Value of TB immunodiagnostic tests in immuno-compromised hosts (case-scenarios)

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Value of TB immunodiagnostic tests in
immuno-compromised hosts (case-scenarios)

• Morten Ruhwald, MD, PhD
• Martina Sester, PhD

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Who is using IGRA?
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Case

• 75 year old Danish woman with Reumatoid Arthritis
• Methotrexate steroid injections
• TNF-a inhibitor candidate
• TST negative
• X-ray normal
• No exposure
• QFT-G (heparin version) indeterminate (low PHA)

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• October 2005
• Infliximab x 4
• Remission of RA symptoms

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• March 2006
• Pleuraeffusion, cough
• Stopped Infliximab 2-3 months
• Microscopy, PCR, culture for TB was neg

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• June 2006
• Worsening of RA symptoms
• Effusion resorbed, no cough
• gt Infliximab x 2

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• August 2006
• Loss of appetite
• Upset stomach
• Ultrasound Ascites
• TST negative
• QFT In Tube test positive
• Ascites
• PCR pos for M.tuberculosis complex
• Resistant to Pyrazinamide?

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• Culture revealed
• M.bovis

Vang-Larsen and Ravn ERJ 2007
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Conclusion

• Bovine TB can reactivate during TNF-a inhibition
• IGRA can diagnose M.bovis infection
• Inconclusive test should be interpreted with
  caution and repeated to rule out technical error

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Outline

• Theory
• Why does immunosuppression influence the IGRA and
  TST
• T-SPOT.TB vs QFT-IT
• Can we monitor immunosuppression with the IGRA?
  And what about TST?
• Common problems - common cases
• HIV
• Autoimmune diseases
• ESRD
• Transplant recipients/drug-mediated
  immunosuppression
• Summary/Conclusions

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How does immunosuppression influence the IGRA and
TST?
IFN-?
APC
T-cell
antigen
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How does immunosuppression influence the IGRA and
TST?
Corticosteroids
Calcineurine Inhibitors
IFN-?
APC
T-cell
antigen
Depleting antibodies
ESRD
Cancer therapy
HIV infection
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TB and immunosuppression
Likelihood of TB reactivation
immunosuppression
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IGRA and TST perform suboptimal in patients with
immunosuppression
T-SPOT.TB
Quantiferon
Likelihood of true positive result
TST
Indeterminate cut off
immunosuppression
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TST and IGRAs in immunocompromised
TST QFT T-SPOT.TB
Adjusts for cell function no no no
Adjusts for cell number no no yes
Modified cut off for immunocompromised (yes) no no
Indeterminate option/positive controls no yes yes
Info on immune status? no (yes) (yes)
Reliablility in immunocompromised little some more than some
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• HIV

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HIV and TB A dangerous liason
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Nunn P et al. Nature Reviews Immunol 2005
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Natural course of HIV infection
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Pantaleo et al NEJM 1993
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HIV infection and test performance
false negative
IGRA indeterminate results
loss of test positivity
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Pantaleo et al NEJM 1993
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Case

• A 25-year-old HIV infected, ART naive, African
  American man
• CD4 count 32 cells/µL, HIV-1 RNA 298,000
  copies/mL.
• Chronic watery diarrhea, subjective fevers,
  chills, and 15 kg weight loss over 1 month
• No respiratory symptoms
• Mantoux skin test 0mm
• QFT-IT negative (Antigen 0.15 IU/ml, Mitogen 0.8
  IU/ml )
• Results of stool and blood cultures and of fungal
  antigen testing were negative for bacterial,
  mycobacterial, and fungal pathogens
• He had no cough and was unable to produce an
  induced sputum specimen

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Modified from Manabe et al JID 2009
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Chest x ray

• A chest radiograph was notable for a linear
  soft-tissue opacity at the right tracheal border,
  reported by the radiologist as being compatible
  with vasculature

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Modified from Manabe et al JID 2009
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• 20 days post ART, the patient presented with a
  fever and cough
• Repeat Mantoux test 24mm
• QFT-IT positive
• Antigen 1.25IU/ml, Mitogen 3.2 IU/ml)
• A chest radiograph
• extensive consolidation of the right upper lobe
  with areas of cavitation and a heterogeneous mass
  extending 9 cm from the right midneck down the
  medial right hemithorax.
• .

