pai m et al. clin. microbiol. rev. 2014;27:3-20

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David Koelle, MD

Allergy and Infectious Diseases, Department of Medicine, UW

Thanks to:Anne Cent, Supervisor, UW Clinical Virology Lab

G P L d T h l i t

“Understanding IGRAs”

Greg Pepper, Lead TechnologistRoxanne Porteus, Technical Secretary

Chris McClurkan, Research TechnologistMasae Kawamura, Qiagen

Janene Stageberg, Oxford Immunotec

Presentation contains quality control/quality improvement data. Do not reproduce without permission.

Concept of a TB spectrumnot just

“LTBI” vs. “ATBI”

ATBI

Positive IGRA

MTBdisease

Pai M et al. Clin. Microbiol. Rev. 2014;27:3-20

LTBI or TST

Themes• QFT/IGRA history and technology

• Real life use and results in a complex medical setting

• Grey zones, pain points, and inappropriate uses

– Arghh those *$)@#*@* intedeterminates• Is there ever any useful medical information in an indet result?

– (+) near the cutoff in a low‐risk patient– What patients are inappropriate for QFT?– Conversions and Reversions from QFT (‐) to QFT (+), vice versa– Does a TST prime for a true or false (+) QFT?

– Blood test for tuberculosis disease?

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How do IGRA work?

Is Interferon Gamma Released? We Assay this.

MTB has 4,000 genes including the RD1 region encoding proteins ESAT6, CFP10

The IGRA TB antigen is peptides from ESAT6, CFP10, ± TB7.7

TB peptides stimulate memory T‐cells in blood that release IFN‐gamma upon exposure to the peptides

FDA approved:Quantiferon Gold or In‐tube (QFT‐IT)T‐SPOT‐TB

IGRA origins lie in veterinary medicine

Randall Reves Univ Colorado

Unique aspects of QuantiFERON‐IT

• Living T‐cells and other cells required

• Advanced collection tubes that are also reaction tubes– Pre bleed

• Specific manufacturing• Shelf life and storage temperature constraints

– Bleed• Shaking requirement “10 karate chops”• No line contaminants from advanced catheters

– After bleed• Time and temperature from bleed to lab• Options to start the lab phase near the bleed site and then transport

Almost every other blood test: lower tech tube, lower stringency at each step

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MTB GENOME

CFP10 and ESAT6test for immune memoryto these MTB proteins

4 X 106 bp

RD1 = Region of Difference 1 deleted in BCG: fundamental advantage over TST deleted in almost all non‐tuberculous mycobacteria

Strains with RD1 DNA and therefore potentially “false” (+) IGRA:

• MTB complex: • M. bovis, M. africanum, M. microti, M. canetti

• NTM:• M. kansasii, M. szulgai, M. marinum

“Fish Finger Disease” Mycobacterium marinum

Tuberculosis‐like lung disease Mycobacterium kansasii

(+) IGRA in addition to MTB

Likes cooler temperatures (fingers…)Aquarium hobbyistsMarine biologistsFisheries industry

“water bug”2nd commonest NTM to affect lung

(#1 MAC #3 abscessus)Underlying lung disease common

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Quantiferon workflow in pictures

Draw 3 X 1 ml To lab < 16 hours 37 oC 16‐24 hour Spin save plasma

Do ELISA for interferon‐gamma

~ 2016: Quantiferon workflow in pictures

Draw 4 X 1 ml To lab < 16 hours 37 oC 16‐24 hour Spin save plasma

Do ELISA for interferon‐gamma

Generations of QuantiFERON• 1st 2001 QuantiFERON‐TB (QFT) for humans

– Used PPD as test antigen just like TST– Same BCG pitfall as TST: possible false (+)

• 2nd 2005 QuantiFERON‐Gold– Switched to MTB peptides– Avoids BCG pitfall

• 3rd 2007 QuantiFERON‐TB Gold In‐Tube (QFT‐GIT)3 2007 QuantiFERON TB Gold In Tube (QFT GIT)– Avoids need to purify lymphocytes from blood– Starts with direct whole blood drawn into special tubes– A whole new set of issues....

