Latent Tuberculosis Infection: An Update on Diagnosis Christopher J. Crnich, MD MS June 13, 2007

TB: Immunopathogenesis Bacillus Destroyed Bacillus Multiplies

Distant Spread (Regional Lymph Nodes, Bloodstream) Stages of TB Infection Distant Spread (Regional Lymph Nodes, Bloodstream) Stage 2 (Logarithmic Growth) Stage 1 Unstimulated macrophages secrete IL-12 in response to intracellular replication of tuberculosis. IL-12 serves to activate NK cells which can kill infected macrophages in the absence of a CMI response. IL-12 also serves to recruit lymphocytes to the area of infection which then become activated when they are presented antigens in the MHC of dendritic cells

Stages of TB Infection Stage 3: Stage 4a: (Early DTH) (Late DTH) Multinucleated Giant Cell Un-activated Macrophage Activated Macrophage Caseous Necrosis Cytotoxic T-cell Good CMI Response Non-replicating bacillus Infected & Dying Macrophage Activated T-cells produce a number of cytokines including IL-2 (autocrine) as well as IFN-gamma and TNF-alpha IFN-gamma primes local macrophages making them capable of destroying phagocytosed bacilli Stage 3: (Early DTH) Stage 4a: (Late DTH)

(Caseous Liquefaction) TB: Reactivation Stage 5 (Caseous Liquefaction) In the presence of a good CMI, infection is controlled and the central casseous necrosis calcifies. For unclear reasons, probably related to reduced CMI, residual bacilli are able to move out of dormancy and cause reactivated infection with liquefaction of central necrosis. Liquefaction is an important step in reactivated disease as the bacillus is now able to multiply extracellularly and achieve a very high bioburden.

TB: Natural History

Rationale behind diagnosis and treatment of LTBI There are individuals who in whom there is: An increased risk of developing active TB over their lifetime (> 10%) Or, in which the consequences of developing TB, even if the risk of active disease would is low, would be catastrophic Treatment of LTBI reduces risk of developing active infection by 65% to 75% Risk of therapy of LTBI is low in studies published since 1991 Hospitalization: 1 in 1,500 Death: 1 in 3,300

Rates of active TB among selected populations ATS, MMWR 2000; 44(RR-6): 1-51

ATS Guidelines for TST ATS, MMWR 2000; 44(RR-6): 1-51 ≥ 5mm induration HIV-positive Recent immigrant from high-prevalence country Patients with no risk for LTBI Recent contacts of patients with active TB Injection drug users Fibrotic changes consistent with old TB Employees/residents of high-risk settings (i.e., prisons, hospitals, LTCF, homeless shelters) Prednisone use ≥15 mg/day for one month or more Employees of mycobacterial laboratories Organ transplant patients Patients with high risk medical conditions (i.e., diabetes mellitus, chronic renal failure, silicosis, malnutrition, jejunal bypass, gastrectomy, lymphoma/leukemia, or carcinoma of head/neck ATS, MMWR 2000; 44(RR-6): 1-51

Effectiveness of Isoniazid for Treatment of LTBI Comstock GW. Int J Tuberc Lung Dis 1999; 3(10): 347 - 50

Risk of hepatitis with LTBI treatment regimens LFTs > 5x ULN Clinical hepatitis Hospitalization rate Mortality rate INH (old) 1.0 5.0 INH (new) 0.1 - 0.15 0.04 RIF/PZA 26.4 18.9 3.0 0.9

TST: Using DTH to Indirectly Detect Latent TB Infection Intradermal PPD causes influx of lymphocytes, macrophages & monocytes Local production of cytokines: TNF-alpha, IFN-gamma, IL-10, IL-12 Release of serotonin & histamine leads to erythema and induration*

Palmer & Edwards, JAMA 1969; 205(3): 167-9 TST and Risk of TB Palmer & Edwards, JAMA 1969; 205(3): 167-9

Comstock et al. AM J Epidemiol 1974; 99(2): 131-8 TST and Risk of TB Comstock et al. AM J Epidemiol 1974; 99(2): 131-8

Limitations of TST Absolute operating characteristics unknown Significant problems with implementation Requires patient follow-up Significant inter-rater variability Prone to rounding up errors Prone to false-negatives (reduced sensitivity) Anergy Booster phenomenon Active tuberculosis (IL-10 effect) Prone to false-positive (reduced specificity) Due to rounding-up error Due to endemic NTM Due to prior BCG vaccination

