Value of TB immunodiagnostic tests
in immuno-compromised hosts
(case-scenarios)
Morten Ruhwald, MD, PhD
Martina Sester, PhD
2
Who is using IGRA?
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)
October 2005
– Infliximab x 4
– Remission of RA symptoms
March 2006
– Pleuraeffusion, cough
– Stopped Infliximab 2-3 months
– Microscopy, PCR, culture for TB was neg
June 2006
– Worsening of RA symptoms
– Effusion resorbed, no cough
– > Infliximab x 2
7
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?
• Culture revealed
M.bovis
Vang-Larsen and Ravn ERJ 2007
Conclusion
• Bovine TB can reactivate during TNF-α inhibition
• IGRA can diagnose M.bovis infection
• Inconclusive test should be interpreted with caution
and repeated to rule out technical error
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
11
How does immunosuppression
influence the IGRA and TST?
IFN-γ
antigen
APC
T-cell
12
How does immunosuppression
influence the IGRA and TST?
Corticosteroids
Calcineurine Inhibitors
IFN-γ
antigen
APC
T-cell
Depleting
antibodies
ESRD
Cancer therapy
HIV infection
13
Likelihood of TB reactivation
TB and immunosuppression
immunosuppression
14
Likelihood of true positive result
IGRA and TST perform suboptimal in patients
with immunosuppression
Indeterminate
cut off
immunosuppression
15
TST and IGRAs in immunocompromised
TST
QFT
T-SPOT.TB
Adjusts for cell function
no
no
no
Adjusts for cell number
no
no
yes
(yes)
no
no
Indeterminate option/positive controls
no
yes
yes
Info on immune status?
no
(yes)
(yes)
little
some
more than some
Modified cut off for immunocompromised
Reliablility in immunocompromised
16
HIV
17
HIV and TB: A dangerous liason
18
Nunn P et al. Nature Reviews Immunol 2005
Natural course of HIV infection
Pantaleo et al NEJM 1993
19
HIV infection and test performance
false negative
IGRA
indeterminate
results
loss of test
positivity
Pantaleo et al NEJM 1993
20
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
Modified from Manabe et al JID 2009
21
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
Modified from Manabe et al JID 2009
22
• 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.
.
Modified from Manabe et al JID 2009
23
• 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
Modified from Manabe et al JID 2009
24
Prevalence of positive TST result correlates with
CD4 count
3711 Swiss HIV-infected
=/< 5mm
Elzi et al CID 2007
25
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
Karam et al PlosONE 2008
26
…and the Quantiferon is somewhere in
the middle
109 HIV infected from Uganda
Quantiferon
T-SPOT.TB
TST
(n=10)
(n=33)
(n=66)
TST: ≥ 5mm
Leidl et al ERJ 2010
≥10mm ≥15mm
27
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
Aabye et al PlosONE 2009
28
Screening and target treatment of HIV+
with a positive test reduces the risk of TB
Elzi et al CID 2007
29
QFT-IT predicts risk of progression to active
TB in HIV infected (but numbers are small!)
830 HIV
positive
37 QFT-IT
positive
738 QFT-IT
negative
3 active TB
0 active TB
Aichelburg et al 2009, Austria
Median follow-up 19 months
Progression rate 8% (3/37)
30
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
31
Candidates for - and
patients on - TNF
antagonist therapy
32
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: 171:23)
33
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
34
Bélard E, Ruhwald M, Ravn P et al in prep. Presented at DDW May 2010
…and leads to fewer TST positives and
more Quantiferon indeterminates
35
Bélard E, Ruhwald M, Ravn P et al in prep. Presented at DDW May 2010
TNF-a
blockage
inhibits
T cell
immunity
Hamdi Arthr ResearchTher 2006, 8 R 114.
Reduced positivity rate of QFT-IT
when treated with of TNF-a antagonists
Matulis 2007, Annals of the Rheumatic Diseases
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
38
ESRD
39
How does immunosuppression
influence the IGRA and TST?
