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L. Masae Kawamura, M.D.Senior Director, Medical and Scientific Affairs, TuberculosisClinician, San Francisco TB Clinic

October, 2018

IGRA update

QuantiFERON®-TB Gold Plus (QFT-Plus)

DISCLAIMER

I am employed by QIAGEN, manufacturer of QFT and other diagnostics, however the views expressed are my

own and not necessarily the views of QIAGEN

Global Tuberculosis Report 2017, WHO

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Tuberculin skin test (TST) IGRA: ELISA IGRA example

Can be fully automated (QFT-Plus)

Highly specific, not affected by BCG

Results with one patient visit

No inter-reader variability (QFT-Plus)

Electronic results (straight to EMR)

Quality-assured laboratory

NO boosting from serial IGRAs

Manual placement, reading and data entry

Results affected by BCG vaccine and NTM

Two patient visits required, high no-show rate

Significant inter-reader variability

Poor surveillance tool

Often no quality control after initial training

Boosted reactions confound serial testing

TST is a not patient or program centered assay, especially in the populations relevant to TB control

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1st generationQuantiFERON®-TB

2nd generationQuantiFERON®-TB Gold

(liquid antigen)

3rd generationQuantiFERON®-TB Gold

(QFT® in tube)

2001: FDA approval 2004: FDA approval 2007: FDA approval

4th generationQuantiFERON®-TB Gold Plus

(QFT®-Plus)

Q4 2014: CE-IVD 2017: FDA approved

• Measured cell-mediated immunity to tuberculin purified protein derivative (PPD)

• Breakthrough: TST becomes a blood test

•

• “Liquid antigen” version• Antigens specific for

M.tb with 99% specificity

• New benchmark: No cross reactivity with BCG

• Logistical advantage –remote incubation

• New benchmark: Scalable and easily automated

• >1500 peer reviewed publicatoins

• >30 million tests sold

• New benchmark: Addition of patented CD8 antigens –potential biomarker of intracellular TB burden

• New flexible blood draw options

Evolution of ELISA IGRA Technology

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QFT Publications prior to QFT-Plus

BCG-vaccinated

Gao L et al, Lancet Infect Dis. 2015 Mar; 15(3):310-9. Epub 2015 FebHowley 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 US Howley MM et al. Pediatr Infect Dis J. 2014 Aug 4.Painter JA et al. PLoS One. 2013 Dec 19;8(12):e82727.

HIVSorborg C et al. Eur Respir J. 2014 Aug;44(2):540-3Cheallaigh 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):282-290Mandalakas 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

PregnancyLaCourse SM et al. J Acquir Immune Defic Syndr. 2017 Jan 30. doi: 10.1097Mathad JS et al. AJRCCM online.Published on 14-January-2016, 10.1164/rccm.201508-1595OCMathad 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, 2016Li CY et al, Journal of Microbiology, Immunology and Infection (2015) 48, 263e268Zellweger et al, Loddenkemper et al., AJRCCM, 12 March 2015Diel 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):282-290Altet N et al, Ann Am Thorac Soc 2015 May;12(5):680-8Soborg 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.

~1500 published studies with key articles showing important utility in testing high risk groups

What we know from >decade of IGRA use: .

IGRAs = Replacing most TST use in TB programs in the US, many countries of W. Europe, Japan, Taiwan, Singapore, andKorea

IGRAs are superior to TST in BCG vaccinated populations in both children and adults (sensitivity, specificity and PPV)

QFT performs consistently in high risk populations  and correlate better to risk than the TST

IGRAs = cost effective compared to TST → eliminate unnecessary radiation exposure, medical exams, and ↓medical error from unnecessary treatment

IGRA operational advantages = convenience for patients and higher quality care 

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There is no gold standard for LTBI (no direct evidence) LEVELS OF PROOF for test accuracy

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Efficacy of preventive therapy

Predictive value for active TB

Correlation to exposure gradient

Sensitivity/spec for active TB

Concordance with TST

Weakest

Stronger

QFT+(n=4455)

TST+(n=6404)

TST+/QFT-(n=3050)

TST-/QFT+(n=1101)

TST+/QFT+(n=3354)

Incidence n 75 62 9 22 53

Cumulative incidence*(95% CI)

1.68%(1.31-2.06)

