Prospettive dell’impiego di test immunologici per lo studio della tubercolosi latente

Delia Goletti

World TB day 2017

Unità di Ricerca Traslazionale, INMI

Roma, 20 marzo 2017

National Institute for Infectious Diseases (INMI) L. Spallanzani, Rome, Italy

HIV: 5,500-6,000

HCV: 1,500-2,000

HBV: 800-1,000

Active TB: 280-300, LTBI: 200-300

Ebola: 2 cases

Outpatient Clinic of Pneumology

yearly

Translational Research Unit

LTBI definition

New tests and new approches

Assays used for LTBI

This talk…

LTBI

x

Worldwide LTBI: size of the problem

LTBI1.7 billion

(Houben, Plos Med 2016)

Active

TB10.4 million

Around 163 fold difference

Wlodarska et al. Clin. Microbiol. Rev., 2015

Infectious spread of M. tuberculosis and resulting disease

LTBI

Schematic approach for programmatic management of latent tuberculosis infection (WHO guidelines)

Getahun et al, ERJ 2015

Flexible approach: WHO recommendations on target populations

High-income and upper middle-

income countries with estimated TB

incidence less than 100

per 100,000 population

Global

Strong • PLHIV

• Household and close contacts (adult and children)

• Patients with silicosis

• Patients on anti-TNF treatment

• Patients receiving dialysis

• Patients under transplantation

• PLHIV

• Household and close

contacts (children, <5

years)

Conditional • Prisoners

• Health workers

• Immigrants from HBC

• Homeless persons

• Illicit drug users Getahun et al, ERJ 2015

Assays used for LTBI diagnosis

This talk…

IGRAs: tests for LTBI diagnosis

ESAT-6, CFP-10

IFN-γ

PBMC Whole Blood

T SPOT.TB QuantiFERON TB Plus

Limitations of the TST (by Mantoux)

Reagent:

Purified protein derivative (PPD) commonly shared among different Mycobacteria (M.tuberculosis, BCG and atypical mycobacteria)

Variability:

Reproducibility in giving the test

Subjectivity in reading the test

Logistics

Repeat visit needed

3 days before result

Positive RD1-IGRA

BCG-vaccination

NTM

Positive M. tuberculosis infection/disease

RD1-IGRA

Accuracy of IGRA and TST in adults

Test Sensitivity for active TB Specificity for TB

Percentage

Sensitivity for active TBSpecificity for

infectionSpecificity for

active TB

TST 6559*/97 75

QFT-IT 80 96 79

T-SPOT.TB 8193 59

*BCG-vaccinated

Goletti et al, J of Rheumathology, 2014;

Sester et Sotgiu et al, ERJ 2010

IGRA in HIV+

Sensitivity:

QFT: 61%

T SPOT-TB: 65%

Specificity:

QFT: 63%

T SPOT-TB: 70%

Santin et al, PloS One 2012

Predictive value of TST and IGRA for incident active tuberculosis in adults

Zellweger et al, AJRCCM 2015

Rangaka et al, TLID 2011

Advantages of IGRA compared to TST

ADVANTAGES

No impact of BCG vaccination

No booster effect

Higher standardization (lab test)

1 day test

DISADVANTAGES

Need of a draw blood

Cost

Logistic (time, transportation)

No standardization of the assay for age, exposure, immune-suppression, work-environment

No discrimination between active TB and LTBI

Impact of immune defects on the test accuracy

Low predictive value for TB

development

CD8+ T-cell specific response and TB

In HIV-uninfected patients: 15% LTBI patients vs 60%

active TB (Rozot, 2013)

In HIV-infected patients: CD8-specific response is

associated with active TB (Chiacchio, 2014)