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Modified from Manabe et al JID 2009
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• Induced sputum
• negative for acid-fast bacilli (AFB)
• Bronchoalveolar lavage
• AFB smear negative
• lymph node (transbronchial needle) aspirate
  obtained at bronchoscopy was positive for AFB
• All sputa and BAL culture were positive for M.
  tuberculosis with in vitro sensitivity to all
  first-line anti-TB drugs
• Conclusion TB-IRIS

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Modified from Manabe et al JID 2009
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Prevalence of positive TST result correlates with
CD4 count
3711 Swiss HIV-infected
/lt 5mm
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Elzi et al CID 2007
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Also the T-SPOT.TB is affected by low CD4 count
in HIV infected, but much less than the TST
247 HIV-infected individuals from Senegal
T-SPOT.TB
TST
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Karam et al PlosONE 2008
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and the Quantiferon is somewhere in the middle
109 HIV infected from Uganda
Quantiferon
T-SPOT.TB
TST
(n10) (n33) (n66)
TST 5mm 10mm 15mm
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Leidl et al ERJ 2010
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IGRA positivity rate is affected by CD4 count,
but the indeterminate option ensures acceptable
sensitivity(using active TB as gold standard)
65 HIV infected TB patients from Tanzania
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Aabye et al PlosONE 2009
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Screening and target treatment of HIV with a
positive test reduces the risk of TB
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Elzi et al CID 2007
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QFT-IT predicts risk of progression to active TB
in HIV infected (but numbers are small!)
Aichelburg et al 2009, Austria Median follow-up
19 months Progression rate 8 (3/37)
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HIV take home messages

• TB is the most common opportunistic infection in
  HIV infected
• HIV leads to loss of CD4 positive T cells
• T-cells are essential for test performance, and
  HIV affects IGRA and TST performance
• In patients with a low CD4 count
• Expect more indeterminate results (especially
  QFT)
• Be aware of the increased risk of false negative
  results
• T-SPOT.TB corrects for the cell count and
  probably has better performance in HIV infected

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• Candidates for - and patients on - TNF antagonist
  therapy

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TNF-antagonist therapy - case
49yrs old male with severe Ankylosing
spondylitis Treated with infliximab and
methotrexate Screened with TST (negative) and
QFT-IT (indeterminate) Regarded as low risk of
LTBI Develops disseminated TB 4 months into
infliximab treatment (Ravn et al Ugeskr Lae 2009
17123)
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Corticosteroids but not other DMARDs severely
affects cellular responsiveness

• 248 patients patients with
• autoimmune diseases
• Before stating up TNF-a blockers
• 156 rheumatoid arthritis or
• spondyloarthropathy
• 93 Crohn's disease
• or ulcerative colitis

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Bélard E, Ruhwald M, Ravn P et al in prep.
Presented at DDW May 2010
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and leads to fewer TST positives and more
Quantiferon indeterminates
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Bélard E, Ruhwald M, Ravn P et al in prep.
Presented at DDW May 2010
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TNF-a blockage inhibits T cell immunity
Hamdi Arthr ResearchTher 2006, 8 R 114.
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Reduced positivity rate of QFT-IT when treated
with of TNF-a antagonists
Matulis 2007, Annals of the Rheumatic Diseases
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DMARDs TNF-a inhibition take home messages

• In patients on immmunosuppressive treatment
• Expect more indeterminate results (especially
  QFT)
• Be aware of the increased risk of false negative
  results (Look at the mitogen response!)
• Be aware of Corticosteroids and TNF-a inhibitors
• Screen for TB before initiating DMARDs

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• ESRD

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How does immunosuppression influence the IGRA and
TST?
Corticosteroids
Calcineurin inhibitors
IFN-?
APC
T-cell
antigen
Depleting antibodies
ESRD
Cancer therapy
HIV infection
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Girndt et al. Kidney Int (1993)44 359
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End stage renal disease - case

• 81 year old man with chronic renal failure
• Dysuria and polacisuria since several weeks
• Short hospital stay because of heart
  insufficiency during the last month
• Admission because of
• loss of appetite
• reduced physical health and
• azotemia (S-Kreatinine 5,2 mg/dl)
• Previous history is unknown at time of admission
• Assessing medical history is complicated by
  language problems (born in Croatia)

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End stage renal disease - case

• Physical examination is inapparent except
• Generally reduced fitness
• Slight pulmonary dry rales at the basal part on
  both sides
• No flank pain, no fever, no swollen lymphnodes
• Laboratory findings
• ESRD urea 135mg/dl  creatinine 5,8mg/dl
• Inflammation with CRP of 93 mg/l
• Urine analysis
• Leukocytes negative  Erythrocytes 25/µl 
  Glucose normal  Proteins 150mg/dl
• Ultrasound
• Right kidney small and hard to identify, stones
  in the pyelon
• Left kidney still of normal size, but already
  narrowed and dense parenchyma
• Liver, spleen etc. inapparent

