Transcript QuantiFERON Summary

Interferon Gamma Release Assays
(IGRAs) for the Diagnosis of TB:
Can We Replace the TST?
Helene M. Calvet, MD
Health Officer and TB Controller
Long Beach Department of Health and Human
Services
Overview
• Diagnosis of active and latent TB
• In-Vitro Interferon Gamma Release Assays
(IGRAs)
• Sensitivity and specificity of IGRAs
• Implementation data from San Francisco
and Long Beach
Definitions
• “Positive PPD”: a tuberculin skin test (TST)
that is indurated:
– >5 mm: HIV+, recent contact of TB case, CXR
c/w old TB, organ transplant or other
immunosuppression
– >10 mm: everybody else (in California)
• Latent TB Infection (LTBI): TB infection
without evidence of clinically active disease
(+PPD, but no symptoms); CXR usually
normal, or may be abnormal, but sputa
negative
• TB Disease: active tuberculous infection of
any organ
Diagnosis of TB
• TB disease
• Latent TB infection (LTBI)
Toolbox for Diagnosis of TB
Disease
Pathology
History
Physical Exam
TST
Sputa
IGRAs
Cultures
PCR
Gold
Standard =
Culture
CXR
Response to therapy
Toolbox for Diagnosis of Latent TB
TST
History
IGRAs
CXR
No Gold Standard!
Comparison of Toolboxes
TB Disease Toolbox
LTBI Toolbox
Abundant tools
Paucity of tools
Gold standard
No gold standard
No need to rely on one test
May need to rely on one test
The TST…A Very Old, and Not-So-Perfect Test
• Discovered by Koch in 1890, skin test agents
standardized in 1976
• False negatives:
–
–
–
–
–
–
Anergy (immunocompromise or malnutrition)
Recent TB infection
Very young age (< 6 months)
Overwhelming TB disease
Recent live virus vaccination
Poor placement or reading of TST
• False positives:
– BCG or nontuberculous mycobacterial infections
– Inaccurate reading of TST
• Requires two visits…substantial proportion do not
come for reading
Tuberculin Skin Testing
Mantoux Method
5 TU of PPD
48 to 72 hours
Interpretation depends
on person’s risk factors
BCG and TST (1)
• General teaching is that reactivity from BCG wanes
after a few years and is unlikely to persist > 10 years,
but may be boosted by PPD.
• Study done in Switzerland* suggests that false
positives due to BCG may be much more common
than we thought:
– 40% of 5000 HCW had positive TST
– Prior BCG strongest risk factor for positive TST among
those less than age 40 with TSTs <18 mm (was not as
strong a risk factor for those > 40 years old and those with
TSTs > 20 mm)
*CID 2005; 40:211 – 217.
BCG and TST (2)
• Review of studies that compared TST responses to
BCG during and after infancy
• Vaccination during infancy estimated to cause
false-positive TST in 6.3% overall, but only 1% of
those tested more than 10 years after vaccination
• Vaccination at 2 years of age or older estimated to
cause false-positive TST in 40% of persons
overall, 20% of those tested 10 years or more after
vaccination
Farhat M et al, Int J Tuberc Lung Dis 2006; 10: 1192-204
In-Vitro Interferon Gamma Release
Assays (IGRAs) for TB
• QuantiFERON® TB Gold (QFT-G): FDA
approved
• QuantiFERON® In-Tube (QFT-IT):
submitted for FDA approval
• T-Spot TB : submitted for FDA approval
QuantiFERON® (QFT) History
• First generation (QuantiFERON ® – TB) test
FDA-approved in 2001
• 2003 CDC guidelines advise use of test in selected
groups only
• Second generation test (QuantiFERON® - TB
Gold) FDA-approved March 2005
• CDC guidelines December 2005* allow use of
QuantiFERON® - TB Gold (QFT-G) in any
situation in which a TST would be used; however,
points out lack of data in many groups (pediatrics,
immunocompromised, etc.)
*MMWR 2005, 54 (RR-15): 49-55
In Vivo and In Vitro Diagnostic
Tests
Presentation of
mycobacterial antigens
IFN-
TNF-
IL-8, etc.
IFN-
Antigen
presenting
cell
Memory
T-cell
Andersen P, et al. Lancet 2000;356:1099
TNF-
IL-8, etc.