Chiacchio et al, J Infection 2014

Rozot et al, J Eur Immunol, 2013

CD8+ T-cell frequency decreases in active TB patients after TB-specific therapy

Day et al, J Immunol 2011

CD8+ T-cell response is associated with a

recent exposure to TB and active TB disease

Nikolova et al, Diagn Microb Inf Dis, 2013

CD4

CD8

QuantiFERON TB Plus

This talk…

QuantiFERON-TB Gold vs QuantiFERON-TB Gold PLUS

QuantiFERON® TB Gold In tube

Blood

collection nil TB antigen mitogen

QuantiFERON® TB Gold Plus

nil TB 1 mitogenTB 2CD4 +

T-cellsCD4+ and

CD8 + T-cellsCD4+

T-cells

ESAT-6 polypeptides

CFP-10 polypeptides

TB7.7 polypeptides

• ESAT-6 polypeptides

• CFP-10 polypeptides

• TB7.7 polypeptides

Peptides

Type

Length

• ESAT-6 polypeptides

• CFP-10 polypeptides

• TB7.7 polypeptides

+

additional 6 short

peptides

Gating strategy

Petruccioli et al, J Infection 2016

TB 1: CD4 response.TB 2: CD4 response in all groups and CD8 response in active TB

Petruccioli et al, submitted

0.00

0.05

0.10

0.15

0.200.20.61.01.41.82.22.63.0

Fre

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of

IFN

+ C

D4

-T c

ells

(%

)

0.00

0.05

0.10

0.15

0.200.20.61.01.41.82.22.63.0

Fre

qu

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D8

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ells

(%

)

p=0.03

CD4 response by cytometry CD8 response by cytometry

TB1-antigenresponders

TB2-antigenresponders

TB LTBI

Remote

LTBI

Recent

TB LTBI

Remote

LTBI

Recent

TB1-antigenresponders

TB2-antigenresponders

TB LTBI

Remote

LTBI

Recent

TB LTBI

Remote

LTBI

Recent

p=0.03

A B

CD4

TB1 N (%)

CD4

TB2 N (%)(N)

CD8

TB1 N (%)

CD8

TB2 N (%)

19 (83) 21 (91) ACTIVE TB (23) 4 (17) 11 (48)

17 (94) 15 (83) LTBI REMOTE (18) 3 (17) 3 (17)

12 (100) 11 (92) LTBI RECENT (12) 4 (33) 3 (25)

48 (90.5) 47 (89) TOTAL (53) 11 (21) 17 (32)

Petruccioli et al, J Infection 2016

0

2

4

6

8

1 0

Nu

mb

er o

f re

sp

on

de

rs

In te rm e d ia te /h ig h s e v e r ity T B

L o w s e v e r ity T B

T B 1 T B 2

C D 4 T -c e ll r e p o n s e

T B 1 T B 2

C D 8 T -c e ll r e p o n s e

In active TB: immune response to TB1 and TB2 antigens according to TB severity

Petruccioli et al, J Infection 2016

Take home message: QFT-Plus in active TB

CD8-T cell response is associated with TB2

CD8-T cell response is associated with severe TB

Multicenter study

Cirillo DM, Barcellini L, Borroni EEmerging Bacterial Pathogen Unit Ospedale San Raffaele, Milano, IT

Ruffo Codecasa L, A.O Niguarda, Milano, IT

Tadolini M, Ospedale Sant’Orsola, Bologna, IT

Goletti D, INMI Lazzaro Spallanzani, Roma, IT

Brunetti E, IRCCS San Matteo, Pavia, IT

Brown J, Free Royal Hospital, London, UK

Aim

To evaluate the accuracy of QFT-Plus in:

Active TB

Recent contacts TST+

Accuracy of QFT-Plus for active TB detection

Active TBpatientsn=119

Negative Positive Indeterminate Sensitivity(excluding

indeterminate)%

TB1 20 96 3 83

TB2 15 101 3 87

QFT-Plus 14 119 3 88

Barcellini et al, ERJ 2016

Negative Positive Indeterminate Specificity%

TB1 104 2 0 98

TB2 104 1 1 98

QFT-Plus 103 3 0 97

Take home message: QFT-Plus in active TB

CD8-T cell response is associated with TB2

CD8-T cell response is associated with severe TB

Sensitivity for active TB detection is likely higher than QFT-IT

QFT-Plus and contact screening

119 recent contacts TST+ baseline

56 QFT-IT positive

+ baseline After 3 months

63 QFT-IT negative 2 QFT-IT conversion

Barcellini et al, ERJ 2016

Contact screening

QFT-GIT results

QFT Plus results

Positive results

per tube

QTF Plus IFN- γ concentrations (IU/ml)*

Negative Positive

TB1 TB2 TB1-Nil TB2-Nil

Negative (n=63)

