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New Ways to Detect TB
all about
Interferon Gamma Release
Assays (IGRAs)
Scott Stienecker MD FACP FSHEA
Medical Director for Epidemiology and Infection
Prevention
Parkview Health
Office 266-1170, Cell 260-438-1426
Acknowledgements
• Some slides provided by: • Michael Knipp, MD
• Qiagen
• Quest
• Oxford Immunotec
• Centers for Disease Control and Prevention (CDC)
• Special thanks to:
• Dr. Richard B. Clark, PhD, D(ABMM), Scientific Director of Microbiology/Virology
Quest Diagnostics Nichols Institute
• Dr. Michael Knipp MD, Parkview Occupational/Employee Health
Famous People Who Reportedly Died of TB
• King Tutankhamen
• Cardinal Richelieu
• Doc Holliday
• Frederick Chopin
• John Keats
• Mozart
• Robert Louis
Stevenson
• Charlotte Bronte
• Eleanor Roosevelt
• Edgar Allan Poe
• George Orwell
• Vivien Leigh
• D.H. Lawrence
Tuberculosis in the U.S.
• 10-15 million people infected with latent TB (4%)*
• 9,951 new cases of active TB in 2013 (-4%); 80% of
cases result from reactivation of latent TB
• Targeted screening and treatment
• 18-20 million skin tests/year
• 50% performed in hospitals
*AJRCCM, 2008, 177, 348
Reported TB Cases United States, 1982–2012*
*Updated as of June 10, 2013.
0
5,000
10,000
15,000
20,000
25,000
30,000
No.
of C
ases
Year
Rate* of TB Cases in the US, 2013
* Per 100,000 population
MMWR, March 21, 2014 / 63(11);229-233
SOURCE: National TB Surveillance System.
Number of TB Cases in U.S.-born vs. Foreign-born Persons, United States, 1993–2012*
*Updated as of June 10, 2013
No.
of
Cases
0
5,000
10,000
15,000
20,000
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93
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94
19
95
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96
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U.S.-born Foreign-born
Complexity Caused by Immigration
• TB rate 13 times greater among foreign-
born
• Foreign-born: 15.6 cases/100,000
• US-born: 1.2 cases/100,000
• 65% of all TB cases occurred in
foreign-born individuals
• Higher number of foreign-born
individuals are BCG vaccinated MMWR, March 23, 2013
Transmission of TB
Family, friends,
workmates, etc.
exposed
Active TB
Infectious
Not infected
Infected, but no symptoms
“Latent TB infection (LTBI)”
If not identified & treated, ~10%-20% develop TB during
their lifetime
Not infectious
TB Surveillance Who should be tested?
High Risk of
Transmission
High Risk of
Progression/Reactivation
(Immunosuppressed)
High Risk of
Disease
•Healthcare workers •HIV •TB suspects
•Foreign-born •Rheumatoid arthritis
•Prisoners •End-stage renal disease
•Military personnel •Elderly
•TB contacts •Children
•Chronic care residents •Cancer chemotherapy
•Organ transplant
•Diabetes
Tuberculin Skin Test (In Routine Use Since 1910)
• Purified protein derivative (PPD) is
injected intradermally
• Polyvalent mixture of >200 TB proteins
• Measure size of reaction after 48-72 h
• Induration (firm area)
• Not erythema (redness)
Tuberculin Skin Test
• Limitations
• Reader variability (requires trained staff)
• Moderate sensitivity (esp. HIV, other immunocompromised patients)
• Variations of results (different anatomical site)
• Need for 2-4 visits
• Reported shortages with some vendor brands
• Poor specificity
• BCG vaccination (up to 80%)
• Non-TB environmental mycobacteria; cross-rx
with up to 30 AFB (up to 2% false +)
Picture courtesy of Neil W. Schluger, MD, Columbia University
Diagnosis of Latent TB Infections in US: A New ERA
• Interferon gamma release assays (IGRA)
• QuantiFERON®
• T-SPOT®.TB
TB Peptide Antigens
ESAT-6, TB7.7, and CFP-10
• Encoded by RD-1 & RD11 genes (not found in BCG strains)—BCG vaccinated patients do not test (+)—provides high specificity
• Induce IFN- responses
• Are TB-specific
• Absent from BCG and most non-TB Mycobacteria
• TB7.7 (QuantiFERON only)
QuantiFERON In Tube (QFT-GIT)
In the field:
• TB-specific antigen, Nil &
mitogen tubes
• Collect blood directly into tubes (Shake 10x to solubilize PHA/TB antigens)
In the lab:
• ELISA for detection of IFN-gamma
Field/Lab:
• Incubate and centrifuge
3. Add plasma and
conjugate to ELISA
plate. Incubate for
120 min. at room
temperature. 4. Wash and add substrate.
