1. Interferon Gamma Release Assays (IGRAs) for the Diagnosis of TB: Can We Replace the TST? Helene M. Calvet, MD Health Officer and TB Controller Long Beach Department of Health and Human Services

2. Overview Diagnosis of active and latent TB In-Vitro Interferon Gamma Release Assays (IGRAs) Sensitivity and specificity of IGRAs Implementation data from San Francisco and Long Beach

3. Definitions “Positive PPD”: a tuberculin skin test (TST) that is indurated: –>5 mm: HIV+, recent contact of TB case, CXR c/w old TB, organ transplant or other immunosuppression –>10 mm: everybody else (in California) Latent TB Infection (LTBI): TB infection without evidence of clinically active disease (+PPD, but no symptoms); CXR usually normal, or may be abnormal, but sputa negative TB Disease: active tuberculous infection of any organ

4. Diagnosis of TB TB disease Latent TB infection (LTBI)

5. Toolbox for Diagnosis of TB Disease History Cultures Sputa CXR Physical Exam PCR Pathology Response to therapy TST IGRAs Gold Standard = Culture

6. Toolbox for Diagnosis of Latent TB TST IGRAs CXR History No Gold Standard!

7. Comparison of Toolboxes TB Disease ToolboxLTBI Toolbox Abundant toolsPaucity of tools Gold standardNo gold standard No need to rely on one testMay need to rely on one test

8. The TST…A Very Old, and Not-So-Perfect Test Discovered by Koch in 1890, skin test agents standardized in 1976 False negatives: –Anergy (immunocompromise or malnutrition) –Recent TB infection –Very young age (< 6 months) –Overwhelming TB disease –Recent live virus vaccination –Poor placement or reading of TST False positives: –BCG or nontuberculous mycobacterial infections –Inaccurate reading of TST Requires two visits…substantial proportion do not come for reading

9. Tuberculin Skin Testing Mantoux Method 48 to 72 hours 5 TU of PPD Interpretation depends on person’s risk factors

10. BCG and TST (1) General teaching is that reactivity from BCG wanes after a few years and is unlikely to persist > 10 years, but may be boosted by PPD. Study done in Switzerland* suggests that false positives due to BCG may be much more common than we thought: –40% of 5000 HCW had positive TST –Prior BCG strongest risk factor for positive TST among those less than age 40 with TSTs 40 years old and those with TSTs > 20 mm) *CID 2005; 40:211 – 217.

11. BCG and TST (2) Review of studies that compared TST responses to BCG during and after infancy Vaccination during infancy estimated to cause false-positive TST in 6.3% overall, but only 1% of those tested more than 10 years after vaccination Vaccination at 2 years of age or older estimated to cause false-positive TST in 40% of persons overall, 20% of those tested 10 years or more after vaccination Farhat M et al, Int J Tuberc Lung Dis 2006; 10: 1192-204

12. In-Vitro Interferon Gamma Release Assays (IGRAs) for TB QuantiFERON®QuantiFERON® TB Gold (QFT-G): FDA approved QuantiFERON® In-Tube (QFT-IT): submitted for FDA approvalQuantiFERON® In-Tube (QFT-IT): submitted for FDA approval T-Spot TB  : submitted for FDA approval

13. QuantiFERON ® (QFT) History First generation (QuantiFERON ® – TB) test FDA-approved in 2001 2003 CDC guidelines advise use of test in selected groups only Second generation test (QuantiFERON ® - TB Gold) FDA-approved March 2005 CDC guidelines December 2005* allow use of QuantiFERON ® - TB Gold (QFT-G) in any situation in which a TST would be used; however, points out lack of data in many groups (pediatrics, immunocompromised, etc.) *MMWR 2005, 54 (RR-15): 49-55

14. In Vivo and In Vitro Diagnostic Tests Antigen presenting cell Memory T-cell Presentation of mycobacterial antigens IFN-  IL-8, etc. TNF-  Andersen P, et al. Lancet 2000;356:1099

15. Why Measure Interferon-  ? TB infection induces T-cell response (CMI) IFN-  is the ‘classic’ CMI cytokine Produced in vitro in response to specific antigen Secreted in measurable and stable amounts Absent from normal circulation Extensive literature showing importance of IFN-  in TB infection

16. QuantiFERON ® -TB Gold Principle Based on the quantitative measurement of IFN-  secreted from stimulated T cells in human whole blood –T-cells reactive to M. tuberculosis - specific antigens are only present in those infected A two stage process: –Incubation of whole blood with TB-specific and control antigens (mitogen) and nil control –Detection of IFN-  using a rapid, single-step, sandwich ELISA