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End stage renal disease - case

• On the following day patient had chronic cough
• X-ray of the chest
• Reticular infiltrates on both sides with
  predominance of the apexes
• CT-scan of the chest
• Chronic fibrotic lesions of the lung with apical
  predominance
• No acute inflammatory signs
• Similar, but less pronounced changes were found
  on a chest X-ray taken two years ago
  (retrospectively)
• Bronchioscopy
• Antrachosis
• Pyogenic bronchitis
• ? Microbiological examination AFB

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End stage renal disease - case

• Dialysis treatment had been initiated
• TST negative
• IGRA
• T-cell reactivity towards ESAT-6/CFP-10 negative
• T-cell reactivity towards PPD positive
  (flow-cytometry)
• Positive for IFN-g, negative for IL-2
  (flow-cytometry)
• ? Standard anti-tuberculosis therapy was applied
  with doses adapted to renal function

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Conclusion - Case

• Despite massive pulmonary changes, TB can be
  oligosymptomatic in patients with renal failure
• TST is often negative in ESRD patients
• IGRA may be negative in ESRD patients
• Modified IGRA may confirm TB infection

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Summary of published literature-ESRD patients-
Study Year Country n TST positive IGRA positive () indeterminates () agreement IGRA with TST (k)
Passalent 2007 USA 203 12 35 (T.SPOT.TB) 6 (T.SPOT.TB) 0.25 (T.SPOT.TB)
Winthrop 2008 USA 100 24 22 (QFT) 28 (T.SPOT.TB) no data 0.43 (QFT) 0.42 (T.SPOT.TB)
Lee 2008 Taiwan 32 62 40 (QFT) 46 (T.SPOT.TB) 6 (QFT) 0.25 (QFT) 0.32 (T.SPOT.TB)
Triverio 2009 Switzerland 62 19 21 (QFT) 29 (T.SPOT.TB) 8 11 0.16 (QFT) 0.32 (T.SPOT.TB)
Seyhan, 2010 Turkey 100 34 43 (QFT) 0 0.26 (QFT)
TBNET study Germany Greece Italy SwitzerlandTurkey 262 26 26.7 (QFT) 27.1 (T.SPOT.TB) 3.8 (QFT) 5.7 (T.SPOT.TB) 0.32 (QFT) 0.28 (T.SPOT.TB)
Agreement between TST and IGRA is only fair to
moderate
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• TRANSPLANT RECIPIENTS/
• DRUG MEDIATED IMMUNOSUPPRESSION

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Case - Identification of M. tuberculosis
infection by IGRA, not by TST-patient with
azathioprine due to Crohns disease-

• Asymptomatic man receiving maintenance
  azathioprine therapy for Crohns disease
• Wife had multidrug-resistant pulmonary
  tuberculosis
• Negative tuberculin skin test result
• Positive ELISPOT assay result
• High-resolution computed tomography of the chest
  showed consolidation with early cavitation
• Bronchoalveolar lavage and culture confirmed
  multidrug-resistant tuberculosis