0.97%(0.73-1.21)

0.30%(0.10-0.49)

2.0%(1.17-2.82)

1.58%(1.16-2.00)

Incidence/100 person years* (95% CI)

0.87(0.68– 1.07)

0.50(0.38–0.63)

0.16(0.05–0.26)

1.03(0.60–1.48)

0.82(0.60–1.04)

*P=<0.0001

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*P=<0.0001

Gao L et al, Lancet Infect Dis 2017; 17: 1053–61

Key study: Incidence of active tuberculosis in individuals with latenttuberculosis infection in rural China (Gao et al, Lancet ID 2017)

2 year follow up study on multi-center cohort: QFT+ or TST+ (≥10mm) 2013: Baseline LTBI prevalence (n=21,022)

• QFT 18% (13-20%)• TST 28% (15-42%)

2014-2015: tracked disease progression by test positivity n=7505

“key populations in communities in rural China…could be targeted for latent infection screening and treatment with an IGRA rather than the TST.”

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4th Generation: QuantiFERON-TB Gold Plus

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SAME test principle-procedure …..SAME technology of QFT

QFT-Plus is an improved version of QFT that is replacing QFT-GIT :• Sensitivity of ~94% in registration trials

• Specificity of >97%

• Innovative CD8+ T-cell stimulating antigens added

– Optimized for CD4+ and CD8+ response

• Improvements in test formulation and manufacturing

Flexible blood draw options• NEW: One Li-heparin tube draw approved for transfer

into QFT-Plus tubes

• NEW: 2 day time limit for blood collection to incubation if

Li-heparin tube stored between 2-8 degrees C

QFT-Plus: potential test evolution – CD8 Antigens

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Studies using single tube collection followed by blood transfer to QFT-tubes and immediate incubation

Indeterminate rates when using single tube Lithium-heparin blood draw

Study N Indeterminate rate

Siegel, JCM 2018QFT-Plus

263Oregon US low riskspecificity trial including 51pts with NTM

1/263 0.4%

QIAGEN clinical trialsPackage insert 2018QFT-Plus

733 212 USA, 322 Japan and 199 from Australia (all no risk for TB)

0/733 Zero

Gallagher David, Synlab publication 2015, QFT-GIT

4615 UK migrant clinics study, multiple sites

10/4615 0.2%

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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.

QuantiFERON® TB Gold In tube QuantiFERON® TB Gold Plus

Title, Location, Date 12

Cells stimulated

Nilcontrol

Mitogen control

Nil control

Mitogen control

noneCD4+ andCD8+ T-Cells

CD4+T-Cells

TBAntigen

TB 1Antigen

TB2Antigen

CD4+T-Cells

none AllAll

Long peptides (MHC class II)•ESAT-6 •CFP-10 •TB7.7+Additional 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

2 Antigen tubes:• More information: 2 data points instead of 1• Allows the calculation of CD8 response

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QFT-Plus: Can it differentiate patients in the spectrum of TB?

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Infection eliminatedLatent TB infection

Subclinical TB disease

Active TB disease

With innate immune response

With acquiredimmune response

TST Negative Positive Positive Positive Usually positive

IGRA Negative Positive Positive Positive Usually positive

Culture Negative Negative Negative Intermittently positive

Positive

Sputum smear Negative Negative Negative Usually negative

Positive or negative

Infectious No No No Sporadically Yes

Symptoms None None None Mild or non Mild to severe

Preferred treatment

None None Preventative therapy

Multidrug therapy

Multidrug therapy

Pai, M. et al. (2016) Tuberculosis Nat. Rev. Dis. Primers doi:10.1038/nrdp.2016.76

The CD8 signal of QFT-Plus, if present, can provide an expanded picture of TB immunity

• MTB specific CD4 T cells produce interferon gamma at all stages• MTB specific CD8 T cells more readily detected as bacterial burden increases

Additional analysis of TB Antigen Tube values – Package Insert data

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 for risk stratification?

Does this information help inform clinical practice?

Is this is recent infection?