17. Species specificity of ESAT-6 and CFP-10 Environmental strains Antigens ESATCFP M abcessus-- M avium-- M branderi-- M celatum-- M chelonae-- M fortuitum-- M gordonii-- M intracellulare-- M kansasii++ M malmoense-- M marinum++ M oenavense-- M scrofulaceum-- M smegmatis-- M szulgai++ M terrae-- M vaccae-- M xenopi-- Tuberculosis complex Antigens ESATCFP M tuberculosis++ M africanum++ M bovis++ BCG substrain gothenburg-- moreau-- tice-- tokyo-- danish-- glaxo-- montreal-- pasteur--

18. Whole Blood IFN-  Assay QuantiFERON-TB Test ESAT-6 CFP 10 Mitogen Control TMB COLOR Stage 1 Whole Blood Culture Stage 2 IFN-gamma ELISA Nil Control Incubate → INF-  from sensitized T- cells Draw blood + heparin Aliquot blood & add antigen Harvest plasma from above settled cells Measure [ IFN-  ] in ‘Sandwich’ ELISA Computerizedinterpretation Cellestis

19. QuantiFERON ® -TB Gold Test Method Advantages and Disadvantages Advantages: –Only one visit required –Objective and reproducible; not operator-dependent –No cross reactivity with BCG, little cross-reactivity with non-tuberculous mycobacteria –Controls for low or no immune response –No chance of ulceration due to brisk skin test reaction Disadvantages: –Blood must be received in lab within 12 hours –Labor intensive for the lab –Not much data for some patient groups

20. T-Spot.TB  : “Six easy Steps” Nil Control Positive Control Infection Oxford Immunotec

21. IGRA Possible Results Positive: indicates TB infection, does not differentiate between active disease and latent infection Negative: no TB infection Indeterminate: not sure

22. What Does an Indeterminate Mean? Indeterminate can occur as a result of low mitogen response (due to patient immunocompromise, poor specimen handling or storage, lab error, etc.) or high nil response (due to patient illness, recent vaccinations, etc.) Estimated rate of indeterminates for QGT-G: approximately 1-2% among HCW, about 5-10% among patients Upon retesting, approximately ½ of the indeterminates come out with a definitive result (unpublished data) Retesting indeterminates once, and if indeterminate again, stop Indeterminate rate much higher among children

23. Estimating Sensitivity and Specificity of IGRAs Sensitivity in people with culture + TB Sen = # positives / # tested Specificity in people at low risk for TB infection Spec = # negative / # tested

24. QFT-G Sensitivity Estimates ReferencePopulation + IFN-  (n) + TST (n) Mori; 2004 Untreated Cult+TB; Japan 89% (118)66% (76) Kang; 2005Pulmonary TB; Korea 81% (54)78% (54) CDC; Unpub.Untreated Cult+TB; US 81% (41) Ravn; 2005Active TB; Denmark 85% (48)Not done Lee, 2006Active TB, Korea 70% (61)67% (58) Menzies* 2007Meta-analysis (9 studies) 80% (393)74% (394) *Menzies, D. et al, Annals of Int Med 2007;146 (5): 340-354

25. TB Suspects: QFT-G Performance Among 242 suspects, 23 of 37 had culture-confirmed tuberculosis and +QFT-G results QFT-G sensitivity: only 64% (95% CI, 48% - 78%), but negative predictive value 89% Sensitivity of the TST was 88% in this review Very poor performance in extrapulmonary TB (14% sensitivity), but numbers were low Conclusion: lowish sensitivity in TB suspects means it ’ s probably not all that useful for that purpose Dewan et al, Clin Infect Dis 2007, 44:69-73.

26. Why Poor Performance in Active TB? CMI response likely diminished in active TB, particularly in those with more advanced disease, malnutrition and older age If their CMI was working well, they wouldn’t have TB disease; so TB disease likely not a great surrogate for LTBI Pooled sensitivity from 10 studies: 75% (71-78%) Lower cutoff may be needed in those who are highly suspect to have TB Pai and Menzies, Clin Infect Dis 2007; 44: 74 - 77

27. QFT-G Specificity Estimates ReferencePopulation + IFN-  (n) + TST (n) Mori; 2004Nursing Students; Japan2% (213)65% (113) Kang; 2005Med Students; Korea4% (99)51% (99) CDC; Unpub. Navy recruits; US.2% (532).9% (532) Menzies* 2007 Meta-analysis (9 studies)3% (711) +BCG: 44% (516) No BCG: 2% (156) *Menzies, D. et al, Annals of Int Med 2007;146 (5): 340-354

28. T-Spot TB Sensitivity Estimates ReferencePopulation + IFN-  (n) + TST (n) Lalvani; 2001Untreated Cult+TB; UK 96% (45)69% (32) Pathan, 2001Active TB, UK 92% (33)N/A Meier, 2005Active TB, Germany 97% (70)N/A Lee, 2006Active TB; Korea 95% (83)67% (58) Menzies* 2007 Meta-analysis (11 studies) 88% (557)74% (394) *Menzies, D. et al, Annals of Int Med 2007;146 (5): 340-354

