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

Disclosures

• Nothing to disclose

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

19

93

19

94

19

95

19

96

19

97

19

98

19

99

20

00

20

01

20

02

20

03

20

04

20

05

20

06

20

07

20

08

20

09

20

10

20

11

20

12

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

Presentation of Mycobacterial Antigens

Image courtesy of Neil Schluger, MD; Columbia University

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

Cows and IGRAs—Who knew?

How to apply a PPD on a hairy animal.

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)

No Cross-Reactivity to BCG and Most NTMs Among IGRAs

20

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

26

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

2013 Annual

Program

Initial Positives by Entity

Entity Number Tested % Initial Positive

HMHOS 49 10.20

LGHOS 201 5.97

NBHOS 45 6.67

OHPVN 31 0.00

PVHHC 190 5.79

PVHOS 731 1.92

PVNOR 1310 2.52

PVPBH 186 3.76

PVPMG 278 4.68

PVSYS 303 3.63

PWLTC 94 8.51

WMHOS 50 4.00

ALL 3468 3.43