DIAGNOSIS OF TB D R. K O N G P O M A R N K O N G C L I N I C F O R C H E S T & I N T E R N A L M E D I C I N E

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1 DIAGNOSIS OF TB D R. K O N G P O M A R N K O N G C L I N I C F O R C H E S T & I N T E R N A L M E D I C I N E

2 PATHOGENESIS OF TB INFECTION AND DISEASE (I)

3 PATHOGENESIS OF TB INFECTION AND DISEASE (II)

4 MODES OF DIAGNOSIS Microbiologic Smear Culture Others ( MODS etc) Molecular Nucleic Amplification Assays (NAA) IFN-γRelease Assays (IGRA) Line-probe PCR for drug sensitivity testing Urinary Assays

5 CURRENT PROBLEMS Active disease Smears are insensitive NTM make up 5% of positive smears locally Cultures take too long ( 12 to 14 days to be positive) Latent TB Infection (LTBI) Mantoux test since 1981 BCG, NTM and boosting can confound issue Low sensitivity and specificity Repeat visits

6 NEW MODALITIES IN THE DIAGNOSIS OF ACTIVE TB NAA Brochoalveolar lavage with Elispot Urinary assays?

7 NUCLEIC AMPLIFICATION TESTS/PCR Rationale Isolation issues Issues with current TB testing MDR-TB Standardized and come in kit forms

8 NAA Amplicor: DNA PCR Amplified / Enhanced MTD / MTA: rrna detection Both FDA approved In 1999, E-MTD approved for use in both smear positive and negative specimens

9 NAA Sensitivity is markedly improved cf. smears MTD: Sensitivity usually range from 83 to 98% in smear + cases. 70 to 81% in smear negative cases. Amplicor slighly less Specificity is 98 to 99% Negative predictive value is more important

10 NAA E-MTD / MTA Improved sensitivity in smear negative cases 1999 study. 489 inmates in Texas prison Overall sensitivity of 95.2% and specificty of 99.1%. 100% in smear positive cases 90.2% and 99.1% in smear negative cases Ontario study (1999) using clinical diagnosis as reference gave nearly 100% specificity and sensitivity for both smear + and sm- cases (Case selection!!)

11 Figure 2. Estimated Predictive Value of Acid-Fast Bacilli Smear and Enhanced Mycobacterium tuberculosis Direct Test by Clinical Suspicion for Tuberculosis Positive predictive value is percentage of patients testing positive who had tuberculosis; negative predictive value, percentage of patients testing negative who did not have tuberculosis. Catanzaro, A. et al. JAMA 2000;283: Copyright restrictions may apply.

12 NAA FOR SHORTENING ISOLATION Campos et. Al. AJRCCM 2008;178: TB suspects in respiratory isolation AFB smears vs in house NAA 46 culture positive cases NAA detected 40 cases and all smear positive cases Sens 0.87 and specificity of 1 Smear sens 0.76 and specificity 0.96 Average RI tooks 5 days

13 CONCLUSION FOR NAA Useful in excluding disease in low suspicion patients and ruling it in in those with intermediate and high suspicion. It is a tool to support diagnosis, not a diagnosis itself. Potential to decrease isolation use

14 BAL & ELISPOT

15

16 DIAGNOSIS OF LTBI LTBI has about 20% chance of breakdown acc to MTX Potentially infectious when it becomes disease In use since 1891 Shortcomings BCG cross reactivity False positives and negatives User variability Variations in reagents and strength Poor sensitivity and specificity

17 PROBLEMS WITH THE MTX (WHO HAS LTBI?) Based on epidemiologic curves Operator issues Reagent issues..us!! Environmental / Locality issues BCG vaccination(repeat BCG vaccinations!) Immunesuppression / anergy No gold standard TB disease Exposure studies

18 GAMMA IFN ASSAYS Basis Mantoux test based on weakened M tuberculosis. Cross reacts with BCG RD1 region of differentiation Antigens nearly unique to M tuberculosis ESAT 6, CFP-10 and TB7.7 Exceptions: M kansasii, M marinum Does not cross react with BCG No gold standard No track record. Breakdown?