Why Measure Interferon-?
•
•
•
•
•
•
TB infection induces T-cell response (CMI)
IFN-  is the ‘classic’ CMI cytokine
Produced in vitro in response to specific antigen
Secreted in measurable and stable amounts
Absent from normal circulation
Extensive literature showing importance of IFN- in
TB infection
QuantiFERON®-TB Gold Principle
• Based on the quantitative measurement of IFN-
secreted from stimulated T cells in human whole blood
– T-cells reactive to M. tuberculosis - specific antigens are only
present in those infected
• A two stage process:
– Incubation of whole blood with TB-specific and control
antigens (mitogen) and nil control
– Detection of IFN- using a rapid, single-step, sandwich
ELISA
Species specificity of ESAT-6 and CFP-10
Tuberculosis
complex
Environmental
strains
Antigens
ESAT
Antigens
ESAT
CFP
+
+
+
-
+
+
+
-
CFP
M tuberculosis
+
+
M africanum
+
+
M bovis
+
+
gothenburg
-
-
moreau
-
-
tice
-
-
tokyo
-
-
danish
-
-
glaxo
-
-
montreal
-
-
pasteur
-
-
BCG substrain
M abcessus
M avium
M branderi
M celatum
M chelonae
M fortuitum
M gordonii
M intracellulare
M kansasii
M malmoense
M marinum
M oenavense
M scrofulaceum
M smegmatis
M szulgai
M terrae
M vaccae
M xenopi
Whole Blood IFN- Assay
QuantiFERON-TB Test
Stage 1 Whole Blood Culture
Nil
ESAT-6 CFP 10Mitogen
Control
Control
Aliquot blood
& add antigen
Draw blood
+ heparin
Stage 2 IFN-gamma ELISA
Incubate → INF-
from sensitized Tcells
COLOR
TMB
Harvest plasma
from above settled
cells
Measure [ IFN- ] in
‘Sandwich’ ELISA
Computerized
interpretation
Cellestis
QuantiFERON®-TB Gold Test Method
Advantages and Disadvantages
• Advantages:
– Only one visit required
– Objective and reproducible; not operator-dependent
– No cross reactivity with BCG, little cross-reactivity with
non-tuberculous mycobacteria
– Controls for low or no immune response
– No chance of ulceration due to brisk skin test reaction
• Disadvantages:
– Blood must be received in lab within 12 hours
– Labor intensive for the lab
– Not much data for some patient groups
T-Spot.TB: “Six easy Steps”
Nil Control
Infection
Infection
Positive Control
Oxford Immunotec
IGRA Possible Results
• Positive: indicates TB infection, does not
differentiate between active disease and
latent infection
• Negative: no TB infection
• Indeterminate: not sure
What Does an Indeterminate Mean?
• Indeterminate can occur as a result of low mitogen
response (due to patient immunocompromise, poor
specimen handling or storage, lab error, etc.) or high
nil response (due to patient illness, recent
vaccinations, etc.)
• Estimated rate of indeterminates for QGT-G:
approximately 1-2% among HCW, about 5-10%
among patients
• Upon retesting, approximately ½ of the
indeterminates come out with a definitive result
(unpublished data)
• Retesting indeterminates once, and if indeterminate
again, stop
• Indeterminate rate much higher among children
Estimating Sensitivity and
Specificity of IGRAs
• Sensitivity in people with culture + TB
Sen = # positives / # tested
• Specificity in people at low risk for TB
infection
Spec = # negative / # tested
QFT-G Sensitivity Estimates
Reference
Population
+ IFN- (n) + TST (n)
Mori; 2004
Untreated Cult+TB;
Japan
89% (118)
66% (76)
Kang; 2005
Pulmonary TB; Korea
81% (54)
78% (54)
CDC; Unpub.