Corticosteroids
Calcineurin inhibitors
IFN-γ
antigen
APC
T-cell
Depleting
antibodies
ESRD
Cancer therapy
HIV infection
40
Girndt et al. Kidney Int (1993):44: 359
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)
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
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 +++
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
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
Summary of published literature
-ESRD patientsStudy
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
Switzerland
Turkey
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
TRANSPLANT RECIPIENTS/
DRUG MEDIATED
IMMUNOSUPPRESSION
47
Case - Identification of M. tuberculosis
infection by IGRA, not by TST
-patient with azathioprine due to Crohn´s disease• Asymptomatic man receiving maintenance
azathioprine therapy for Crohn´s 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
Identification of M. tuberculosis
infection by IGRA, not by TST
-patient with azathioprine due to Crohn´s disease-
Richeldi et al 2004 Ann Int Med 140: 709
How does immunosuppression
influence the IGRA and TST?
Corticosteroids
Calcineurin inhibitors
IFN-γ
antigen
APC
T-cell
Depleting
antibodies
ESRD
Cancer therapy
HIV infection
50
IGRA
„T-cell interferon-g release assays“
PPD
ESAT-6/CFP-10/TB7.7
Negative controls
Positive controls, i.e. PHA/SEB
Cytokineinduction
Cytokineinduction
Cytokineinduction
Flow-Cytometry
ELISPOT Assay
T-SPOT.TB
ELISA
QuantiFERON
8h
24h
24h
≥1ml
8-10ml
3ml
Dose-dependent decrease in
specific T-cell reactivity in the presence of CNI
renal Tx
lung Tx
% 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
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
IGRA are unaffected by maintenance levels of
immunosuppressive drugs
prevalence
52/107
48.6%
68/127
53.5%
0.23%
0.18%
controls
61/117
52.1%
hemodialysis
0.22%
renal Tx
Sester et al. Kidney Int (2004) 65: 1826; Sester et al. Nephrol Dial Transplant (2006) 21: 3258
Decrease in specific immunity
early after transplantation
Sester et al Eur Resp J (2009) 34: 702
Lower frequencies of PPD reactive
T cells in lung transplant recipients
53.1%
16.3%
n=49
>12 months post Tx
Maintenance
immunosuppression
Low and high
DL
Sester et al Eur Resp J (2009) 34: 702
European Multicenterstudy
Bulgaria
Greece
Sweden
R. Markova
I. Gerogianni
J. Bruchfeld/I. Julander
Denmark
P. Ravn
Germany
F. Behrens
C. Lange/M. Ernst
M. Sester
D. Wagner
T. Wolf
Netherlands
F. van Leth
Portugal
R. Duarte
Switzerland
J.P. Janssens/P. Soccal
Turkey
F. Eyuboglu/A.G. Dilektasi
A. Yalcin
Romania
D. Bumbacea
O. Caliman-Sturdza
U.K.
A. Lalvani
H. Milburn
Italy
D. Cirillo/A. Matteelli
D. Goletti/E. Girardi
G.B. Migliori
Spain
J. Dominguez/I. Latorre
Australia
J. Rothel
Patients
•
•
End-stage renal disease
Solid organ transplantation
–
•
•
•
lung, liver, kidney, kidney-pancreas
Stem cell transplantation
Rheumatoid arthritis
HIV infection
–
•
•
T-SPOT.TB
1600
individuals
high/low CD4 counts
Immunocompetent low-risk controls
Immunocompromised patients with active TB
QuantiFERON
TB Gold in
tube
FUTURE
PERSPECTIVES
59
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
60
Use the mitogen response
Patient A
Patient B
5.0
0.6
Cut off
Cut off
0.3
Antigen
Mitogen
negative
0.3
Antigen
Mitogen
negative
 Who would be more likely to be falsely negative?
61
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
62
Increase of specific T-cell frequencies in BAL
blood
blood
BAL
BAL
Barry et al. J Inf Dis (2003) 187: 243
Jafari et al Am J Resp Crit Care Med (2006) 174: 1048
Jafari et al Am J Resp Crit Care Med (2009) 180: 666
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
64
Loss of IL-2 is indicative of active TB
T-TB
A-TB
IFNg/IL-2 pos. <56%
Specificity: 100%
Sensitivity: 70%
CONCLUSIONS
66
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
Practical take home messages
• Screen for LTBI/TB before starting initiating
immunosuppressive treatment
• Don’t 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
68