19 GAMMA INTERFERON ASSAYS Quantiferron, Elispot Initial data suggests usefulness in detecting LTBI Advantages 1 blood draw No cross reactivity with BCG No boosting phenomenon ( known) Disadvantages Expensive Advanced equipment No Gold standard and no track record NEED TO RISK STRATIFY! Spontaneous reversions

20 QUANTIFERON -GOLD QFN using ESAT-6 and CFP-10 antigens ELISA assay New in tube formulation QTF Gold TB in tube

21 ELISPOT More sensitive, specific and convenient Mononuclear cells from single blood sample stimulated with ESAT-6 Elispot can detect 1 in 60,000 IFN- secreting cells Positive in 96% of TB patients ( vs 50% in Quantiferon) and 85% of presumed LTBI Negative in 26 BCG vaccinated subjects

22 T SPOT TB PBMC separated and washed Incubated in antibody ( to IFN) coated wells Antigens added and incubated Cells and supernatatnt removed Antibodies added to highlight the IFN secreted Machine counted Negative Ctrl CFP-10 ESAT-6 Positive control

23 IGRA SENSITIVITY AND SPECIFICITY Mori et,al. AJRCCM 2004; 170: Japanese nursing students, 152 TB suspects of which 119 were proven TB. No history of TB exposure All BCG vaccinated Using Quantiferon-GOLD ROC curves to establish cutoff 64.6% were MTX positive ( specificity of 35.4%) but only 1.9% were ESAT-6 or CFP-10 positive

24 Mori et.al AJRCCM 2004; 170:59-64

25 Mori et.al Overall sensitivity was 89% and specificty 98.1% Issues Shows high specificity Sensitivity in LTBI not established Expect better sensitivity with LTBI as -IFN levels appear suppressed in active disease cf with LTBI.

26 Ewer et.al. Lancet 2003; 361: School outbreak in UK 1128 students screened with Heaf test. Detected 69 TB cases and 254 cases of LTBI 535 students enrolled in RD1 Elispot study Detailed contact information obtained Estimate of OR for Elispot with increased exposure

27 Ewer et.al Lancet 2003; 361:

28 Ewer et.al Lancet 2003; 361:

29 Results Elispot & TST + RR 17.6 Elispot + but TST - RR 11.7 Elispot - and TST + RR 2.97 TST+ related to BCG status. Elispot wasn t

30 Ferrara et.al AJRCCM 2005; 172: QFT-Gold study in a hospital of 318 patients over 8 months. Tb suspects 68 indeterminate results Indeterminate results significantly overrepresented in TSTpatients Indeterminate results more common in patients on immunosuppressive treatment Indeterminate results may be useful.

31 Ferrara et.al AJRCCM 2005; 172:

32 -IFN ASSAYS -IFN Assays appear promising as replacement for TST Improved sensitivity would include a group previously undetected.? Immunesuppressed In immune suppressed, are they less likely to develop Tb and hence TST negative? Overall, it could save cost if the kits were cheaper Recent FDA approval to replace TST!

33 -IFN ASSAYS RD-1 assays showed superior sensitivity and specificity Unaffected by BCG Pooled sensitivity for T-spot, QFT-GIT, TST was 90%, 83% and 89%. Specificity: 88%, 99% and 85%

34 PREDICTING BREAKDOWN Diel et.al. AJRCCM 2011;183:88-95

35 PREDICTING BREAKDOWN Diel et.al. AJRCCM 2011;183: contacts between 5/05 till 4/ completed QFT-GIT (20.8%) and TST( 63.3% at 5mm, 25.4% at 10 mm) 903 contacts not given chemoprevention. 19 TB cases 100% of cases QFT positive. Progression rate of 12.9% For TST. 89.5% and 3.1% (5mm). 52.6% and 4.8% (10mm)

36 PREDICITNG BREAKDOWN

37 IGRA IN SUBGROUPS Immune suppressed Does not appear affected by diabetes T-spot appears more sensitive for HIV and generally immunesuppressedcases Children Use of TNF blockers

38 CONCLUSION IGRA offer superior sensitivity and specificity for diagnosing LTBI Show potential in predicting risk of TB disease progression Convenience Currently already recommended as replacement for MTX

39 MDR-TB SENSITIVITY TESTING