Untreated Cult+TB; US
81% (41)
81% (41)
Ravn; 2005
Active TB; Denmark
85% (48)
Not done
Lee, 2006
Active TB, Korea
70% (61)
67% (58)
Menzies* 2007 Meta-analysis (9 studies) 80% (393)
74% (394)
*Menzies, D. et al, Annals of Int Med 2007;146 (5): 340-354
TB Suspects: QFT-G Performance
• Among 242 suspects, 23 of 37 had culture-confirmed
tuberculosis and +QFT-G results
• QFT-G sensitivity: only 64% (95% CI, 48% - 78%),
but negative predictive value 89%
• Sensitivity of the TST was 88% in this review
• Very poor performance in extrapulmonary TB (14%
sensitivity), but numbers were low
• Conclusion: lowish sensitivity in TB suspects means
it’s probably not all that useful for that purpose
Dewan et al, Clin Infect Dis 2007, 44:69-73.
Why Poor Performance in Active TB?
• CMI response likely diminished in active TB,
particularly in those with more advanced disease,
malnutrition and older age
• If their CMI was working well, they wouldn’t
have TB disease; so TB disease likely not a great
surrogate for LTBI
• Pooled sensitivity from 10 studies: 75% (71-78%)
• Lower cutoff may be needed in those who are
highly suspect to have TB
Pai and Menzies, Clin Infect Dis 2007; 44: 74 - 77
QFT-G Specificity Estimates
Reference
Population
+ IFN- (n)
+ TST (n)
Mori; 2004
Nursing Students; Japan
2% (213)
65% (113)
Kang; 2005
Med Students; Korea
4% (99)
51% (99)
CDC;
Unpub.
Navy recruits; US
.2% (532)
.9% (532)
Menzies*
2007
Meta-analysis (9 studies)
3% (711)
+BCG: 44% (516)
No BCG: 2% (156)
*Menzies, D. et al, Annals of Int Med 2007;146 (5): 340-354
T-Spot TB Sensitivity Estimates
Reference
Population
+ IFN-
(n)
+ TST (n)
Lalvani; 2001 Untreated Cult+TB; UK
96% (45)
69% (32)
Pathan, 2001
Active TB, UK
92% (33)
N/A
Meier, 2005
Active TB, Germany
97% (70)
N/A
Lee, 2006
Active TB; Korea
95% (83)
67% (58)
Menzies*
2007
Meta-analysis (11 studies)
88% (557) 74% (394)
*Menzies, D. et al, Annals of Int Med 2007;146 (5): 340-354
T-Spot TB Specificity Estimates
Population
+ IFN-
(n)
Spec.
Lalvani; 2001
Low risk BCG-vaccinated
subjects, UK
0% (26)
100%
Pathan, 2001
Low risk BCG-vaccinated
subjects, UK
0% (32)
100%
Lalvani, 2001
Low risk BCG-vaccinated
subjects, India
0% (40)
100%
Menzies* 2007
Meta-analysis (4 studies)
Reference
+ BCG: 44% (516)
8% (229)
No BCG: 2% (156)
*Menzies, D. et al, Annals of Int Med 2007;146 (5): 340-354
T-SPOT.TB vs. QFT-TB Gold!
Comparison of T-Spot.TB and QFTTB Gold
• Site - Italy
• Design - Prospective study of 393 consecutively
enrolled patients with LTBI or suspected TB
• Agreement with the TST and the two IGRAs was
similar, but T-Spot TB more likely to yield positive
results in close contacts
• Indeterminate results were more common with QFTTB Gold than T-Spot.TB (11% vs 3%).
• Indeterminate results were more likely in young
children (< 5 yrs) and immunosuppressive treatment
Ferrara G, et al. Lancet. 2006;367:1328-1334
Comparison of Test Results
Among Contacts
TST
All contacts
QFT-G
62 (54%)
T-SPOT.TB
25 (22%)
39 (34%)
<0.0001
<0.0001
Non-BCG vac.
contacts
36 (43%)
21 (25%)
0.0001
0.0923
Ferrara G, et al. Lancet. 2006;367:1328-1334
29 (35%)
Comparison of T-Spot.TB and
QFT-TB Gold
Test results
Active TB (%)
Low risk for TB
No. of subjects
87
131
TST + (≥ 10 mm)
58 (66.7)
28 (21.4)
QFT-G positive
61 (70.1)
11 (8.4)
T-SPOT.TB positive
83 (95.4)
20 (15.3)
Lee JY, et al. ERJ. 2006
Three Ways to Interpret This Data
• QFT-G is less sensitive than TST and TSpot TB
OR
• QFT-G is a lot more specific than TST and
T-Spot TB
OR
• A little bit of both
Diagnosis of TB: The Truth*?