Are they more likely to progress?

n=733 n=588 n=357

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Horne D, Narita M et al, IJTLD, 22(6):617–621, 2018

PowerPoint Style Guide 15

Multicenter study of QuantiFERON-TB Gold Plus in patients with active tuberculosis

QFT-GIT Sensitivity 94.3% (vs. 93.02%) (P=0.16)• No statistical difference in sensitivity • Agreement was 98.7%, Kappa 0.89 (CI .75–1.00)• QFT-GIT quantitative values higher than QFT-Plus

Other findings: • TB2 greater than TB1: 99/157 or 63%• TB1 greater than TB2: 39/157 or 25%• TB1 and TB2 equal: 16/157 or 10.2%• Median difference between TB2 and TB1

(nil subtracted): 0.14 IU/ml

WARNING: Lack of a CD8 response does not rule out TB disease or subclinical TB or TB that will progress

However, the presence and strength of the CD8 response may help determine risk of progression or

disease in the context of the patient scenario

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Note: There is no currently validated quantitative cut point or threshold for a

significant CD8 response

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Independent evidence:Sensitivity-Specificity

Sensitivity of QFT-Plus – Published data (culture confirmed TB)

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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 % (0/24) 95.8% (23/24)

YI et al, Scientific Reports 2016 3.1% (5/162) 91% (147/162)

Takasaki, J Infect Chemother 2017 0% (0/99) 99% (98/99)

Horne, IJTLD 2018 4.3% 7/164 89% (146/164)

Fukushima, Kekkaku 2018 1.3% 1/77 93.5% (72/77)

TOTAL N=642 2.5% 16/642 91.5% (588/642)

Sensitivity meta-analyses (culture proven TB) QFT - GIT

Sester et al. ERJ. 2011 80%

Diel et al. Chest. 2010 84%

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Specificity of QFT-Plus – published data

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.

Publication N Specificity (negative/N)

BARCELLINI et al, ERJ 2016 106 97.2% (103/166)

YI et al, Scientific Reports 2016 212 97.6% (207/212)

Moon et el, JCM 2017 625 US HCW no risk identified

97.1% (607/625)

Takasaki, J Infect Chemother2017

106 98% (104/106)

Siegel et al, IJTLD, 2018 262 (includes 51 with pulmonary NTM)

98.1% (257/262)

TOTAL 1311 97.3% (1021/1049)

Specificity of QFT-Plus – Independent dataConservative positive definition in persons with no risk: concordant positive of bothTB1 and TB2

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.

Publication N Specificity (negative/N)

BARCELLINI et al, ERJ 2016 106 99.1% (105/106)

Moon et el, JCM 2017 625 US HCW no risk identified

99% (619/625)

Takasaki, J Infect Chemother2017

106 99% (105/106)

TOTAL 837 99% 829/837

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

FDA approved US Package Insert

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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)

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Evidence:CD8 antigens in action

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QuantiFERON-TB Gold Plus: Potential to better identify risk for progression

QuantiFERON-TB Gold Plus: Gaining attention in 2016 WHO TB report

Barcellini L, Borroni E, Brown J, Brunetti E, Campisi D, Castellotti PF et al. First evaluation of QuantiFERON-TB Gold Plus performance in contact screening. Eur Respir J. 2016:ERJ-00510-02016.

CD8 and CD4 T-cell response

“Current IGRA assays primarily detect a CD4 T-cell response. However, a new generation assay, the QuantiFERON-TB Plus (QFT-Plus, Qiagen, Hilden, Germany), has been developed to stimulate gamma interferon production by both CD4 and CD8 T-cells. First results indicate that the CD8 T-cell response may be able to identify people at greater risk of progression to active TB”.

TB2:TB1 differential as a surrogate measure for CD8 stimulation

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15% of contacts had TB2-TB1 values >0.6 IU/mL (25% of those QFT+)

• Significantly associated with proximity to the index case

◦ p = 0.0029

• Significantly associated with European origin

◦ p = 0.043

Overall stronger risk association compared to QFT-GIT

“…QFT-Plus in contact screening has improved performance compared to QFT-GIT...”