Edwards et al. Am Rev Respir Dis 1969; 99(4): Suppl 1- 132

Edwards et al. Am Rev Respir Dis 1969; 99(4): Suppl 1- 132

False-Positives Due to NTM Am Rev Respir Dis 1992; 146(3): 752-6 Cross-reactivity with PPD-B explained 50% of the reactions to PPD-S in the 5-9mm range Cross-reactivity with PPD-B only explained 4% of the reactions to PPD-S in the 10+ range. PPD-B reactions in excess of 15mm seen in only 1.9% of subjects (and many of these may have been due to cross-reactivity with PPD-S) Reactivity to PPD-B decreased the longer the individual had resided in Montreal

False Positive Due to NTM - 784 students and HCWs in Northern and Southern medical centers tested with both PPD-S and MAS, 64 had PPD reactions in excess of 5mm. - Diagonal line separates PPD-dominant from MAS-dominant cases. Looking at this figure, only 4 (out of 23, 17%) cases of persons with a PPD in excess of 15mm would be attributable to NTM (and none with a reaction >20mm), however, a significant proportion of reactions between 10-14 are attributable to NTM. - This study suggests that there may be a role for IGRAs in testing of HCWs with skin reactions between 10-14mm but not 15+ Von Reyn et al. Int J Tuberc Lung Dis 2001; 5(12): 1122-8

False Positives Due to BCG Size Cutoff RR 95% CI P-value 5 mm >6 years 4.7 1.6 - 13.7 0.004 2-6 years 4.4 1.3 - 13.4 0.014 10 mm 2.1 1.5 - 3.0 <0.001 15 mm 2.7 0.6 - 11.7 NS Wang et al. Thorax 2002; 57(9): 804-9

False Positives Due to BCG Wang et al. Thorax 2002; 57(9): 804-9

False Positives Due to BCG Tissot et al. Clin Infect Dis 2005; 40: 211-7

False Positives Due to BCG RR 95% CI P-value BCG in infancy 2161/9712 1730/9004 1.2 1.1 - 1.2 <0.001 <15 years 736/5828 111/2138 2.4 2.0 - 3.0 >15 years 1343/2843 1538/3748 BCG after infancy 1028/2890 902/5192 2.1 1.9 - 2.2 41/141 11/378 10 5.3 - 18.9 918/2443 810/1696 0.8 0.7 - 0.8 Wang et al. Thorax 2002; 57(9): 804-9

Testing for LTBI: Historical Perspective Diagnosis of LTBI, 1907 Diagnosis of LTBI, 2007? Clemens von Pirquet is an allergist who came up with the idea to inject Koch’s bacillus intradermally to detect the presence of tuberculosis. Von Pirquet’s method was modified by Charles Mantoux and it became a standard diagnostic test for tuberculosis in 1907 Florence Seibert isolated a number of non-species dependent mycobacterial molecules from tuberculin by serially precipitation of Koch’s tuberculin in ammoniumsulfate, using a protein-free culture medium (PPD) PPD-S adopted as reference tuberculin in 1951, a new lot PPD-S2 was prepared and standardized in the 1990’s

Mazurek et al. MMWR Recomm Reports 2005; 54(RR15): 49-55

Interferon- Release Assays (IGRA) + APC T-Cell TB Antigen Interferon-

IGRA’s Antigen Are Specific ESAT = early secreted antigenic target-6 CFP = culture filtrate protein-10

Commercial IGRA’s ELISA-based tests ELISPOT-based tests Quantiferon-TB Quantiferon-TB Gold Quantiferon Gold In-Tube Assay ELISPOT-based tests T-SPOT.TB

QuantiFERON TB - Gold TB Neg. TB Pos. TB Pos. Indeterm. Indeterm. Nil Mitogen ESAT-6 CFP-10

QuantiFERON TB - Gold Mazurek et al. MMWR 2005; 54(RR-15): 49-55 ESAT-nil or CFP-nil or Both Nil Mitogen Result Interpretation ≥ 0.35 IU/mL and >50% above nil Any Positive TB likely < 0.35 IU/mL ≤ 0.7 ≥ 0.5 Negative TB unlikely* < 0.5 Indeterminate QFT-G cannot be interpreted ≤ 50% above nil > 0.7 Mazurek et al. MMWR 2005; 54(RR-15): 49-55