• 4th ~ 2016 “QFT‐Plus”– MTB antigens different and in 2 tubes

• 4 tubes overall: 4 numbers (let us confuse people even more)– Won’t help “false positive, low MT‐nil, low‐risk person” specificity issue

– May improve detection of ATBI and splitting ATBI from LTBI– 2015 CE‐labeled for Europe; 2016 US FDA submission planned

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– (+) near the cutoff in a low‐risk patient– What patients are inappropriate for QFT?– Conversions from QFT (‐) to QFT (+)– Reversions from QFT (+) to QFT (‐)– Does a TST prime for a true or false (+) QFT?– Blood test for tuberculosis disease?

QFT‐Plus• Nil (grey) = as per current product

• TB1 (green) = ESAT‐6 and CFP‐10 CD4 peptides

• TB2 (yellow) = ESAT‐6 and CFP‐10 CD4 and CD8 peptides

• Mitogen (purple) = as per current product

14• In‐lab phase as per current product

QFT‐Plus: potential role for diagnosis of ATBI?

TB2 – TB1 (nil subtracted)Observed ∼0.5 IU/ml difference between TB1 and TB2 in active disease

151. QuantiFERON‐TB Gold Plus Package Insert. October 2014. 1083163EN.

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QFT‐IT at UW Medical Center system

me of QFT results

Month starting on indicated date

Total volu

All QFT test ordered 2009‐April 2015

Test sites

800

QFT-IT at UW 2009-2014: Major Users

of te

st re

sults

Immune suppressed High suspicion LTBI or TB diseaseOccupational health settingInpatient/EDExternal usersUW/HMC/UWPN outpt clinics

80

Num

ber o

7

All QFT results 2009‐end April 2015

T t it R lt ll t tTest sites Results all tests

Results all outpatient tests Results all inpatient tests

All QFT results 2009‐end April 2015

T t it R lt ll t tTest sites Results all tests

Results all outpatient tests Results all inpatient tests

Results trends: all tests

results


Percen

t of

8


results


Percen

t of

Indet rate averagingaround 7%: quite highfor a medical test


results


Percen

t of

high indetIndet rate averagingaround 7%: quite highfor a medical test

Outpatient tests

results


Percen

t of

high indet

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Inpatient tests

results


Percen

t of

high indet





– (+) near the cutoff in a low‐risk patient – What patients/uses are inappropriate for QFT?– Conversions from QFT (‐) to QFT (+)– Reversions from QFT (+) to QFT (‐)– Does a TST prime for a true or false (+) QFT?– Blood test for tuberculosis disease?

Sources of variability in the QuantiFERON-TB Gold In-Tube assay.


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!!!!

Issue 1 Endotoxin 2012

Lipopolysaccharide (=endotoxin = LPS) in the TB antigen tube

product recall Sep 27 2012 bad tubes Jan 6‐ Aug 2 2012

also happened with high altitude tubes early 2013

also happened in 2009 in Japan and Europe

LPS acts on TLR4 to activate immunity → high IFN gamma

Uh‐oh clinical impact: potential false (+)

Issue 2: mitogen too weak 2013‐2014 (?)

A mitogen is a substance thatcauses mitosis and also cell

activation, in this case including ll i bl d

Potential clinical impact: too many indeterminate tests

memory T cells in blood

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• PHA = Phytohemagglutinin a bean protein

– uncooked red kidney beans (highest concentrations)

– white kidney beans (cannellini), green, broad beans

Meet your MIT: PHA

• It is a protein and biologically fragile

• It is multivalent and effects correlate with aggregation

• Agglutinates (cross‐links) “accessory cells” e.g. monocytes to T‐cells


Patientvariables


n=78 SCCA samples with abs lymph within 5 d of QFT-IT

neg or pos indet

Seattle Cancer Care Alliance QFT data: low lymphocyte counts correlate with indeterminate results

n=65 n=13abs lymph

0 0 10-1000 35 10

1000-2000 22 22000-3000 6 03000-4000 1 0

>4000 1 0

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Other variables associated with QFT indeterminate results