“[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]

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

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NEW! A multicentre verification study of the QFT-Plus

PowerPoint Style Guide 25

ED Pieterman et al, Tuberculosis 108 (2018) 136–142

Study objective: comparison QFT-Plus vs QFT-GIT in testing at-risk groupsN=1031 16 laboratories in Netherlands and Belgium

Reason for screening

N Positive by QFT-Plus

TB suspicion 263 41/263 15%

Contact investigation

127 54/127 42%

Screening before immunotherapy

337 18/337 5%

Occ health periodic check*

189 9/189 5%

Other 57 13/57 23%

Unknown 58 12/58 21%*HCWs working with TB patients or possibly Mtb infected materials

Percent with CD8 signal >0.6 IU/ml TB2-TB1

17%

33%

11%

33%

15%

33%“Themost striking observation of this study was that one third of all positive tested contact screening subjects had a true difference in IFN‐γ release between TB1 and TB2…”

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Evidence:QFT-Plus adding value in

immunocompromised persons

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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 +

Telisinghe et al. (2016)The sensitivity of the QuantiFERON®-TB Gold Plus assay in Zambian adults with active tuberculosis. Int. J. Tuberc. Lung Dis. 21(6)

108 Pulmonary TB patients

68 HIV-positive39 smear+, 29 Xpert+

40 HIV-negative34 smear+, 6 Xpert+

QFT-Plus results

58 Positive

6 Negative

4 Indeterminate

32 Positive

5 Negative

3 Indeterminate

85%

80%

6%

8%

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

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

• HIV positive: 29% of negative/indeterminate results on TB1 were positive on TB2.

• QFT Plus negative/indeterminate (n=18) were associated with nutritional status (76% underweight) and not associated with HIV status

• 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

QFT-Plus performance in PLHIV (high-burden setting) cont….

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3-way comparison: QFT-Plus, QFT-GIT and T-Spot in active TB

K. Fukushima et al, Kekkaku, Vol. 93, No. 10: 517-523, 2018

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• N= 77 culture or LAMP/PCR proven pulmonary TB patients • Average 80 years old• T-SPOT cut point =8 spots (blood age >8hrs with additive T-cell Xtend)

Assay Sensitivity (%) Indeterminate or invalid (%)

QFT-Plus** 72/77 (93.5) 1 (1.3)

QFT-GIT 70/77 (90.9) 3 (3.9)

T-SPOT 57/77 (74.0)* 6 (7.8)

*statistically significant p<0.05 from both QFTs

QFT-plus had the highest sensitivity in all age groups and was significantly higher than Elispot in active TB, elderly patients (p<0.005) and those with low CD4

Sensitivity by assay N=20 <age 80 N=57 >age80 N=23 CD4<200

QFT-Plus (%) 95 93 82.6

QFT-GIT (%) 95 89.5 73.9*

T-Spot (%) 80 71.9 65.2*

**2 of the 3 QFT-GIT indeterminates were positive for QFT-plus due to positive TB2 tube

Quantiferon-TB Gold Plus is a More Sensitive Screening Tool than Quantiferon-TB Gold In-Tube for Latent Tuberculosis Infection among Older Adults in Long-Term Care Facility

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NEW! ELISA-IGRA study in the elderly

Study Design: QFT-GIT/QFT-Plus comparison study• LTBI sensitivity, specificity, PPV and NPV derived from reproducible positive results

Key findings:• QFT‐Plus had significantly higher LTBI sensitivity and NPV and equal specificity  

and PPV compared to QFT‐GIT• QFT‐GIT had decreased in sensitivity over age 75, whereas no loss in sensitivity 

was seen with QFT‐Plus• Positive TB2 test results significantly increased the sensitivity of QFT‐Plus, from 

86.4% to 100.0% (P=0.004)

Jung‐Yien Chien et al, J. Clin. Microbiol. doi:10.1128/JCM.00427‐18, Posted Online 23 May 2018

N=229 51% maleMedian age 80 yrs

(60-102 yrs)

Positiveresults

Sensitivity Specificity PPV NPV

QFT-GIT 28.8% 89.4% 95.7% 89.4% 95.7%QFT-Plus 32.3% 100%* 95.1% 89.2% 100%*

*P<0.05

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Infect Chemother. 2018 Feb;24(2):110-116. doi: 10.1016/j.jiac.2017.09.012

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Japanese IGRA Comparison LTBI trial among patients with rheumatoid arthritis

Assay type Positive (%) Negative (%) Indeterminate (%)

QFT-Plus 15 (9.7) 138 (89.6) 1 (0.7)

TSpot-TB 7 (4.5) 143 (92.9) 4 (2.6)

*P>0.05 Estimated Japanese LTBI prevalence for age 60-79 yrs (2015, Matsumoto): 10.6% -15%