Mazurek et al. MMWR Recomm Reports 2005; 54(RR15): 49-55

Performance of IGRA’s Studies of patients with active TB Animal studies Cattle (M. bovis) Studies of patients with active TB Level of exposure studies Performance in low-prevalence populations

Developed in Cattle – An Excellent Model for Human TB Bovine TB is an excellent model for human TB Immune response to infection is very similar Most infected cattle have LTBI Active TB disease normally found only in old or undernourished animals M. bovis PPD injected intradermally and read 72 hrs later M. avium PPD is used as well for Comparative Testing

SID and IGRA in Cattle Wood et al. Vet Microbiol 1992; 31(1): 71-9 Necroscopy + - SID 15 58 73 7 1284 1291 22 1342 1362 Sens = 68.2 / Spec = 95.7 / PPV = 20.5 / NPV = 99.5 Necroscopy + - IGRA 18 30 48 4 1312 1316 22 1342 1362 Sens = 81.8 / Spec = 97.8 / PPV = 37.5 / NPV = 99.7 Wood et al. Vet Microbiol 1992; 31(1): 71-9

Menzies et al. Ann Intern Med 2007; 146(5): 340-54 IGRA’s in Active TB Test Studies (n) Sensitivity 95% CI TST 5 mm 9 0.74 0.66 - 0.82 10 mm 4 0.72 0.50 - 0.95 15 mm 1 0.40 0.25 - 0.56 QuantiFERON QFT-G 0.80 0.73 - 0.87 In-Tube 3 0.67 0.56 - 0.78 T-SPOT.TB ESAT-6 0.93 0.91 - 0.96 ESAT-6/CFP-10 0.87 0.78 - 0.95 Only three studies comparing QFT and T-SPOT.TB have been published. In general, T-SPOT.TB more sensitive than QFT-G In patients with active TB. Menzies et al. Ann Intern Med 2007; 146(5): 340-54

IGRAs: Exposure Level Kang et al. JAMA 2005; 293(22): 2756-61

IGRA’s: Exposure Level Ewer et al. Lancet 2003; 361(9364): 1168-73

IGRA’s: Exposure Level Exposure Gradient High Moderate Low Very Low (N) 48 1,019 99 98 QFT 44% 23% 4% 2% TST 71% 26% 51% 351 541 461 560 T-SPOT.TB 45% 37% 25% 19% 60% 34% 22% Menzies et al. Ann Intern Med 2007; 146(5): 340-54

IGRA’s: Exposure Level Arend et al. Am J Respir Crit Care Med 2007; 175(6): 618-27

IGRA’s Reduce BCG Effect Study of 60 medical students and 17 patients with confirmed TB. QFT-PPD assessed before and after BCG vaccination. Likewise, IFN-production in whole blood upon exposure to ESAT-6 antigen was assessed before and after vaccination. Johnson et al. Clin Diagn Lab Immunol 1999; 6(6): 934-7

Menzies et al. Ann Intern Med 2007; 146(5): 340-54 Specificity of IGRA’s Menzies et al. Ann Intern Med 2007; 146(5): 340-54

IGRA’s: The Good IGRA’s are more sensitive than TST in patient’s with active TB T-SPOT.TB is more “sensitive” than QFT-G but associated with lower “specificity” QFT-G In-Tube may be less sensitive than QFT-G Although all tests are imperfect in the diagnosis of TB

Lee et al. Eur Respir J 2006; 28(1): 24-30 QFT-G vs. T-SPOT.TB Active TB Low-Risk Lee et al. Eur Respir J 2006; 28(1): 24-30

Lee et al. Eur Respir J 2006; 28(1): 24-30 QFT-G vs. T-SPOT.TB Total of 87 patients with active TB, T-SPOT identified 83 (Sens = 96.6), QFT-G only 61 (70.1), and TST only 58 (67). There was more overlap between the TST and T-SPOT than between the QFT-G and TST. 131 subjects in low-risk group. This slide also highlights discordance between IGRA’s, more on this later. Active TB Low-Risk Lee et al. Eur Respir J 2006; 28(1): 24-30

