low mitogen High Nil

Sample & Assay Technologies For Internal Use Only Clinical performance of QuantiFERON ® -TB Gold A sensitive test would accurately identify people with infection, whether latent or active (maximize true positive results) A specific test would accurately identify people who are uninfected (maximize true negative results)

Sample & Assay Technologies For Internal Use Only Real World Experiences NYC Dept. of Health San Francisco Dept. of Health University of Illinois Chicago Cleveland Clinic

Sample & Assay Technologies For Internal Use Only

Sample & Assay Technologies For Internal Use Only

Sample & Assay Technologies For Internal Use Only

Sample & Assay Technologies For Internal Use Only 2 nd Global symposium on IGRA’s (Dubrovnik, Croatia, June 2009)

Sample & Assay Technologies For Internal Use Only

Sample & Assay Technologies For Internal Use Only Performance of IGRAs and the TST: An up-to-date TB Test Meta-Analysis RDiel, R Loddenkemper and A Nienhaus Evidence based comparison of commercial interferon-gamma release assays for detecting active tuberculosis – a meta-analysis. Chest, 2009, Published on Dec 18, 2009 in electronic format;

Sample & Assay Technologies For Internal Use Only Key findings from meta-analysis: IGRA and TST specificity *QFT significantly more specific than both the TST and T-Spot (p<0.0001) p<0.0001

Sample & Assay Technologies For Internal Use Only Key findings: IGRA and TST Specificity p< How does this translate into false-positives per 1,000 tested people without TB?

Sample & Assay Technologies For Internal Use Only IGRA Indeterminate rates from Diel et al, Chest, 2009 QFTNumber of subjects Number Indeterminate % indeterminate Immune competent16, % Immune suppressed5, % T-Spot.TB Immune competent9, % Immune suppressed2, % For both IGRAs there are significantly more indeterminate results in those immune suppressed

Sample & Assay Technologies For Internal Use Only

Sample & Assay Technologies For Internal Use Only Negative and positive predictive value of a whole-blood IGRA for developing active TB - an update Diel R, Loddenkemper R, Niemann S, Meywald-Walter K, Nienhaus A Am J Respir Crit Care Med [Epub Aug 27, 2010] M C An analysis of 954 tuberculosis contacts comparing QuantiFERON ® TB Gold (QFT ® ) and tuberculin skin test (TST) Cellestis Synopsis

Sample & Assay Technologies For Internal Use Only Predictive Value of QFT (Diel et al AJRCCM Aug 2010) 954 close contacts 142 QFT-positive/ TST-positive 142 QFT-positive/ TST-positive Chemoprophylaxis RIF and/or INH No active TB 51 QFT-positive (49 TST-positive) 51 QFT-positive (49 TST-positive) Mean follow-up >3.5 yr TST cut-off >5mm Not treated 17 developed active TB 343 TST negative 5 QFT-positive TST-negative 5 QFT-positive TST-negative Not treated 2 developed active TB 413 TST positive Not treated No active TB 198 QFT-positive 756 QFT-negative Contact Investigation – Summary

Sample & Assay Technologies For Internal Use Only Predictive Value of QFT (Diel et al AJRCCM Aug 2010) Chemoprophylaxis RIF and/or INH No active TB 51 QFT-positive (49 TST-positive) 51 QFT-positive (49 TST-positive) Not treated 343 TST negative 5 QFT-positive TST-negative 5 QFT-positive TST-negative Not treated 2 developed active TB 413 TST positive Not treated No active TB 198 QFT-positive 756 QFT-negative QFT-negative contacts Predictive Value of QFT (Diel et al AJRCCM Aug 2010) Contact Investigation – Results

Sample & Assay Technologies For Internal Use Only Predictive Value of QFT (Diel et al AJRCCM Aug 2010) 343 TST negative 413 TST positive Not treated 756 QFT-negative QFT-negative contacts 55% of QFT-negative were TST-positive No progression to active TB at 3.5 years In this study, QFT demonstrates 100% NPV* * Negative Predictive Value (NPV) Contact Investigation – Results No active TB No Active TB

Sample & Assay Technologies For Internal Use Only Predictive Value of QFT (Diel et al AJRCCM Aug 2010) 142 QFT-positive/ TST-positive 142 QFT-positive/ TST-positive Chemoprophylaxis RIF and/or INH No active TB 51 QFT-positive (49 TST-positive) 51 QFT-positive (49 TST-positive) Not treated 17 developed active TB 5 QFT-positive TST-negative 5 QFT-positive TST-negative Not treated 2 developed active TB 413 TST positive Not treated No active TB 198 QFT-positive 756 QFT-negative QFT-positive contacts Contact Investigation – Results

Sample & Assay Technologies For Internal Use Only Predictive Value of QFT (Diel et al AJRCCM Aug 2010) 142 QFT-positive/ TST-positive 142 QFT-positive/ TST-positive Chemoprophylaxis RIF and/or INH No active TB 51 QFT-positive (49 TST-positive) 51 QFT-positive (49 TST-positive) Not treated 17 developed active TB 5 QFT-positive TST-negative 5 QFT-positive TST-negative Not treated 2 developed active TB 198 QFT-positive Contact Investigation – Results QFT-positive contacts All 19 untreated contacts who progressed to active TB were QFT-positive. TST missed progression; 11% >5mm 47% >10mm

