The 4th

generation IGRA

L. Masae Kawamura, M.D.Senior Director, Medical and Scientific Affairs, TB Diagnostics, QIAGEN

August 2017

QuantiFERON-TB Gold Plus

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Disclaimers - QuantiFERON-TB Gold Plus (QFT Plus)

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QFT-Plus is CE-IVD marked and FDA approved.

It will be available in the US this fall.

QFT-Plus is an in vitro diagnostic aid for detection of Mycobacterium

tuberculosis infection (including disease) and is intended for use in

conjunction with risk assessment, radiography, and other medical and

diagnostic evaluations. QFT-Plus results alone cannot distinguish active TB

disease from latent infection. QFT-Plus Package Inserts, available in multiple

languages, as well as up-to-date licensing information and product-specific

disclaimers can be found at www.QuantiFERON.com.

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Tuberculin skin test (TST) QuantiFERON-TB (QFT)

Can be fully automated

Highly specific, not affected by BCG

Results with one patient visit

No inter-reader variability

Electronic results (straight to EMR)

Quality-assured laboratory test

Manual placement, reading and data entry

Two patient visits required, high no-show rate

Significant inter-reader variability

Poor surveillance tool

Often no quality control after initial training

In-vivo test---causes boosting

TST is not patient or program-centered and least effective in most vulnerable:

“hard to reach” groups

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Evolution of QFT Technology

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QuantiFERON-TB to QuantiFERON-TB Gold

1st Generation 2nd Generation 3rd Generation

QuantiFERON-TB Gold In tube

• 2007 U.S. FDA approval

• Scalable and easily

automated

• Logistical advantage –

remote incubation

• >1200 peer reviewed

publicatoins

• >30 million tests sold

QuantiFERON-TB Gold

• 2004 U.S. FDA approval

• “Liquid antigen” version

• Antigens specific for M.

tuberculosis complex

organisms to measure cell-

mediated immunity

• 99% specificity

• No cross reactivity with BCG

QuantiFERON-TB

• 2001 U.S. FDA approval

• Measured cell-mediated

immunity to the same

tuberculin purified protein

derivative (PPD) used for

the tuberculin skin test (TST;

M. avium)

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QFT-Plus was built on the shoulders of QFT-GIT

BCG-vaccinated

Gao L et al, Lancet Infect Dis. 2015 Mar; 15(3):310-9. Epub 2015 Feb

Howley MM et al. Pediatr Infect Dis J. 2014 Aug 4.

Painter JA et al. PLoS One. 2013 Dec 19;8(12):e82727.

Riazi S et al. Allergy Asthma Proc. 2012 May-Jun;33(3):217-26.

Migrants to USHowley MM et al. Pediatr Infect Dis J. 2014 Aug 4.

Painter JA et al. PLoS One. 2013 Dec 19;8(12):e82727.

HIV

Sorborg C et al. Eur Respir J. 2014 Aug;44(2):540-3

Cheallaigh CN et al. PLoS One. 2013;8(1):e53330.

Aichelburg MC et al. Clin Infect Dis. 2009 Apr 1;48(7):954-62.

Raby E et al. PLoS One. 2008 Jun 18;3(6):e2489.

BiologicsHsia EC et al. Arthritis Rheum. 2012 Jul;64(7):2068-77.

Ponce De Leon D et al. J Rheumatol. 2008 May;35(5):776-81.

Matulis G et al. Ann Rheum Dis. 2008 Jan;67(1):84-90.

Pediatrics

Jason Andrews et al, Lancet Respir Med. 2017 Apr;5(4):282-290

Mandalakas A et al, AJRCCM, Vol 191: 7 2015

. Garazzino S et al. Pediatr Infect Dis J. 2014 Sep;33(9):e226-31Howley MM et al. Pediatr Infect Dis J. 2014 Aug 4.