QFT-G
T-Spot TB?
TST
T-Spot TB
QFT-G?
TST
Truth
Latent
Active
QFT-G
TST
T-Spot TB
Sensitivity
Specificity
* My opinion only, based on impression of available data
QuantiFERON Testing: The San
Francisco Experience
• Implemented QuantiFERON-TB (QFT-1)
screening in 4 community clinic sites (2
homeless, 1 methadone, 1 immigrant) and TB
clinic in November 2003
• Switched to QuantiFERON-TB Gold (QFT-G) in
March 2005
• Over 6100 samples run between March 2005 and
February 2006
QFT-G Test Results by Age Category
March 2005 – February 2006
100%
Percent
80%
60%
40%
20%
0%
Indeterminate
Negative
Positive (%)
<5
(3)
5 to 14
(1)
15 to
24
(6)
25 to
44
(7)
45 to
64
(12)
65 to
84
(31)
TB Infection Prevalence by Test
and Clinic Type
Homeless
TB
Clinic
Methadone
Immigrant
TST
(2001-2003)
26%
>50%
10%
37%
QFT-1
(11/04-2/05)
17 %
n=1848
48 %
n=292
18 %
n=346
37 %
n=344
QFT-Gold
(3/05-2/06)
6%
n=3594
26 %
N=693
4%
n=546
12 %
n=626
77%
48%
60%
66%
Decline in
positive rate
from TST
QFT Implementation – LB Experience
• Lab was experienced with the technology after
doing a contact investigation study in a dialysis
center using QFT in 2003
– Over 120 patients and staff tested twice
– QFT-Gold found to be more closely associated with
contact to case, less affected by albumin, good
reproducibility
• Waited for FDA approval of QFT-Gold and
completion of lab reconstruction before
implementing
• Performed validation study Nov. 2005 started
running patient samples in Jan. 2006
QFT in the Lab
First Experience
• From January through June 2006, 193
samples run on patients in TB clinic
• Some testing also done in Occupational
Health
• Of 193 samples in TB, 137 (71% foreignborn, 130 (67%) with history of BCG
• Many samples done as “confirmatory”
testing for positive TST
Results in First 6 Months
• 108 (56%) negative, 57 (30%) positive and 25
(13%) indeterminate
• Excluding indeterminates, 65% negative and 35%
positive
• Of those with known prior TSTs (n = 100,
excluding indeterminates)
– 33 (33%) positive QFT-G
– 67 (67%) negative QFT-G
• Average size of prior TST by QFT-G result
category
– QFT-G positive: median 18 mm, (range 10 -30)
– QFT-G negative: median 14 mm (range 10 – 30)
QFT-G in Occupational Health (OH)
• Police recruit class, May ’06:
– 80 healthy young folks
– 30% positive, 14% indeterminate
– Much higher positivity rate than historical, but no prior
tests
• Firefighter screening, September ‘06
– 384 healthy, low risk firefighters with prior negative
TSTs
– 19% positive, 5% indeterminates, 76% negative
• What the heck was going on?
Tube B or Not Tube B…?
• QFT-G users had been reporting unusual results
for several months
• High nil values noted in many of the positive
samples in LB firefighters in September
• October 3 letter from Cellestis notifying users of
increased numbers of indeterminates and false
positives (usually with high nil values) associated
with certain lots of Becton Dickinson Na heparin
tubes; recommended switching to lithium heparin
tubes
High Nil Value
• High nil value does not always lead to an
indeterminate result
– If reactivity to ESAT-6 or CFP-10 is > 50% higher than
nil, computer calls it positive
• Not sure cause for this outcome: blood drawing
technique, inadequate mixing, lab phenomenon,
contaminated tubes etc.
• SF also had experience with this, and change of
blood drawing venue took care of it
• Planned to repeat Fireman testing using both Na
heparin and lithium heparin tubes
Repeat Testing on Firefighters
• 93 firefighters retested January 2007 with both
lithium and sodium heparin tubes
• 1/92 (1%) positive on both (subsequently found to
have hx of prior +TST), 86/92 (93%) negative on
both, 5/92 (5%) discordant
• Among 5 discordant, all positive on lithium
heparin but negative on sodium heparin; repeat of
assay gave same result on sodium heparin, but
varying results (+, -, indeterminate) on lithium
heparin = likely gray zone, or borderline, values
• So, is lithium heparin really better or not?