Richeldi et al 2004 Ann Int Med 140 709
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Identification of M. tuberculosis infection by
IGRA, not by TST-patient with azathioprine due
to Crohns disease-
Richeldi et al 2004 Ann Int Med 140 709
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How does immunosuppression influence the IGRA and
TST?
Corticosteroids
Calcineurin inhibitors
IFN-?
APC
T-cell
antigen
Depleting antibodies
ESRD
Cancer therapy
HIV infection
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IGRAT-cell interferon-g release assays
PPD ESAT-6/CFP-10/TB7.7 Negative
controls Positive controls, i.e. PHA/SEB
Cytokine- induction
Cytokine- induction
Cytokine- induction
ELISPOT Assay T-SPOT.TB
Flow-Cytometry
ELISA QuantiFERON
8h
24h
24h
1ml
8-10ml
3ml
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Dose-dependent decrease in specific T-cell
reactivity in the presence of CNI
reactive T cells
MFI (amount of cytokine/cell)
Stimulus PPD, same data for ESAT-6/CFP-10
reactivity
Sester et al Eur Resp J (2009) 34 702
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IGRA are unaffected by maintenance levels of
immunosuppressive drugs
Long-term transplant recipients Stimulus PPD
Sester et al. Kidney Int (2004) 65 1826 Sester
et al. Nephrol Dial Transplant (2006) 21 3258
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IGRA are unaffected by maintenance levels of
immunosuppressive drugs
prevalence
52/107 48.6
61/117 52.1
68/127 53.5
0.23
0.22
0.18
controls
hemodialysis
renal Tx
Sester et al. Kidney Int (2004) 65 1826 Sester
et al. Nephrol Dial Transplant (2006) 21 3258
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Decrease in specific immunity early after
transplantation
Sester et al Eur Resp J (2009) 34 702
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Lower frequencies of PPD reactiveT cells in lung
transplant recipients
53.1
16.3
n49 gt12 months post Tx
Maintenanceimmunosuppression Low and high
DL
Sester et al Eur Resp J (2009) 34 702
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European Multicenterstudy
Greece I. Gerogianni
Bulgaria R. Markova
Sweden J. Bruchfeld/I. Julander
Denmark P. Ravn
Netherlands F. van Leth
Switzerland J.P. Janssens/P. Soccal
Germany F. Behrens C. Lange/M. Ernst M. Sester D.
Wagner T. Wolf
Portugal R. Duarte
Turkey F. Eyuboglu/A.G. Dilektasi A. Yalcin
Romania D. Bumbacea O. Caliman-Sturdza

• U.K.
• Lalvani
• H. Milburn

Italy D. Cirillo/A. Matteelli D. Goletti/E.
Girardi G.B. Migliori
Spain J. Dominguez/I. Latorre
Australia J. Rothel
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Patients

• End-stage renal disease
• Solid organ transplantation
• lung, liver, kidney, kidney-pancreas
• Stem cell transplantation
• Rheumatoid arthritis
• HIV infection
• high/low CD4 counts
• Immunocompetent low-risk controls
• Immunocompromised patients with active TB

1600 individuals
QuantiFERONTB Gold in tube
T-SPOT.TB
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• FUTURE PERSPECTIVES

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Improving IGRAs

• We should consider interpreting IGRA
  incorporating the mitogen response
• Mitogen-adjusted algorithm?
• Alternative biomarkers (e.g. IP-10 or
  multiplexing markers)
• Modified IGRAs for assessing disease activity
• Comparison of blood and local compartments
• Changes in cytokine profiling/phenotype

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Use the mitogen response
Patient A
Patient B
5.0
0.6
Cut off
Cut off
0.3
0.3
Antigen
Mitogen
Antigen
Mitogen
negative
negative
? Who would be more likely to be falsely negative?
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Improving IGRAs

• We should consider interpreting IGRA
  incorporating the mitogen response
• Mitogen-adjusted algorithm?
• Alternative biomarkers (e.g. IP-10 or
  multiplexing markers)
• Modified IGRAs for assessing disease activity
• Comparison of blood and local compartments
• Changes in cytokine profiling/phenotype

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Increase of specific T-cell frequencies in BAL
blood
blood
BAL
BAL
Jafari et al Am J Resp Crit Care Med (2006) 174
1048
Barry et al. J Inf Dis (2003) 187 243
Jafari et al Am J Resp Crit Care Med (2009) 180
666
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Improving IGRAs

• We should consider interpreting IGRA
  incorporating the mitogen response
• Mitogen-adjusted algorithm?
• Alternative biomarkers (e.g. IP-10 or
  multiplexing markers)
• Modified IGRAs for assessing disease activity
• Comparison of blood and local compartments
• Changes in cytokine profiling/phenotype

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Loss of IL-2 is indicative of active TB
T-TB
A-TB
IFNg/IL-2 pos. lt56 Specificity
100 Sensitivity 70
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• CONCLUSIONS

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IGRA and immunosuppression

• Indeterminate results as indicator for severe
  immunosuppression
• IGRAs are much less influenced by
  immunosuppression than TST
• T-SPOT.TB have less indeterminate results and may
  be more sensitive than Quantiferon TB Gold
• More studies are needed to assess
• the effect of the extent of immunosuppression on
  IGRA results
• the prognostic value of IGRA for development of
  active TB

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Practical take home messages

• Screen for LTBI/TB before starting initiating
  immunosuppressive treatment
• Dont take no for a definite answer
• Look at the mitogen response when interpreting
  IGRA results
• Need for lowering the cut off for patients with
  immunosuppression?
• Inacceptable sensitivity and specificity for
  active tuberculosis