Sollai et al. BMC Infectious Diseases 2014, 14 (Suppl 1):S6

Pregnancy

LaCourse SM et al. J Acquir Immune Defic Syndr. 2017 Jan 30. doi: 10.1097

Mathad JS et al. AJRCCM online.Published on 14-January-2016, 10.1164/rccm.201508-1595OC

Mathad JS et al. PLoS One. 2014 Mar 21;9(3):e92308.

Lighter-Fisher J, Surette AM. Obstet Gynecol. 2012 Jun;119(6):1088-95.

Contacts

Matsumoto K et al, Kekkaku Vol 91, No.2:45-48, 2016

Li CY et al, Journal of Microbiology, Immunology and Infection (2015) 48, 263e268

Zellweger et al, Loddenkemper et al., AJRCCM, 12 March 2015

Diel R et al. Am J Respir Crit Care Med. 2011 Jan 1;183(1):88-95.

Arend SM et al. Am J Respir Crit Care Med. 2007 Mar 15;175(6):618-27.

Risk of Progression

Gao L et al, Lancet Infect Dis. 2017 Jul 14. pii: S1473-3099(17)30402-4. doi: 10.1016/S1473-3099(17)30402-4

Jason Andrews et al, Lancet Respir Med. 2017 Apr;5(4):282-290

Altet N et al, Ann Am Thorac Soc 2015 May;12(5):680-8

Soborg C et al. Eur Respir J. 2014 Aug;44(2):540-3.

Diel R et al. Chest. 2012 Jul;142(1):63-75.

End Stage Renal Rogerson TE et al. Am J Kidney Dis. 2013 Jan;61(1):33-43.

>1200 published studies with key articles showing important utility in testing high risk groups

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Customers wanted more......

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Desired characteristics

Enhanced performance

Improved performance in high-risk groups

Potential to provide additional clinical information

Harmonization of workflow options globally

• Increased sensitivity

• Sustained high specificity

• People who are immunocompromised

• People living with HIV/AIDS

• Risk-based algorithms

• Better assist patient assessment and management

• 1-tube blood collection (optional)

• 4-point standard curve

Test Progression

QFT-GIT 12.9%

TST > 5 mm 3.1%

Improve prediction of

disease?

1. Diel, R., et al. (2011). Am J Respir Crit Care Med 183, 88

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New CD8+ antigens: WHY?

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CD8+ T cells and role in TB immunity:

MTB-specific CD8+ T cells secrete IFN- and other soluble factors to (1–3):

• Suppress MTB growth

• Kill infected cells

• Directly lyse intracellular MTB

BIOMARKER for intracellular burden

TB-specific CD8+ T cells that produce IFN- have been:

• More frequently detected in those with active TB disease vs. latent infection (4, 5)

• Associated with recent exposure to TB (6)

• Detectable in active TB subjects with HIV co-infection and young children (7, 8)

• Observed to decline when patients are exposed to anti-tuberculosis treatment (9)

References: 1. Turner, J. et al. (1996) Immunology 87, 339. 2. Brookes, R.H. et al. (2003) Eur. J. Immunol. 33, 3293. 3. Stenger, S. et al. (1998)

Science 282, 121. 4. Day, C.L. et al. (2011) J. Immunol. 187, 2222. 5. Rozot, V. et al. (2013) Eur. J. Immunol. 43, 1568. 6. Nikolova, M. et al. (2013)

Diagn. Microbiol. Infect. Dis. 75, 277. 7. Chiacchio, T. et al. (2014) J. Infect. http://dx.doi.org/10.1016/j.jinf.2014.06.009. 8. Lanicioni, C. et al. (2012)

Am. J. Respir. Crit. Care Med. 185, 206. 9. Nyendak M. Et al. (2014) PLoS ONE 8, e81564. Epub.

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QFT-Plus Product Format

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QFT-Plus uses the same test principle, procedure and reliable technology of QFT

ELISA remains mainly the same.

• Same procedure

• New labeling

• New instructions for use

• New plate layout

– 4 wells per patient

• New QFT-Plus analysis software

Tubes have the same phlebotomy practice and handling process.

