1. Traditional and Novel Diagnostic Tests of TB Infection Toru Mori, MD, PhD Research Institute of Tuberculosis/JATA National Institute of Infectious Diseases, Japan 1st Asia Pacific Region Conference of IUATLD Kuala Lumpur, Aug 2-5, 2007

2. Roles of TB Infection Diagnosis Indication for treatment of LTBI Adjunct to diagnosis of TB Disease Decision making for contact actions Monitoring of infection control Epidemiological surveillance and research

3. Tuberculin Reaction in TB Patients （ Bacteriologically Confirmed Patients, PPD 0.05mcg ） Ｎｏ． 602 Mean 16.1mm S.D. 4.6mm (Mori et al, 1975)

4. Tuberculin Reactions of Uninfected Infants (Infants 0 to 3 Years ） Total 10,710 10mm+ 2.9% 20mm+ 0.29% 30mm+ 0.02% 0.0 (Okinawa Pref, 1982)

5. Tuberculin Reaction after 6 Months of BCG Vaccination Tuberculin Reaction after 6 Months of BCG Vaccination (Infants, Erythema, N=103) Mean 21.3mm >=10mm 91.3% >=30mm 13.6% (Mori, 1980)

6. Problems of TST in Infection Dx Confounded by BCG history Confounded by environmental mycobacteria Booster phenomenon Variability in administering and reading Needs two visits

7. Specific Antigens ESAT-6/CFP-10 Tuberculosis complex M. tuberculosis M. africanum M. bovis (Other than BCG) M. leprae M. leprae Environmental strains M. kansasii M. marinum M. szulgai M. flavescens M. gastrii M. bovis BCG All substrains Environmental strains M. intracellulare M. avium Present in Absent from

8. Interferon-gamma Release Assays (IGRAs) Enzyme-linked Immunosorbent assay QuantiFERON-TB Gold T-SPOT.TB Enzyme-linkedimmunospot PBMC separated Whole blood Plasma separated Stimulation with Antigens

9. Responses to CFP-10 & ESAT-6 for each study group Nursing students Cut-off (Mori et al, 2004)

10. TST Distribution and QuantiFERON (+) (BCG-vaccinated Healthy Subjects, N=220, *QTF(+)) **** (Mori et al, 2004)

11. QTF Positivity according to Mantoux test Size QTF Positivity according to Mantoux test Size (TB Patients, Over-all QFT-Positivity = 82%, p for Linear trend =0.002) (Mori et al, 2004)

12. Sensitivity, TB Patients QFT-G, ESAT-6+CFP-10 Pooled 0.81 (0.76-0.84) Chi-sq=17.1, df=8, p=0.03 I 2 = 53%

13. Sensitivity, TB Patients TST Pooled 0.73 (0.67- 0.78) Chi-sq=15.2, df=7, p=0.03 I 2 = 54% 1.00 (0.54 - 1.00) 0.83 (0.63 - 0.95) 0.83 (0.61 - 0.95) 0.78 (0.64 - 0.88) 0.74 (0.63 - 0.82) 0.70 (0.46 - 0.88) 0.66 (0.54 - 0.76) 0.33 (0.07 - 0.70)

14. QFT-G & QFT-GIT Compared Untreated TB Patients QFT-GIT PositiveNegativeTotal QFTGQFTG Positive 77 (81.9%) 1 (1.1%) 78 (83.0%) Negative 10 (10.6%) 6 (6.4%) 16 (17.0%) Total 87 (92.6%) 7 (7.4%) 94 (100%) Sensitivity QFT-G=83.0% QFT-GIT=92.6% (p=0.006) kappa=0.466 (Harada et al, submitted)

15. Sensitivity, TB Patients QFT-G-IT, ESAT-6+CFP-10+TB7.7 Pooled 0.78 (0.71- 0.83) Chi-sq=27.5, df=4, p=0.000 I 2 = 89%

16. Specificity, BCG-vaccinated Low-risk QFT-G, ESAT-6+CFP-10 (+TB7.7) Pooled 0.97 (0.95- 0.98) Chi-sq=13.5, df=5, p=0.019 I 2 = 63% 1.00 (0.82-1.00) 0.99 (0.96-1.00) 0.98 (0.95-0.99) 0.97 (0.87-1.00) 0.96 (0.90-0.99) 0.92 (0.85-0.96)