29. T-Spot TB Specificity Estimates ReferencePopulation + IFN-  (n) Spec. Lalvani; 2001 Low risk BCG-vaccinated subjects, UK 0% (26)100% Pathan, 2001 Low risk BCG-vaccinated subjects, UK 0% (32)100% Lalvani, 2001 Low risk BCG-vaccinated subjects, India 0% (40)100% Menzies* 2007Meta-analysis (4 studies) 8% (229) + BCG: 44% (516) No BCG: 2% (156) *Menzies, D. et al, Annals of Int Med 2007;146 (5): 340-354

30. T-SPOT.TB vs. QFT-TB Gold!

31. Comparison of T-Spot.TB and QFT- TB Gold Site - Italy Design - Prospective study of 393 consecutively enrolled patients with LTBI or suspected TB Agreement with the TST and the two IGRAs was similar, but T-Spot TB more likely to yield positive results in close contacts Indeterminate results were more common with QFT- TB Gold than T-Spot.TB (11% vs 3%). Indeterminate results were more likely in young children (< 5 yrs) and immunosuppressive treatment Ferrara G, et al. Lancet. 2006;367:1328-1334

32. Comparison of Test Results Among Contacts TST QFT-GT-SPOT.TB All contacts62 (54%) 25 (22%) 39 (34%) <0.0001 Non-BCG vac.36 (43%) 21 (25%) 29 (35%) contacts 0.0001 0.0923 Ferrara G, et al. Lancet. 2006;367:1328-1334

33. Comparison of T-Spot.TB and QFT-TB Gold Test resultsActive TB (%)Low risk for TB No. of subjects87131 TST + (≥ 10 mm)58 (66.7)28 (21.4) QFT-G positive61 (70.1)11 (8.4) T-SPOT.TB positive83 (95.4)20 (15.3) Lee JY, et al. ERJ. 2006

34. Three Ways to Interpret This Data QFT-G is less sensitive than TST and T- Spot TB OR QFT-G is a lot more specific than TST and T-Spot TB OR A little bit of both

35. Diagnosis of TB: The Truth*? Truth Sensitivity Specificity TST QFT-G QFT-G?T-Spot TB Active Latent T-Spot TB? QFT-G TST T-Spot TB * My opinion only, based on impression of available data

36. QuantiFERON Testing: The San Francisco Experience Implemented QuantiFERON-TB (QFT-1) screening in 4 community clinic sites (2 homeless, 1 methadone, 1 immigrant) and TB clinic in November 2003 Switched to QuantiFERON-TB Gold (QFT-G) in March 2005 Over 6100 samples run between March 2005 and February 2006

37. QFT-G Test Results by Age Category March 2005 – February 2006 (3) (1) (6) (7) (12) (31)

38. TB Infection Prevalence by Test and Clinic Type Homeless TB Clinic MethadoneImmigrant TST (2001-2003) 26%>50%10%37% QFT-1 (11/04-2/05) 17 % n=1848 48 % n=292 18 % n=346 37 % n=344 QFT-Gold (3/05-2/06) Decline in positive rate from TST 6 % n=3594 77% 26 % N=693 48% 4 % n=546 60% 12 % n=626 66%

39. QFT Implementation – LB Experience Lab was experienced with the technology after doing a contact investigation study in a dialysis center using QFT in 2003 –Over 120 patients and staff tested twice –QFT-Gold found to be more closely associated with contact to case, less affected by albumin, good reproducibility Waited for FDA approval of QFT-Gold and completion of lab reconstruction before implementing Performed validation study Nov. 2005 started running patient samples in Jan. 2006

40. QFT in the Lab

41. First Experience From January through June 2006, 193 samples run on patients in TB clinic Some testing also done in Occupational Health Of 193 samples in TB, 137 (71% foreign- born, 130 (67%) with history of BCG Many samples done as “confirmatory” testing for positive TST

42. Results in First 6 Months 108 (56%) negative, 57 (30%) positive and 25 (13%) indeterminate Excluding indeterminates, 65% negative and 35% positive Of those with known prior TSTs (n = 100, excluding indeterminates) –33 (33%) positive QFT-G –67 (67%) negative QFT-G Average size of prior TST by QFT-G result category –QFT-G positive: median 18 mm, (range 10 -30) –QFT-G negative: median 14 mm (range 10 – 30)

43. QFT-G in Occupational Health (OH) Police recruit class, May ’06: –80 healthy young folks –30% positive, 14% indeterminate –Much higher positivity rate than historical, but no prior tests Firefighter screening, September ‘06 –384 healthy, low risk firefighters with prior negative TSTs –19% positive, 5% indeterminates, 76% negative What the heck was going on?