• Custom QFT-Plus tubes:

– Nil – same grey color

– TB1 – green, CD4 only

– TB2 – yellow: optimized to detect both CD4 and CD8!

– Mitogen – same purple

• Standard lithium heparin tube (optional)

– 16 hours at room temp / 48hr refrigerated from draw to incubation

Flexible sample collection, but still allows remote incubation

Familiar technology for the lab

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QuantiFERON® TB Gold In tube QuantiFERON® TB Gold Plus

Title, Location, Date 12

Cells stimulated

Nil

controlMitogen

control

Nil

controlMitogen

control

noneCD4+ and

CD8+

T-Cells

CD4+

T-Cells

TB

Antigen

TB 1

AntigenTB2

Antigen

CD4+

T-Cells

none AllAll

Long peptides (MHC class II)

•ESAT-6

•CFP-10

•TB7.7+Additional 6 short peptides(MHC class I)

Long peptides (MHC class II)

•ESAT-6

•CFP-10

•TB7.7

Long peptides (MHC class II)

•ESAT-6

•CFP-10

•TB7.7

Polypeptide

Antigens

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Proof of concept studies detected the involvement of CD8 T cells

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Synergistic increase in interferon gamma production when short and long peptides together but not in everyone

What could the delta between TB1 and TB2 tell us?

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1

2

3

4

5

LTBI Active TB

IFN

-γ(I

U/m

l) CD8

involvementEarly observations:

Levels generally higher and

Delta from MTB-specific

CD8+ T cell activation more

pronounced in active TB

CD4 optimised peptides CD4 & CD8 optimised peptides

FOR INTERNAL USE ONLY QFT Product Training

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Sensitivity of QFT-Plus- FDA approved US Package Insert

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QFT-Plus has markedly improved sensitivity at >94% versus 89% of QFT previously

Sensitivity results by region

Sensitivity results by TB antigen tube

*Important note: 9 positive by TB2 only, including 3 that would have been indeterminate by TB1 only.

Study Region N QFT PlusQFT-Plus

Indeterminate

USA (3 sites) 5386.7%

(77.4-94.7)0

Japan (3 sites) 35294.43%

(91.5-96.4)11

Australia (1 site) 29100%

(88.3-100)0

Total 434 94.09% 11 (2.5%)

Result TB1 TB2* QFT-Plus

Positive 388 397 398

Negative 32 25 25

Indeterminate 14 11 11

Sensitivity (95% CI) 92.4% (89.4-94.6) 93.9% (91.1-95.8) 94.09 (91.4-96.0)

FOR INTERNAL USE ONLY QFT Product Training

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Sensitivity of QFT-Plus – Independent data

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Sensitivity (culture confirmed TB) results from Independent QFT-Plus studies

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Publication Indeterminate Sensitivity

BARCELLINI et al, ERJ 2016 2.6% (3/116) 87.93% (102/116)

Includes 4/4 HIV infected

HOFFMAN et al, CMI 2016 0 95.8% (23/24)

YI et al, Scientific Reports 3.1% (5/162) 91% (147/162)

1. QuantiFERON-TB Gold Plus (QFT-Plus) ELISA Package Insert. Rev. 02. February 2015.1083163.

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QFT-Plus: Clinical performance – Specificity Studies

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Overall No loss of specificity with QFT-Plus

Total of 733 low-risk subjects

Site N QFT QFT-Plus QFT-Plus

(concordant positive)

% Specificity

(95% CI)

% Specificity

(95% CI)

% Specificity

(95% CI)

Australia 3 199 95.9 %(92.3 – 97.9)

95.5%(91.6 - 97.6)

97.9 %(94.6 - 99.4)

Japan 1 216 98.6 %(96.0 - 99.7)

97.7 %(94.7 - 99.2)

99.1 %(96.7 - 99.9)

Japan 3 106 99.1 %(94.9 - 99.8)

98.1 %(93.4 - 99.5)