*

N=154 Average age 66.5 years• Compared results by assay type and CD4 and CD8 response• 63% on steroids, 53% on biologic agents, 76% on methotrexate

• QFT-Plus positive rate significantly higher than TSPOT but lower than expected age group prevalence

• Higher QFT-Plus + rate was not driven by CD8 tube (TB2) –suggests these patients are not newly infected or in the process of developing active TB

• Positive rates impacted by CD4 and CD8 count of both assays (Indication: careful interpretation of negative results )

Igari H, Ishikawa S, Nakazawa T, Oya Y, Futami H, Tsuyuzaki M, Suzuki K, Matsumura R

One-Year Experience with QuantiFERON-TB Gold Plus in Patients with Immunosuppressive Conditions (Spain)

Study Design: Assessment of QFT-Plus by comparing results from 2 periods of head to head testing of patients with QFT-GIT and T-Spot.TB (5/15 to 6/16) vs QFT-Plus and TSTB (7/16 to 7/17)

Risk factors: HIV, hematologic diseases, inflammatory bowel disease, rheumatic disease, candidates for biological therapy

A. Fernandez-Blazquez et al, Am J Respir Crit Care Med 2018;197:A5549, www.atsjournals.org

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FINDINGS• Significantly higher agreement between QFT-Plus and TSTB • Significantly lower indeterminate rates by both QFTs compared to TSTB

Authors conclusion:“ The performance of QTF-Plus improves that of QTF-GIT, achieving substantial strength of

agreement with TSTB.”

% Positive % Indeterminate Agreement Kappa

QFT-GIT 14.33% 3.26 83.19%, 0.53

TSTB 17.02% 8.47

QFT-Plus 15.02 2.05 87.57%* 0.61*

TSTB 15.37 5.26

p<.00001

p<.00001

p<..0007N=1535

N=1464

Per

iod

1P

erio

d 2

Poster ATS 2018

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QuantiFERON TB Gold Plus in renal transplantation receiving immune suppressive agents (Japan)

Study Design: 113 renal transplantation patients tested with QFT-Plus. Percentage of CD4 T-cell or CD8 T-cell was determined by flow cytometry

N=113 Mean age 49 yrs (44-63)

H Igari et al, Am J Respir Crit Care Med 2018;197:A5548, www.atsjournals.org

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FINDINGS Patients with • CD8 T-cell >=600/μL and CD4/CD8 ratio <1.0 had significantly higher positive rates.• CD8 T-cell counts showed a positive correlation with IFN-gamma production in TB-1 and

TB-2, however, CD4 counts did not. • QFT-Plus had significantly lower indeterminate rates compared to elispot

Authors conclusion:“QFT-Plus positive rate of 5.3% is acceptable in consideration of Japanese TB statistics.”

“Our result demonstrated the CD8 T-cell dominant immune response to TB specific antigen in the patients of renal transplantation”

% Positive % Indeterminate

QFT-Plus 5.3 0

TB2 4.4

TB1 5.3

Poster ATS 2018

QFT-Plus is an improved version of QFT-GIT and moving the field of LTBI forward

• CD8 responses correspond to new infection in contacts, exposure and TB burden in active TB (Barcellini 2016, Petruccioli2017, Knierer 2017)

• Compared to QFT_GIT, QFT-Plus with higher sensitivity in the elderly (Chien 2018, Fukushima-2018 in press)

• Unlike QFT-GIT, QFT-Plus results are not impacted by HIV status and less impacted by CD4 count (Telisinghe 2017, Walles 2018 pregnancy study, Fukushima 2018)

• In direct comparison studies in active TB and rheumatoid arthritis patients, QFT-Plus is significantly more sensitive than elispot (2018 Fukushima 2018, Igari 2018)

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Summary

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QFT-Plus moving the field of LTBI forward: Research questions

• CD8 responses as a biomarker of new infection…biomarker of incipient TB?

• Will TB2 and CD8 response have clinical utility?

• The CD8 signal: Is there a quantitative threshold that is significant?

• 2 antigen tubes=2 results in one test:

1. What is its utility in maximizing specificity in low risk persons being tested?

2. Will test conversion that signifying new infection be more evident?

• Will QFT-Plus perform better in young children as it does in the extreme elderly?