• HIV (+) CD4 < 200

• Liver failure

• Immune suppressive drugs


PRE‐LAB


Site of draw is definitely associated with indeterminate rateCombined UWMC/HMC/UWPN/outside user data

Neg

Pos11%

Indet6%

Outpatient

Indet11%

ED/ER

N=14779

N=169

83%

Neg65%Pos

6%

Indet29%

Inpatient Neg70%

Pos19%

11%

N=1528

13

0

100

200

300

400

500

600

no of results

minutes between blood draw and temp upshift

negative

100

120

140

Indeterminate

Delay between blood draw and incubation @ 37 oCseems not to account for IND issue at UW


0

5

10

15

20

25

30

35

40

45

no of results


Positive

0

20

40

60

80

no of results

minutes between blood draw and temp upshiftTime between blood draw and 37 oC, min

Time between blood draw and 37 oC, min



in‐LAB

Those )%*#) intedeterminates

• Optimally, an indeterminate result tells you that MTB infection data cannot be obtained from the QFT‐IT test– Low lymphocyte count– Low lymphocyte count

– Low lymphocyte activation potential

• Optimally, an improvement over the current TST in which “anergy” cannot be diagnosed

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Results from UWMC pre‐transplant outpatient clinic

PHA problem summer 2013

Pre‐transplant getting a little better….

The other IGRA

T‐SPOT‐TB

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Plusses and minuses of T‐SPOT‐TB• Plusses

– Compensates for lymphopenia (low lymphocytes per ml in patient blood sample as in some immune suppressed persons)

– Separates lymphocytes from liver/kidney toxins: might help

– Interpretive criteria recognizes “gray zone” reality • Four “results”: pos borderline=equivocal neg (plus• Four results : pos, borderline=equivocal, neg (plus indeterminate=invalid)

– Interpretive criteria recognize reality that pre‐test LTBI probability influences the positivity cutoff (Bayesian probability)

• Developing world: high pre‐test probability: high likelihood that a few spots are a true positive: fewer spots needed to call positive

• Developed world: low pre‐test probability: low likelihood that a few spots are a true positive: more spots needed to call positive

Plusses and minuses of T‐SPOT‐TB• Minuses and miscellany

– Still requires living lymphocytes: same issues as QFT– Definition of a “spot” is subjective– Expensive in‐lab phase

• Specialized equipment and staff/training• Seldom implemented in local/regional labs

– 8 results! • developed world origin (+) (-) borderline invalid

• developing world origin (+) (-) borderline invalid

• New central lab near Fedex headquarters one‐stop operation– Our cutoff time is 2 PM for draw at UWMC

• QFT‐IT and T‐SPOT TB each FDA approved/CDC recommended






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500

600

700

y

TB‐ nil, Pos samples

How positive is a positive? UW experience 2009‐2014

0

100

200

300

400

< 0.35 0.35 ‐ 1 1 ‐ 2 2 ‐ 3 3 ‐ 4 4 ‐ 5 5 ‐ 6 6 ‐ 7 7 ‐ 8 8 ‐ 9 > 9

Freq

uency

Frequency

Arguable low pos/borderlineClinical significance requires context

Unequivocally positive

Does IGRA (+) predict progression to tuberculosis disease in mid‐high incidence countries: NO

Unadjusted incidence rates for development of tuberculosis disease in the short term (median f/u 3 years), stratified by IGRA result. Rate estimates on X axis are per

1,000 person years of follow up. Pai M et al. Clin. Microbiol. Rev. 2014;27:3-20






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Seroconversions / seroreversions