17. Index Case ： A Student, aged 22 years Ill for 2 mos, Heavily Smear Positive, Secondary cases: 12 Close contacts: 220 QFT(+) 32.7 ％ (+-) 15.9 ％ (-) 52.3 ％ Other contacts: 135 QFT(+) 0.7 ％ (+-) 0.7 ％ (-) 98.5 ％ （ Funayama et al, 2005) A TB Outbreak in a University QFT-Negative N=148 QFT-Positive N=72

18. Results Age in mos Total (N=195) QFTTST <36 mos (N=113)>36mos (N=82) ++ 14 28 +- 415 -+ 71219 -- 8855143 QFT(+)% 15.9 (9.2 – 22.7)18.3 (9.9 – 26.7)16.9 (11.7 – 22.2) TST(+)% 18.6 (11.4 – 25.8)31.7 (21.6 – 41.8)24.1 (18.1 – 30.1) Agreement 0.9030.8410.877 κ 0.6600.5870.626 （ Submitted ） QFT/TST on TB Contact Children QFT/TST on TB Contact Children （ Cambodia, 0 ～ 5 yo ， N=195 ， Okada et al, 2006)

19. Indian TB Suspects Study (1-12 yrs, N=106, In-Tube QFT-G, Dogra, 2006) QFT+QFT-Total Agreement κ-coeff. Cut- off:>=5mm TST+8816 0.895 0.53 (0.34-0.71) TST-38689 Total1194105 Cut- off:>=10mm TST+824 0.952 0.73 (0.53-0.92) TST-39295 Total119499 Cut- off:>=15mm TST+404 0.933 0.50 (0.33-0.66) TST-794101 Total1194105

20. Indian TB Suspects Study (cont’d) (1-12 yrs, N=106, In-Tube QFT-G, Dogra, 2006) TST(>=10mm)QFT (0.35IU/mL) No.Pos /Tested (%) OR, adjusted No.Pos /Tested (%) OR, adjusted Age 1-4 yrs2/42 (5)1 1 5-9 yrs2/33 (6)1.16 (0.14,9.49)3/33 (9)2.02 (0.30,13.5) 9-12 yrs6/30 (20)5.69 (0.95,33.8)6/30 (20)5.92(1.02,34.5) Contact No 7/89 (8)18/89 (9)1 Yes 3/16 (19)2.48 (0.51,11.9)3/16 (19)2.00 (0.42,9.35)

21. QFT Level at TB Detection among Contacts （ Comparison with LTBI, Harada et al, 2007, submitted) Adj.OR95%CIp Sex0.8450.3172.2520.736 Age group 1.6331.0542.530.028 TST0.7170.4461.1520.169 QFT (CFP/ESAT) 1.8491.2092.8290.005 N=35 N=76 Factors contributing to TB Onset (Multivariate analysis)

22. TB Risk according to Parameter Value （ Induration size for TST, Quartile grade for QFT ） -14 -19 -24 25+ TST ESAT-6CFP-10ESAT/CFP* * Chi 2 for trends p=0.028 （ Harada et al, submitted)

23. QFT in Healthy General Population (Japan, Rural Community, N=1,559) (Mori et al, 2007)

24. Age-specific Prevalence of TB Infection Age-specific Prevalence of TB Infection （ Japan, Estimated, Years 1950 & 2005) (Mori, 2005)

25. QFT in Healthy General Population QFT in Healthy General Population (Comparison with Estimated Prevalence of TB Infection) (Mori et al, 2007)

26. QFT according to Types of X-ray TB Findings (Predicted: Expected from Rate of those with No TB Finding, Age adjusted ） （ 1,359 ）（ 51 ）（ 45 ）（ 31 ） ［ Example ］ Certain ： Fibrotic lesion Probable: Calcification ・ Pleural adhesion ． Possible ： Apical cap 1 1.93 1.60 2.54 (Mori et al, 2007)