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The future

Sample to Insight

Using QFT-Plus results: case scenarios

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Golden Rules: NEVER use a TST or IGRA to:

“Rule out” disease in TB suspects (symptoms, abnormal CXR or physical finding suspicious of TB)

“Rule out” LTBI in immunocompromised individuals in the setting of high exposure and high risk of disease progression (e.g., HIV, children under 5, transplant patients, those on immunosuppressive drugs)

NO TEST CAN REPLACE CLINICAL JUDGMENT!!!!

Sample to Insight

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Scenario 1: In a S. American country, a rheumatologist has 3+ smear positive active cavitary tuberculosis and coughing for 2 months

She takes the contact investigation upon herself. Her strain is pan-

susceptible

She has 2 daughters (age 1 and 3), a baby sitter and husband in her home and visits with her sister frequently.

QFT-Plus is performed

TB EXPOSURE SCENARIO: CONTACT INVESTIGATION

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Sample to Insight

39

Scenario 1: In a S. American country, a rheumatologist has active cavitary tuberculosis and coughing for 2 months

She has 2 daughters (age 1 and 3), a baby sitter and husband in her home and visits with her

sister frequently.

QFT-Plus is performed 6/18Husband: PositiveBabysitter: Positive

Sister: Positive

2 daughters: NegativeBabysitter’s 6 yr old child: Negative

(occasional contact)

CXRs performed on all household contacts: NORMAL

Isoniazid started on husband, babysitter, sister and 2 children (All asymptomatic with normal

examinations)

TB EXPOSURE SCENARIO: CONTACT INVESTIGATION

Is primary prophylaxis of the QFT-negative daughters warranted? YES, consistent with US and WHO guidelines

What next?

40

Scenario 1: Examining the QFT resultsInitial QFT June 2018 and repeated Sept 2018

TB EXPOSURE SCENARIO: CONTACT INVESTIGATION

Daughter age 3

Check list:

Control values Consistency of TB1 and TB2 Change in values between tests? Conversion? Weak or strong?

Findings:• Nil close to zero• Healthy mitogen responses• Healthy conversion from 0.02 to

>2 iu/ml

Diagnosis: LTBI, recent conversionClinical management: Complete 9 months INH

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QuantiFERON-TB Gold Plus (QFT-Plus)

41

Interpretation of QFT-Plus results (1)

Nil (IU/ml) TB1 minusNil (IU/ml)

TB2 minus Nil (IU/ml)

Mitogen minus Nil (IU/ml)

QFT-Plus result

Report/Interpretation

≤8.0 ≥0.35 and ≥25% of Nil

Any Any Positive M. tuberculosis infection likely

Any ≥0.35 and ≥25% of Nil

<0.35 OR ≥0.35 and <25% of Nil

≥0.5 Negative M. tuberculosis infection NOT likely

<0.5 Indeterminate Likelihood of M.tuberculosisinfection cannot be determined

>8.0 Any

Note: Cutoffs have not changed from QFT: revalidated 0.35 as most clinically relevant.

Positive results by TB1, TB2, or both are considered positive.

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

Systematically assessing both qualitative and quantitative results

1. Review control values to: Assess the quality of the host response Assess potential technical error What to expect in a healthy patient with NO risk of false negative

or indeterminate? Nil value close to zero Mitogen value near 10 IU/ml or above

2. Review quantitative values of antigen (TB1 and TB2) to: Assess values in light of control results Assess potential technical error (eg. TB1>>>TB2) What to expect? TB1 and TB2 values should be close in value, or TB2>TB1

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43

Scenario 1: Examining the QFT resultsInitial QFT June 2018 and repeated Sept 2018

TB EXPOSURE SCENARIO: CONTACT INVESTIGATION

Daughter age 1

Check list:

Control values Consistency of TB1 and TB2 Change in values between tests?

Findings:• Nil closer to zero than 8.0 IU/ml• Healthy mitogen responses• Missing: 3 of 4 TB1 and TB2 values

Diagnosis: LTBI unlikelyClinical management: 1. Obtain all TB1 and TB2 quantitative

values2. If values close to zero, stop INH 3. If values close to cut point consider

continuing INH? ---conservative approach in setting with known transmission and highly vulnerable contact

Documented transmission hence, Important to ensure

TST and IGRA test interpretation:

false-negative or indeterminate resultsHost factors affecting TST and likely IGRAs

HIV- low CD4, no ARVs

Recent TB infection (<8 weeks)

Infections (viral, fungal, bacterial)

Other illness affecting lymphoid organs

Live virus vaccination

Immunosuppressive drugs

Overwhelming TB

Malnutrition

Age (<2 years, elderly)

Does our patient have any of these factors?