Read absorbance after 30 min 5. Software calculates
results and prints report
IFN- stable
refrigerated for at
least 4 weeks
Stage 1 – Blood incubation and harvesting
Stage 2 – Human IFN-γ ELISA
The ELISA stage is easily automated on existing machines.
2. Centrifuge tubes at 2,000-
3,000 x g for 5 minutes. 1. Collect 1 mL of blood (x3). Incubate 36-38ºC (16-24 h).
ESAT-6 CFP-10 TB 7.7
Nil Control
Mitogen Control
QuantiFERON-TB Gold In-Tube
QuantiFERON Results at Quest Diagnostics
• Reports contain a qualitative result and 3 quantitative results
• Possible qualitative results are
• Detected (positive)
• Not Detected (negative)
• Indeterminate
• Three quantitative results
• Nil (0-0.5 IU/ml)
• Mitogen – Nil ( 0.5 IU/ml)
• TB Antigen – Nil ( >0.35 IU/ml = Positive)
T-SPOT.TB Method
BD Sodium Citrate Vacutainer® CPT™
BD Vacutainer®
Lithium Heparin PST™
Greiner Bio-One Lithium Heparin
Vacuette®
T-SPOT.TB Results Interpretation
European:
>6 spots = Positive
US:
>8 spots = Positive
5-7 spots = Equivocal
<4 spots = Negative
Data sources: T-SPOT.TB PI-TB-US-V3 and CDC IGRA Guidelines MMWR (Jun,
2010) p.4
INVALID:
>10 spots in Nil control OR <20 spots in
Mitogen control (and not Positive or Equivocal)
27
MANUAL COUNTING OF SPOTS
IGRA - Indeterminate Results
Host Factors • Compromised immune status of test individual (ie, HIV CD4 counts <200
cells/mm3)
• Insufficient lymphocytes
• Inability of patient’s lymphocytes to generate gamma interferon
• Extremes of age (<5 and >80 years old)
• Lack of response to PHA by some individuals (<0.1%)
Advantages of IGRAs
• Requires single patient visit
• Results available within 24 h
• Not subject to reader interpretation
• Not affected by prior BCG vaccination
• Reduces number of X-rays
• Reduces INH usage and resulting liver
toxicity
• More sensitive and specific than TST
“In healthy persons who have a low likelihood both of TB infection and of progression to active TB, if infected, a single (+) IGRA result should not be taken as reliable evidence of TB.
Repeat testing, with either initial test or a different test, may be considered on a case by case basis.”
CDC, 2010
Equivocal/Wobble Zones
• Grey-zone for T-Spot.TB (5-7 spots)
established
• Grey-zone for QFT (not established)
--0.35-0.69 IU/mL (Pai et al., Marder et
al.)
--0.25-0.45 IU/mL (Graviss et al.)
--0.2-0.7 IU/mL (Metcalfe et al.)
--<1.11 IU/mL (Thanassi et al.)
Grouping of TB Ag minus Nil Values HCWs with no Risk Factors for TB
TB Ag-Nil Definition
<0.35 Negative
0.35-0.7 Low Positive
0.7-1.0 Intermediate Positive
>1.0 High Positive
David C. Marder, MD, University Illinois Chicago
Possible Management Algorithm for Persons With No History of TB Exposure
• TB Ag-Nil levels that are >0.35 should be considered for
being repeated.
• The test should be repeated within 1-3 months of the initial test.
• A third test is generally not necessary, but if it is done, it should
be repeated within 3 months of the initial test.
• Clinical treatment decisions should be made within 3 months of
an initial positive test.
David C. Marder, MD, UIC
Possible Management Algorithm for Persons With No History of TB Exposure (continued)
• All TB Ag-Nil values that remain above 1.0 for 2 tests should be
considered persistently infected and thus treated for LTBI.
• TB Ag-Nil levels that remain between 0.7 and 1.0 for two tests should be
monitored at 3-6 month intervals.
• TB Ag-Nil levels that remain between 0.35 and 0.7 for 2 tests should be
considered lowly infected and continued to be monitored annually (more
often if clinically indicated).
• All HCWs should continue to be tested according to their surveillance
cycle whether they are treated or not.
David C. Marder, MD, UIC
Parkview TB-Environmental Control
Assessment
• Medium Risk Facility at Randallia, LaGrange, and the
Oaks
• Surveillance system to assess effectiveness of
Environmental Controls
• Previously, used PPD
• 2012—changed to Quantiferon Gold
Why the Change from TB Skin Tests?