Sample & Assay Technologies For Internal Use Only Predictive Value of QFT (Diel et al AJRCCM Aug 2010) QFT identified 100% (19/19) of contacts who progressed to active TB >5mm cut-off missed 11% (2/19) 10mm cut-off missed 47% (9/19) By using QFT, at least 60 fewer contacts required treatment Number of Contacts Needing Treatment to Prevent Progression to Active TB

Sample & Assay Technologies For Internal Use Only QFT demonstrated 100% NPV in this study  No contacts who tested QFT-negative developed TB Lower program costs by only treating those who really need it Recommendations & Guidelines suggest QFT can be used as a replacement for the TST  US: Centers for Disease Control & Prevention  Japan: Kekkaku 2010 Be Confident Using QFT Predictive Value of QFT (Diel et al AJRCCM Aug 2010)

Sample & Assay Technologies For Internal Use Only Sahni et al Infect. Control Hosp. Epidemiol. 2,048 QFT results on HCWs 90 were QFT positive INH acceptance compared to when using the TST Acceptance increased from 11% to 52% Reduces the “I am positive because of BCG” effect

Sample & Assay Technologies For Internal Use Only CDC Guidelines IGRAs may be used in place of (and not in addition to) TST in all situations in which CDC recommends TST Which IGRA or TST to be used should be based on the context for testing, test availability, and overall cost effectiveness of testing. Neither IGRAs, nor TST should be used for testing persons who have a low risk of TB infection IGRA is preferred –for testing persons from groups that historically have poor rates of return for TST reading. –for testing persons who have received BCG TST is preferred –for testing children younger than 5 years old ‘ QFT-G can be used in all circumstances in which the TST is currently used’ …now able to detect TB with greater specificity than previously possible’

Sample & Assay Technologies For Internal Use Only TBoss Program Overview

Sample & Assay Technologies For Internal Use Only Role of Public Health Public Health in charge. CDC involved only in case of outbreaks Reference – Maryam Haddad, CDC

Sample & Assay Technologies For Internal Use Only Role of Cellestis Have Program Coordinator on site who works closely with DOH Work through checklist to mobilize resources needed Ensure –QFT kits are available on site –Blood draw logistics in place including trained phlebotomists, blood collection kit (butterflies etc…) –Identify preferred laboratory for QFT testing –Coordinate collection, tube handling and shipment to testing laboratory –Be a liaison with lab and DOH to ensure data integration

Sample & Assay Technologies For Internal Use Only Reimbursement for Diagnostic Use CPT Code: Tuberculosis test, cell mediated immunity measurement of gamma interferon antigen response listed under all state Medicare laboratory fee schedule Medicare: $92.00 for most states Medicaid: up to $86.59 some states not yet covered Private Payers: Aetna: QFT a medically necessary preventive service for LTBI screening in recent immigrants, injection-drug users, residents and employees of prisons and jails, HCW and military United Healthcare: enrollees who are at increased risk for tuberculosis in all benefit plans Blue Cross/Blue Shield: approved in some states

Sample & Assay Technologies For Internal Use Only True’ cost of a TST program Lambert et.al. Infect. Control Hosp. Epidemiol. (2003) Annual cost of implementing and maintaining a TST program (4 hospital sites and 2 health departments): Hospital: cost per HCW = $41 to $362 Health Dept: cost per HCW = $176 to $26 4 TST supply costs accounted for less than 1.5% of the total cost of the TST program for all sites QuantiFERON ® -TB GOLD is cost effective!

Sample & Assay Technologies For Internal Use Only Cost-effectiveness of Interferon Gamma Release Assays vs Tuberculin Skin Tests in Health Care Workers Marie A. de Perio, MD; Joel Tsevat, MD, MPH; Gary A. Roselle, MD; Stephen M. Kralovic, MD, MPH; Mark H. Eckman, MD, MS Result: the QFT-GIT is the most effective and least costly strategy. Conclusion: Use of the QFT-G and QFT-GIT leads to superior clinical outcomes and lower costs than the TST and should be considered in screening non–BCG vaccinated and BCG-vaccinated new HCWs for LTBI. Arch Intern Med. 2009;169(2):

Sample & Assay Technologies For Internal Use Only Significant improvement in diagnosis of TB infection Significant improvement in diagnosis of TB infection Eliminate BCG and NTM false positivesEliminate BCG and NTM false positives QFT-Gold positive result is highly predictive of TB infectionQFT-Gold positive result is highly predictive of TB infection More sensitive for active TBMore sensitive for active TB Eliminates subjectivityEliminates subjectivity Improves testing compliance Improves testing compliance Contact investigations, Homeless, Jail inmates, HCW’sContact investigations, Homeless, Jail inmates, HCW’s Minimizes inappropriate treatment and toxicity Minimizes inappropriate treatment and toxicity QuantiFERON ® -TB GOLD = Better Healthcare!

Sample & Assay Technologies For Internal Use Only QUESTIONS?