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45

Scenario 1: Examining the QFT resultsInitial QFT June 2018 and repeated Sept 2018

TB EXPOSURE SCENARIO: CONTACT INVESTIGATION

Husband

Check list:

Control values Consistency of TB1 and TB2 Change in values between tests?

Findings: Positive, valid but unusual• Nil value high but closer to zero than

8 IU/ml (cut point for nil indeterminate)

• Mitogen response lower than expected

• TB1 and TB2 values >25% the nil value and perfect duplication of results

Diagnosis: LTBI Clinical management: 1. No change in INH treatment

May indicate:

Excessive levels of circulating IFN-(e.g., another infection)

Presence of heterophile antibody

Incorrect sample handling

Over vigorous shaking at high temperatures

Disturbing material on surface of gel when sampling for ELISA

Very rare

In clinical studies*, < 0.25% of subjects had IFN- > 8 IU/ml for Nil

High Nil (> 8 IU/ml) Mitogen – Nil < 0.5 IU/ml

Indeterminate QFT results – Causes

May indicate:

Incorrect sample handling

> 16 hours from blood specimen draw to incubation

Transportation / incubation at incorrect temperature

Inadequate shaking of tubes- most common cause

Overfilling of tubes

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Summary: Highly infectious index case with contacts under age 5, known transmission (QFT conversion of 3 yr old, totaling 4/5+ contacts)

Conservative approach: ensure reviewing quantitative as well as qualitative results, especially when the patient is vulnerable to false-negative results. Any immune response may be helpful in making a clinical decision

Test conversion was not subtle: both TB1 and TB2 values went from near zero to >1.9 IU/ml indicating true conversion

Doctors are not robots: Decision to test is a decision to think! (John Bernardo MD, Boston)

Use clinical patient information, risks and a systematic approach to review quantitative results to make final management decisions

Key points

Seeking help and consultation

48

This doctor has been using QFT-Plus to

monitor progress of active TB treatment. He notices fluctuations and wants to

know why.

What’s missing from the data that was sent?

Nil and Mitogen control values

Clinical information about TB risk, co-morbidities, nutritional status, and clinical response to

treatment

What’s available in the literature?

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Effect of tuberculosis treatment on newly developed QuantiFERON-TB® Gold Plus

Kamada, Arisu et al, National Hospital Organization Hokkaido Medical Center, Sapporo, JapanP1174, 2016 ATS

• Significant decreases of interferon gamma responses were observed during treatment of active TB in both TB1 and TB2 tubes in the first 3 months.

• The delta (TB2-TB1) between tubes was statistically significant in the latter half and throughout treatment (P=<.05).

“The finding suggests CD8+ responses (represented by the difference between the 2 tubes) declined with TB treatment”

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Quantitative results (IU/mL) During Treatment N=38

Seeking help and consultation

50

This doctor has been using QFT-Plus to

monitor progress of active TB treatment. He notices fluctuations and wants to

know why.

Is it appropriate to use QFT-Plus for monitoring

treatment?

NO!Like the TST, IGRAs

should not be used for treatment monitoring until

there is more evidence

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Summary

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• Although better than TST, IGRAs are not panaceas: Doctors must remain doctors and use their clinical assessment

• Interpretation and management of IGRA results is optimized when the following is taken into consideration:

• Quantitative values of controls and antigen tubes

• Risk of infection and population prevalence of LTBI

• Risk of disease progression or potential bad outcome if LTBI is missed

• Risk vs. benefit of treatment 

• The CD8 response may be helpful in assessing where a patient falls in the TB spectrum of  infection to disease

• QFT‐Plus is moving the field forward in LTBI: providing more information than prior IGRAs with the new CD8 antigens and 2nd antigen tube

• New evidence continues to be favorable for QFT‐Plus, especially in immunocompromised populations

THANK YOU FOR YOUR ATTENTION!

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