100.0 %(96.6 - 100.0)

USA 4 212 99.1 %(96.6 - 99.9)

98.1 %(95.2 - 99.5)

99.1 %

(96.6 - 99.9)

Overall 733 98.1 %

(96.9 - 99.0)

97.3 %

(95.9 - 98.4)

98.9 %

(97.9 - 99.5)

FOR INTERNAL USE ONLY QFT Product Training

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Additional analysis of TB Antigen Tube values – Package Insert data

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TB2 Minus TB1 (i.e. CD8/CD4 – CD4)

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TB2 – TB1 (Nil subtracted)

Surrogate for isolated CD8 response

Potential for additional valuable information in risk stratification?

Does this

information help

inform clinical

practice?

We suspect this is

recent infection

Are they more

likely to progress?

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QFT PLUS Publications

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Title/Authors

First independent evaluation of QuantiFERON-TB Plus performance

BARCELLINI et al

Published – ERJ 2016

Equal sensitivity of the new generation QuantiFERON-TB Gold plus in direct comparison with the previous

test version QuantiFERON-TB Gold IT.

HOFFMANN et al.

Published – CMI 2016

First evaluation of QuantiFERON-TB Gold Plus performance in contact screening

BARCELLINI et al

Published – ERJ 2016

Preliminary data on precision of QuantiFERON-TB Plus (QFT-Plus) performance.

GALLAGHER et al

Published- ERJ 2016

CD8 response is associated to active TB and to the response to TB2 in the QuantiFERON-TB Plus kit.

PETRUCCIOLI et al

Published– J of Infection 2016

Evaluation of QuantiFERON-TB Gold Plus for Detection of Mycobacterium tuberculosis

infection in Japan

YI et al

Published-Scientific Reports

2016

QuantiFERON-TB® Gold Plus as a potential tuberculosis treatment monitoring tool

KAMADA et al

Published –ERJ 2017

The sensitivity of the QuantiFERON®-TB Gold Plus assay in Zambian adults with active tuberculosis

TELISINGHE et al

Published–IJTLD 2017

QFT-Plus: a plus in variability? – Evaluation of new generation IGRA in serial testing of students with a

migration background in Germany

KNIERER et al

Published – J of Occ Med and

Tox

Evaluation of QuantiFERON®-TB Gold-Plus in Healthcare Workers in a Low-Incidence Setting.

MOON et alJCM 2017

Prevalence of latent tuberculosis infection among foreign students in Lübeck, Germany tested with

QuantiFERON-TB Gold In-Tube and QuantiFERON-TB Gold Plus

MORALES et al

Journal of Occ Med and

Toxicology 2017

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QFT PLUS 2016-2017 Key Publications

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Area of study Authors Finding

Sensitivity (culture proven

TB)Italy, England, Germany,

Japan

BARCELLINI et al,(n=116)

HOFFMANN et al. (n=24)

YI et al (n=162)

PETRUCCIOLI et al, (n=49)

88%-96% (indeterminate results

excluded)

Package insert: 94%

Specificity (low-no risk of

exposure) Italy, USA

BARCELLINI et al (n=106)

MOON et al (n=626)97-99% (99% if using conservative

definition of both Ag tubes concordant

positives)

Contact investigation

QFT-GIT vs. QFT-PlusItaly

BARCELLINI et al,

N=119 TST+ contacts

(5mm cut point)

QFT-Plus results more closely

associated with exposure risk. 15%

with CD8 signal

HIV infected persons with

culture proven TBZambia

TELISINGHE et al

n=108 (63% HIV+)QFT-Plus sensitivity not impacted by

HIV status. HIV+ (85%) vs HIV

negative (80%) Sensitivity 89% if

indeterminate results excluded

Treatment monitoringJapan

KAMADA et al

n=38, pansusceptible TBCD8 but not CD4 response

decreasing through end of treatment

Laboratory Precision QFT-

GIT vs. QFT-Plus UK

GALLAGHER et al

n=20 samples from 2 donorsCV: 9.60% (QFT-Plus) vs.18.25%

(QFT-GIT)