IGRA’s: The Good IGRA’s are more sensitive than TST in patient’s with active TB T-SPOT.TB is more “sensitive” than QFT-G but associated with lower “specificity” QFT-G In-Tube may be less sensitive than QFT-G Although all tests are imperfect in the diagnosis of TB

IGRA’s: The Good IGRA’s are more sensitive than TST in patient’s with active TB T-SPOT.TB is more “sensitive” than QFT-G but associated with lower “specificity” QFT-G In-Tube may be less sensitive than QFT-G IGRA’s correlate better with level of exposure than TST in contact tracing studies Although all tests are imperfect in the diagnosis of TB

IGRA’s: The Good IGRA’s are more sensitive than TST in patient’s with active TB T-SPOT.TB is more “sensitive” than QFT-G but associated with lower “specificity” QFT-G In-Tube may be less sensitive than QFT-G IGRA’s correlate better with level of exposure than TST in contact tracing studies IGRA’s do not appear to be influenced by prior BCG status, TST clearly is Although all tests are imperfect in the diagnosis of TB

IGRA’s: The Bad No data on predictive ability for the development of TB Few published effectiveness studies Limited data on performance in immunosuppressed patients Limited data on performance in children Problem with indeterminate results

Rangaka et al. Am J Respir Crit Care Med 2007 (in press) IGRA’s and HIV Study of 160 South African patients, 74 HIV(+) and 86 HIV(-) Statistically significant differences for TST and HIV status but not significant with the IGRA’s Rangaka et al. Am J Respir Crit Care Med 2007 (in press)

Brock et al. Respir Res 2006; 7: 56-64 IGRA’s and HIV With decreasing CD$, there is a lower response to PHA and a higher likelihood of an indeterminate response Brock et al. Respir Res 2006; 7: 56-64

Dogra et al. J Infect 2007; 54(3): 267-76 IGRA’s in Children TST Result QFT Result Agreement(%) Kappa (95% CI) QFT(+) QFT(-) Total TST cut-point ≥ 5mm TST(+) 8 16 89.5 0.53 (0.34 - 0.71) TST(-) 3 86 89 11 94 105 TST cut-point ≥ 10mm 2 10 95.2 0.73 (0.53 - 0.92) 92 95 TST cut-point ≥ 15mm 4 93.3 0.50 (0.33 - 0.66) 7 101 Study performed in children with a suspicion of TB hospitalized in an Indian hospital 133 screened and 105 children enrolled and underwent both TST and QGT-IT All 105 children had an interpretable test Dogra et al. J Infect 2007; 54(3): 267-76

Connell et al. Thorax; 61(7): 616-20 IGRA’s in Children QFT-G Diagnosis (-) (+) Indeterminate Total Uninfected 38 (76%) 12 (24%) 50 LTBI 26 (62%) 11 (26%) 5 (12%) 42 TB 9 (100%) 9 64 20 17 101 Study performed in 106 children hospitalized in an Australian hospital with a high risk of TB (exposure, symptoms, or immigration within 5 years) 17% of QFT-G tests indeterminate (12 because of increased nil background and 5 because of inadequate mitogen response) 13 of 26 subjects with LTBI and discordant results on QFT-G had skin induration in excess of 15 mm and 21 of 42 subjects with LTBI had not had BCG Connell et al. Thorax; 61(7): 616-20

Indeterminacy with IGRA’s 318 persons tested with QFT-G at an Italian facility 70% of tests ordered for suspicion of TB or contact with TB 10% ordered for screening prior to immunosuppressive therapy 68/318 (21.4%) of tests were indeterminate QFT-G Result Variable Indeterminate Determinate OR (95% CI) P-value Immunosuppressive Therapy 26 39 3.35 (1.84 - 6.08) <0.001 Cancer 8 33 1.07 (0.49 - 2.30) 0.855 Age <3 or >80 11 1.46 (0.68 - 3.12) 0.324 HIV (+) 2 5 2.53 (0.55 - 11.6) 0.230 Renal Failure 4 6 2.26 (0.62 - 8.25) 0.214 Ferrara et al. Am J Respir Crit Care Med 2005; 172(5): 631-5

Indeterminacy by Type of IGRA 10% of QFT-G tests indeterminate versus 3.1% for T-SPOT.TB (p < 0.0001) Ferrara et al. Lancet 2006; 367(9519): 1328-34