• The test measures Immune memory to TB specific antigens, and
therefore, offers Improved specificity
• The results are not subject to reader interpretation
• Results are not affected by the booster phenomenon (i.e., increased
sensitivity on subsequent test, leading to false-positive results)
• Accuracy is not affected by prior BCG vaccination
• Requires only 1 patient visit (2-4 required for TST) 2 step Tb Skin Test
is not necessary
• Cost savings from reduced data tracking time and avoidance of
unnecessary chest x-rays due to false positive reactions.
• The literature suggests an average cost savings of Quantiferon Gold is
about $10 per person being screened for TB.
2012 Program
• Total of 4395 Tests Performed
• New Positives were defined as those with a positive Quantiferon Gold Test
result without history of positive PPD
• Serial Testing was completed for all employess with a positive test.
• Reduced Positive Reactor List by 75%
• All Staff at PV Hospital, PRMC, The Oaks, and PV Lagrange were tested. In
addition, there was department specific testing completed.
Improved Specificity
Positive Reactor Data • In 2012 there were 277 active employees that were deemed as positive
reactors in 2011
• All were tested/serial tested in 2012 to establish a future baseline of positive
reactors
• Results show that 65 of the 277 past positive reactors were positive after the
first test IGRA test.
• Serial Testing further reduced the number of positive reactors from 65 to 59
for a total reduction of about 77%
Elimination of Subjective interpretation
• Review of the IGRA positive data from the compliance time period of
September 1st, 2012 to December 31st, 2012 showed 27 new positive
reactors.
• Review of the same data time frame in 2011 showed a single new positive
reactor.
• Of the new 2012 positives, 2 were treated for non-work related latent
tuberculosis
• 66% of the new positives had their negative 2011 Tb test read outside of
Occupational/Employee Health
Increased Pre-Hire Compliance Rates
• In 2011 there were 671 Pre-Hire exams between September 1st and
December 31st . Each exam required a two step TST yet only 976 tests were
administered. 28% required follow-up work
• During the same time period in 2012 there were 380 Pre-Hire exams and 389
IGRA tests administered. (16 positive tests that required re-testing meaning
that there were at least 7 indeterminate results) 6% required follow-up work
2012 Data
Summary
(Total Tests)
2012 Tests Sept.-Feb. Total 4395
2012 Initial Positives Total 2012 Retest Positives Total 202 29
Tb-nil 0.35-0.69 Total Retest Results Total
(Weak Positive) 67 Negatives 12
Weak Positives 3
Moderate Positives 2
Strong Positives 1
Terminated 1
Past Positive 12
Pre-Hire 2
No-show 5
Tb-nil 0.70-0.99 Total Retest Results Total
(Moderate Positive) 30 Negatives 7
Weak Positives 2
Moderate Positives 1
Strong Positives 2
Terminated 0
Past Positive 13
Pre-Hire 1
No-show 2
Tb-nil 1.0-10.0 Total Retest Results Total
(Strong Positive) 105 Negatives 29
Weak Positives 2
Moderate Positives 3
Strong Positives 13
Terminated 3
Past Positive 42
Pre-Hire Strong x 2 1
Pre-Hire 5
No-show 17
Strong X 2 (New) 3
2013 TB Program
• 4,620 Total Tests from February 1st 2013 to February 1st 2014 (Pre-
Hire and Annual).
• Modified criteria of “True/New” positive to be only those with results
of Strong Positive confirmed by serial testing. (This modification was
adjusted on the 2012 data on the next page)
• All “Moderate” and “Weak” positives were reviewed and
recommended to continue in the annual testing pool, not requiring
serial testing for 2013 screening as long as they were asymptomatic
regarding Active Tuberculosis symptoms
• Criteria for testing was modified to PV Randallia, PV Lagrange,
Oaks, and those individuals at PRMC who worked a total of 10
shifts in 2012. In addition, departmental criteria were added.
2013 Data
Summary
(Total Tests)
2013 Tests Feb.-Feb. Total
4620
2013 Initial Positives Total 2013 Retest Positives Total
148 16
Tb-nil 0.35-0.69 Total Retest Results Total
(Weak Positive) 64 Negatives 5
Weak Positives 3
Moderate Positives 2
Strong Positives 0
Terminated 2
Past Positive 14
Pre-Hire 18
No Retest 54
Tb-nil 0.70-0.99 Total Retest Results Total
(Moderate Positive) 26 Negatives 3
Weak Positives 1
Moderate Positives 0
Strong Positives 0
Terminated 1
Past Positive 11
Pre-Hire 6
No Retest 22
Tb-nil 1.0-10.0 Total Retest Results Total
(Strong Positive) 58 Negatives 13
Weak Positives 2
Moderate Positives 0
Strong Positives 8
Terminated 3
Past Positive 24
Pre-Hire Strong x 2 4
Pre-Hire 18
No Retest 34
Strong X 2 (New) 2