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First evaluation of QuantiFERON-TB Gold Plus performance in contact screening

Study design: Prospective recruitment of TST-positive adult contacts (TST ≥5 mm)

• Average age of 39 (30-79)

• 26%(n=61) non-European born, 78.85% (n=82) BCG vaccinated

• 9.24% (n=11) were immunocompromised (HIV and other)

• Contact screening based on NICE TB guidelines 2011 and Italian guidelines

Results

RESULTS

Barcellini et al, Eur Respir J. 2016 Jul 7. pii: ERJ-00510-2016. doi:

10.1183/13993003.00510-2016. Epub ahead of print]

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Barcellini et al, 2016: Results continued…

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Strong overall agreement: ĸ = 0.8

◦ Discordant results N=12

All 12 results were negative QFT and positive QFT-Plus

All but one discordant result had positive TST result >10 mm

Two conversions occurred with QFT upon retesting at 10–12 weeks.

Both initially positive by QFT-Plus

QFT-Plus with stronger risk association

• QFT-Plus showed a stronger risk association to aggregate exposure time than QFT:

Odds ratio 6 QFT vs. 14 QFT-Plus

• QFT-Plus showed a stronger risk association to index case proximity than QFT:

Odds ratio 4 QFT vs. 6 QFT-Plus

“…our data show that QFT-Plus in contact screening has improved

performance compared to QFT-GIT...”

Barcellini et al, Eur Respir J. 2016 Jul 7. pii: ERJ-00510-2016. doi:

10.1183/13993003.00510-2016. Epub ahead of print]

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TB2:TB1 differential as a surrogate measure for CD8 stimulation

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15% of QFT+ contacts had TB2-TB1 values >0.6 IU/mL

• Significantly associated with proximity to the index case

◦ p = 0.0029

• Significantly associated with European origin

◦ p = 0.043

“[QFT-Plus performance] suggests a role for the differential value

between the two tubes as a proxy for recent infection.”

Barcellini et al, Eur Respir J. 2016 Jul 7. pii: ERJ-00510-2016. doi:

10.1183/13993003.00510-2016. Epub ahead of print]

Barcellini et al, 2016: Results continued…

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1st evaluation of QFT-Plus performance in PLHIV (high-burden setting)

Study design: Prospective recruitment of Zambian patients with pulmonaryTB• Mean age of 32, 73% male; 63% HIV infected, BMI <18.5 (>50%)

• 108 consecutive smear or Xpert +

Results

RESULTS

Telisinghe et al, INT J TUBERC LUNG DIS 21(6):690–696

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QFT-Plus results not affected by HIV status

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• Median IFN γ was higher in TB2 than in TB1 irrespective of HIV status

• Among HIV negative, 20% of negative/indeterminate results on TB1 were positive on TB2,

and among PLHIV, 29% of patients with negative/indeterminate results on TB1 were

positive on TB2.

• QFT Plus negative/indeterminate (n=18) were underweight (76%), and 56% of those

patients were HIV positive

• Compared to prior study in their institution with similar cohorts (Raby 2007) QFT not

affected by HIV status and less affected by low CD4 count (results in % below)

Telisinghe et al, INT J TUBERC LUNG DIS 21(6):690–696

Telisinghe et al, 2017: Results continued

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QFT-Plus: the latest evolution of QFT technology

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QFT-Plus uses the same test principle, procedure and reliable technology of QFT

QFT-Plus is an improved version of QFT, offering:

Higher sensitivity than available metaanalysis

Maintenance of high specificity

Innovative CD8+ T-cell technology

– Optimized for CD4+ and CD8+ response

– Independent studies showing CD8+ correlation to

new infection and burden of TB

– Numerous studies ongoing around the world

Improvements in test formulation and manufacturing

QFT-Plus: setting a new benchmark in TB infection testing

CD8 and CD4 T-cell response