IGRA’s: The Ugly Test variability Conversions Reversions Discordance between different types of IGRA’s QFT-G versus T-SPOT.TB QFT-G versus QFT-IT

Pai et al. Am J Respir Crit Care Med 2006; 174(3): 349-55 IGRA vs. TST Conversion 22 nurses or medical students had a TST or QFT-IT conversion on follow-up. The 6 cases in which there was an increase in the TST by at least 10mm was always associated with large increases in QFT-IT results. Pai et al. Am J Respir Crit Care Med 2006; 174(3): 349-55

Wilkinson et al. J Infect Dis 2006; 193(3): 354-9 IGRA Reversions Wilkinson et al. J Infect Dis 2006; 193(3): 354-9

Are Reversions Due to Wobble Around Cut-Point? Baseline IFN- level No. Retested No. That Received INH (%) Total Reversions Incidence of Reversions (%) P-value for Trend 0.35 - 0.69 11 1 (9%) 6 6/11 (55%) < 0.01 0.70 - 1.00 2 0 (0) 1 1/2 (50%) 1.10 - 5.00 9 2 (33) 1/9 (11) > 5.00 16 9 (56) 1/16 (6) Total 38 13 (34) 9/38 (24) Pai et al. Am J Respir Crit Care Med 2006; 174(3): 349-55

Or Is It Something More Fundamentally Immunological? TST+ QFT-G+ ELISPOT+ LST+ No. (%) INH Treatment No 19 17/19 (95) 8/19 (42) 8/17 (24) 16/17 (94) Yes 8 8/8 (100) 1/8 (12.5) 3/7 (43) 6/7 (86) 27 subjects with diagnosis of LTBI on the basis of TST during a contact investigation and screening in high risk groups. 2 of these subjects had induration of 8 and 9 mm respectively and were therefore not listed as positive. Leyton et al. Clin Vaccine Immunol 2007; (in press)

Summary IGRA reversions are common Reversions most likely when: There is discordance with the TST (TST-/IGRA+) IGRA near cut-point (0.35 IU/mL for QFT-G and 20 SFC/106 WBCs) Reasons for reversions likely a combination of: Decay of peripheral effector cells Natural biologic wobble Variation due to laboratory procedures

Ferrara et al. Lancet 2006; 367(9519): 1328-34 IGRA Agreement T-SPOT.TB + - I QFT-G 94 6 100 40 191 9 240 10 30 3 43 144 227 12 383 Agreement = 0.75 Kappa = 0.53 Ferrara et al. Lancet 2006; 367(9519): 1328-34

Mahomed et al. Int J Tuberc Lung Dis 2006; 10(3): 310-6 IGRA Agreement QFT-GIT + - QFT-G 123 14 137 78 143 221 201 157 358 Agreement = 0.74 Kappa = 0.50 Mahomed et al. Int J Tuberc Lung Dis 2006; 10(3): 310-6

Summary Discordance between different IGRA tests is very common Reasons? QFT-GIT has an additional antigen (TbA 7.7 in addition to ESAT-6 & CFP-10) Intrinsic differences due to features of the tests (ELISA versus ELISPOT) Misspecification of cut-points

Improving Concordance Arend et al. Am J Respir Crit Care Med 2007; 175(6): 618-27

My Interpretation IGRA’s can be a useful adjunctive test in: Patients with active TB Contact investigations IGRA’s cannot be used as a rule-out test in these situations, particularly in: Immunosuppressed patients Children

My Interpretation IGRA’s are not a gold standard for determining the presence of LTBI There is still no way to determine which test is correct when there is discordance IGRA’s have a high rate of conversions and reversions There is significant discordance between different types of IGRA’s The only way to establish the true value of IGRA’s is to perform longitudinal studies to determine who progresses to active TB

My Interpretation IGRA’s should not replace the TST Using IGRA’s alone for serially surveillance of HCWs may paradoxically increase treatment for LTBI I believe we should still be using the TST If not, looking at absolute increases in IFN level rather than using a threshhold should be done IGRA’s can be a useful supplement to the TST in select populations Patients with h/o BCG Patients with TST between 10-15mm and no risk factors (TST in excess of 18mm should definitely be considered LTBI)