QFT Reproducibility
 Reproducibility good, but conversions and
reversions possible
 Actual breakpoints of conversion & reversion not
well defined (aside from change from positive to
negative or back)
 Since normal variations not known, thresholds not
established (as for TST)
 Conversion and reversion more likely with results near
gray zone, or for results discordant with the TST.
 What is the natural history of T-cell response to
TB infection??
Facilitating Interpretation
 Might it be better to report continuous variables
(actual IFN readings) instead of dichotomous
variables (positive/negative)?
 Could assist clinician in interpreting the results
 Might give more data in serial tests to help determine
conversion versus normal variation
 Recent recurrent problems with false positives
(associated with high nil values) have led the state
to suggest that labs should call all specimens with
high nil value indeterminate, regardless of the
ESAT-6 and CFP-10 values
QFT-G: My Concerns about Sensitivity
• There definitely are false positive skin tests, but are
there really that many??
• The test relies on effector lymphocytes, which have
recently encountered antigen in vivo, to produce
interferon within hours
• Memory cells, which have encountered antigen in
the distant past, would require several days in the
presence of antigen to produce interferon
• Could we be missing distant, latent infection that is
not immunologically active? (I think we may be)
….and/or…
• Could this be telling us who has infection that is
requiring more of an immunological response, and
thus possibly at increased risk of progression??
The IGRA Research Agenda
• Group of experts met March 2006
• Comprehensive Research Agenda Generated
• 58 key questions in 7 general areas






Biological issues
Test performance in high-risk populations
Test reproducibility & serial testing
Responses during treatment
Epidemiological & field applications
Health systems & economic research
Pai, M et al, Lancet Infect Dis 2007; 7: 428-38
Key Questions (1)
• What are appropriate cut-off points for different
groups?
• What is the normal variation in T-cell responses?
• What is the risk of active disease in those with
positive and negative IGRA results? How does
this relate to risk associated with positive TST?
• Is there a cut-off point that will predict incipient
active TB?
• How should conversions and reversions be defined
(incrementally), and how often do they occur?
Pai, M et al, Lancet Infect Dis 2007; 7: 428-38
Key Questions (2)
 What is the biological basis for discordance
between IGRAs and TST?
 What is the role of IGRAs in monitoring response
to therapy of active or latent TB?
 Can IGRAs be used to revise estimates of
worldwide TB infection and the lifetime risk of
TB disease?
 How useful is the combined approach of using
TST to screen and IGRA to confirm?
Pai, M et al, Lancet Infect Dis 2007; 7: 428-38
Things to Think About in Implementing
QFT-G in Occupational Health
• Realize the limitations and benefits of the test!
– Lowish sensitivity for LTBI, but probably highest specificity,
especially in BCG-vaccinated population
•
•
•
•
•
Cost of tests and turn-around time
Educating everybody involved about this test
Specimen collection, handling and transport
Obtaining appropriate waivers for use in HCW screening
Changing all documentation that has TST on it (screening
cards, charts, forms, etc.) to include TST results
• What to do with indeterminates
• Mode of implementation: stepwise, or across –the –board?
• Relationship with your lab; tracking indeterminates,
identifying problems
IGRAs: Summary
• QFT-G:
– Like the TST, not a perfect test!
– Very specific (usually), but somewhat lacking in
sensitivity
– Revised cut-offs and quantitative reporting may be
helpful
– 3rd generation submitted for FDA-approval; will be
easier for clinicians (does not need to be to lab in 12
hours), easier for lab
• T-Spot TB:
–
–
–
–
–
Superior sensitivity to QFT-G, but …
Possibly less specific than QFT-G
Much more difficult from lab standpoint
More expensive
Not yet commercially available
IGRAs: Summary (2)
• IGRAs represent an exciting new
opportunity to learn more about a very old
disease
• IGRAs are not perfect tests, so clinicians
need to be aware of their limitations
• Can we replace the TST? – Not yet!
Questions?