27. Change in QFT after Treatment of LTBI (1) ESAT-6 CFP-10 0.999 >> 0.272 IU/ml (p= > 0.124 IU/ml (p=004) Geometric Mean (Higuchi et al, 2007)

28. Change in QFT after Treatment of LTBI (2) (TB Outbreak in a mental hospital, with >15 secondary cases) 6 mos treatment completed ( 7(25%) reverted ) N=28 ESAT-6 0.999 0.272 CFP-10 0.346 0.124 Interrupted (< 3 mos) ( No reversion ) N=5 ESAT-6 0.743 0.729 CFP-10 0.595 0.572 1.5 Years after Treatment ( No net reversion ) N=17 ESAT-6 0.381 0.442 CFP-10 0.087 0.192 (Higuchi et al, 2007)

29. QFT Profile in TB Patients during and after Chemotherapy (N=50, Aoki et al, 2006)

30. IGRAs in Special Settings 1. Children QFT(+) in 30 clinically diagnosed TB patients aged 0-14 years in Japan ( Takamatsu et al, 2007 ); 77% (62 – 92%) For 41 patients aged 0-5 years in Italy ( Russo, 2007 ); 88.0- 95.6% In India ( Dogra et al, 2006 ) and Cambodia ( Okada et al, 2007 ), QFT-G gave results comparable with TST in family contact children. In Nigeria QFT-GIT detected more LTBIs than TST, regardless of age (0-4/5-9/10/14) ( Nakaoka et al, 2007 ) IFN-G response to mitogen is lower in young infants, causing more “Indeterminate” results. ( Harada et al, 2007 )

31. IFN-G Release to Mitogen according to Age (Age: 0-95 years, N=12,856) Mean value Frequency of “Indeterminate” (Harada et al, unpublished)

32. IGRAs in Special Settings 2. Aged subjects (Nursing home residents, N=61, Mean age =79.9 yrs, Chi- square for linear trend=2.68, p=0.101) (Suzuki, Harada et al) In other sample, the “indeterminate” results are commoner in the aged subjects; 2.0% for 60+years vs 0.6 for 20-59 years. (p=0.00)

33. QFT-G vs TST by Age in TB Patients

34. IGRAs in Special Settings 3. Immunocompromized hosts In rheumatic patietns receiving immuno- suppressive therapy IFN-gamma assay was superior compared to the TST for detection of LTBI. ( Matulis et al, 2007 ) In HIV+ patients, those with a CD4 count ≤ 200 were more likely to have an indeterminate test 200 were more likely to have an indeterminate test result. Further studies are needed to assess utility of IGRAs. ( Talati et al, 2007 ) In hematological malignancy patients, IGRA produced more positives than TST. ( Losi et al, 2007 )

35. QTF-TB2G as Compared with TST Strengths –Specific –Sensitive –Reliable –Needs one visit only –No booster effect Weaknesses –Cost –Needs whole blood –Labor intensive –Stimulation <12hrs –Technical/Instrum ent

36. Uses of QFT TB Control of Healthcare Workers On Employment ： Replace Two-Step TST When exposed to TB: without TST Contact Investigation With / without TST Clinical Diagnosis Differentiate TB / NTM / Tumor.... Prescribe Rx of LTBI in High Risk Subjects (Jap Soc TB, 2006)

37. Further Research Needs Performances Time from Infection to Positive Conversion Relationship with Risk of Clinical Breakdown Influence of TB Treatment, Response in “Old” Infections Difference in Response between different antigens Difference from TST: Effect of prolonged incubation (effector vs memory cells?) Children & Infants Procedures Test with Smaller amount of Blood （ for Children ） Time until Stimulation→QFT-3G Costs

38. Acknowledgment Collaborators –Dr. Harada N (RIT/JATA), –Dr. Higuchi K (RIT/JATA), –Dr. Takamatsu (Osaka Municipal Respiratory & Allergy Center) Funded partly by the Emerging and Reemerging Diseases Study Grant, Ministry of Health, Labor & Welfare, Japan.