T‐cell conversions / T‐cell reversions

463 unique persons had > 1 test

563 unique within‐person comparisons

386 persons had 2 tests58 persons had 3 tests16 persons had 4 tests2 persons had 5 tests1 person had 6 tests

Time between tests mean/median 287/258 days

200

time elapsed between QFT‐IT re‐tests 2009‐2014

173

84

47

120

59

2418 20

11 8 5 1 0 0 10

20

40

60

80

100

120

140

160

180

200

numbe

r of paired tests

days between tests

IRA Conversions and Reversions are a real problem in low risk HCW

Results of paired QFT‐IT tests UWMC/HMC 2009‐present

308

200

250

300

350

‐person test pairs

2336

55 61

2

40

1

45

0

50

100

150

neg toneg

neg topos

neg toind

pos toneg

pos topos

pos toind

ind toneg

ind topos

ind toind

Num

ber of with

in

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Red countries where BCG vaccine is given after infancy or multiple times (at present or in past)

IGRA maybe better


TST: less of a problem still fine in persons from yellow countries





– (+) near the cutoff in a low‐risk patient– What patients are inappropriate for QFT?– Conversions from QFT (‐) to QFT (+)– Reversions from QFT (+) to QFT (‐)– Does a TST prime for a true or false (+) QFT?– Blood test for tuberculosis disease or risk of tuberculosis disease?

“YES”





– (+) near the cutoff in a low‐risk patient– What patients are inappropriate for QFT?– Conversions from QFT (‐) to QFT (+)– Reversions from QFT (+) to QFT (‐)– Does a TST prime for a true or false (+) QFT?– Blood test for tuberculosis disease or risk of tuberculosis disease?

NO!

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Case 1• 68 yr old man Haitian, in US 24 years, with cough and fever. Community clinic does CXR (abnormal) and refers. You admit, isolate, collect sputum (AFB smear pending) and treat for community acquired pneumonia. Will a QFT be useful and why/why not?

1) useful: if (+) he has TB disease and needs empiric RIPE– 1) useful: if (+) he has TB disease and needs empiric RIPE– 2) useful: if (+) he has LTBI and needs Rx at some point– 3) useful: if (+) must screen family & contacts– 4) useful: if (‐) he does not have TB disease, d/c isolation– 5) useless: high pre‐test probability of TB immune memory; IGRA cannot distinguish LTBI from TB disease; cost‐benefit of LTBI therapy may be low

Case 2– 26 year old nurse starting at the hospital. No int’l travel or known TB exposure and always had (‐) TST, last one year ago. Rubella, VZV, HBV immune. QFT shows

• Nil 0.06• MTB 0.46• Mit >10

D– Do you– 1) perform TST now– 2) repeat QFT now– 3) CXR and history to r/o TB disease, if normal, LTBI Rx– 4) perform T‐SPOT‐TB now– 5) no more testing, allow to begin duty, re‐test later

OUR CASE

Nil 0.06 nil is <8 so far so goodMTB 0.46 TB minus nil is 0.4, which is > 0.35 and > 25% of nilMit >10 mitogen minus nil > 0.5, mit result does not change interp

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Conclusions: IGRA• Ask for the numbers and not just the “interpretation”

• LTBI testing most appropriate in med‐high pre‐test probability of LTBI• IGRA preferred if h/o multiple or post‐infancy BCG• IGRA equivalent to TST if no BCG• TST perhaps fine if one infancy BCG

– Will person benefit from Rx of LTBI if diagnosed?• Lower pre‐test probability but irreversible/severe immune suppression coming upWill R f LTBI if di d?• Will you Rx for LTBI if diagnosed?

• ED/ER and much inpatient use may not be appropriate

• High‐tech pre‐analytical phase: challenges, product defects

• HCW screening plagued by high “low (+)” rate, and high conversion and reversion

– Possible higher cutoffs may be appropriate– ?confirm with TST or repeat IGRA: CDC suggestion– Must place in context of pre‐test probability of MTB infection

pai m et al. clin. microbiol. rev. 2014;27:3-20

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