44. Tube B or Not Tube B…? QFT-G users had been reporting unusual results for several months High nil values noted in many of the positive samples in LB firefighters in September October 3 letter from Cellestis notifying users of increased numbers of indeterminates and false positives (usually with high nil values) associated with certain lots of Becton Dickinson Na heparin tubes; recommended switching to lithium heparin tubes

45. High Nil Value High nil value does not always lead to an indeterminate result –If reactivity to ESAT-6 or CFP-10 is > 50% higher than nil, computer calls it positive Not sure cause for this outcome: blood drawing technique, inadequate mixing, lab phenomenon, contaminated tubes etc. SF also had experience with this, and change of blood drawing venue took care of it Planned to repeat Fireman testing using both Na heparin and lithium heparin tubes

46. Repeat Testing on Firefighters 93 firefighters retested January 2007 with both lithium and sodium heparin tubes 1/92 (1%) positive on both (subsequently found to have hx of prior +TST), 86/92 (93%) negative on both, 5/92 (5%) discordant Among 5 discordant, all positive on lithium heparin but negative on sodium heparin; repeat of assay gave same result on sodium heparin, but varying results (+, -, indeterminate) on lithium heparin = likely gray zone, or borderline, values So, is lithium heparin really better or not?

47. QFT Reproducibility  Reproducibility good, but conversions and reversions possible  Actual breakpoints of conversion & reversion not well defined (aside from change from positive to negative or back)  Since normal variations not known, thresholds not established (as for TST)  Conversion and reversion more likely with results near gray zone, or for results discordant with the TST.  What is the natural history of T-cell response to TB infection??

48. Facilitating Interpretation  Might it be better to report continuous variables (actual IFN readings) instead of dichotomous variables (positive/negative)?  Could assist clinician in interpreting the results  Might give more data in serial tests to help determine conversion versus normal variation  Recent recurrent problems with false positives (associated with high nil values) have led the state to suggest that labs should call all specimens with high nil value indeterminate, regardless of the ESAT-6 and CFP-10 values

49. QFT-G: My Concerns about Sensitivity There definitely are false positive skin tests, but are there really that many?? The test relies on effector lymphocytes, which have recently encountered antigen in vivo, to produce interferon within hours Memory cells, which have encountered antigen in the distant past, would require several days in the presence of antigen to produce interferon Could we be missing distant, latent infection that is not immunologically active? (I think we may be) ….and/or… Could this be telling us who has infection that is requiring more of an immunological response, and thus possibly at increased risk of progression??

50. The IGRA Research Agenda Group of experts met March 2006 Comprehensive Research Agenda Generated 58 key questions in 7 general areas  Biological issues  Test performance in high-risk populations  Test reproducibility & serial testing  Responses during treatment  Epidemiological & field applications  Health systems & economic research Pai, M et al, Lancet Infect Dis 2007; 7: 428-38

51. Key Questions (1) What are appropriate cut-off points for different groups? What is the normal variation in T-cell responses? What is the risk of active disease in those with positive and negative IGRA results? How does this relate to risk associated with positive TST? Is there a cut-off point that will predict incipient active TB? How should conversions and reversions be defined (incrementally), and how often do they occur? Pai, M et al, Lancet Infect Dis 2007; 7: 428-38

52. Key Questions (2)  What is the biological basis for discordance between IGRAs and TST?  What is the role of IGRAs in monitoring response to therapy of active or latent TB?  Can IGRAs be used to revise estimates of worldwide TB infection and the lifetime risk of TB disease?  How useful is the combined approach of using TST to screen and IGRA to confirm? Pai, M et al, Lancet Infect Dis 2007; 7: 428-38

53. Things to Think About in Implementing QFT-G in Occupational Health Realize the limitations and benefits of the test! –Lowish sensitivity for LTBI, but probably highest specificity, especially in BCG-vaccinated population Cost of tests and turn-around time Educating everybody involved about this test Specimen collection, handling and transport Obtaining appropriate waivers for use in HCW screening Changing all documentation that has TST on it (screening cards, charts, forms, etc.) to include TST results What to do with indeterminates Mode of implementation: stepwise, or across –the –board? Relationship with your lab; tracking indeterminates, identifying problems

54. IGRAs: Summary QFT-G: –Like the TST, not a perfect test! –Very specific (usually), but somewhat lacking in sensitivity –Revised cut-offs and quantitative reporting may be helpful –3rd generation submitted for FDA-approval; will be easier for clinicians (does not need to be to lab in 12 hours), easier for lab T-Spot TB: –Superior sensitivity to QFT-G, but … –Possibly less specific than QFT-G –Much more difficult from lab standpoint –More expensive –Not yet commercially available

55. IGRAs: Summary (2) IGRAs represent an exciting new opportunity to learn more about a very old disease IGRAs are not perfect tests, so clinicians need to be aware of their limitations Can we replace the TST? – Not yet!

56. Questions?