51 (80.9%) 12 (19.0%)

10° 10

§ 0.01 (-0.01;0.17) 0.04 (0;0.23)

Positive (n=56)

0 56 (100%)

56 56 10.60 (2.94;16.57)

11.00 (3.32;17.75)

Total (n=119) 51 (42.8%) 68 (57.1%)

66 66 0.74 (0.01;9.65) 0.67 (0.04;8.94)

High overall agreement, Cohen’s kappa of 0.8 (95% CI 0.69-0.91)

Discordant results were found in 12 subjects: they all

scored negative to the QFT-GIT and positive to the QFT-

Plus

Barcellini et al, ERJ 2016

Contact screening: Independent predictors of QFT-Plus and QFT-GIT positivity

QFT-GIT Positive QFT Plus Positive

OR (95% CI) p-value OR (95% CI) p-value

Age 1.04 (1.00; 1.07) 0.048

Estimated incidence of TB per 100,000 person-year in country of birth°

0-10 1

>10 3.14 (1.11; 8.88) 0.031

Time spent with the index case (hours per day)

1-12 1 1

>12 4.63 (2.05;10.47)

0.0002 6.41 (2.56; 16.06) 7.38e-05

Barcellini et al, ERJ 2016

Take home message: QFT-Plus in recent TB contacts

QFT-Plus seems to have a higher sensitivity compared to QFT-IT for LTBI detection

Potentially useful for monitoring LTBI preventive therapy

Would the use of QFT-Plus change ourcurrent practice?

Currently available data suggests that the QFT-Plus

improved sensitivity (88%) compared to the QFT-GIT and a

low indeterminate rate.

Unlikely to be useful in the diagnosis of active TB

Should aid in the detection of latent TB infection and

preventive treatment monitoring

Future directions

As our first data suggests, it should be investigated in larger multicentre trials:

The QFT-Plus performance in HIV co-infected individuals and other subgroups of immune-compromised patients, as those under biological treatment

The QFT-Plus performance in children

The QFT-Plus Positive Predictive Value (PPV) for TB progression

If any TB2-TB1 value can discriminate different states of the infection-disease

Agenda

Will this change practice?

C-Tb skin, a novel specific skin test based on ESAT-6 and CFP10 antigens

The authors investigated the safety and diagnostic potential of C-Tb compared with established tests in the contact-tracing setting.

C-Tb skin test: accuracy results

BCG-unvaccinated BCG-vaccinated

Ruhwald et al, Lancet Respiratory Medicine, 2017

C-Tb skin test: accuracy results. Similar PPV of the QFT-IT

Ruhwald et al, Lancet Respiratory Medicine, 2017

From Tom Ottenhoff 2014

Active TB development

From Tom Scriba, 2016

cumulative incidence: 2%

IPT effectiveness: 50%

80% IGRA+

Aa blood RNA signature for TB disease risk

Cross-validation performance of the tuberculosis risk signature in the ACS training set by days before tuberculosis diagnosis

Zak at al, Lancet, 2016

Test for identifying TB progressors

Petruccioli et al, ERJ 2016

Positive Predictive Value according to Sens/Spec for risk of progression

cumulative incidence: 2%

IPT effectiveness: 50%

Petruccioli et al, ERJ 2016

Number Needed to Test & Treat according to Sens/Spec for risk of progression

NNTT captures clinician/PH perspective (If treating all test+,

how many do I need to test and treat to prevent one case?)

cumulative incidence: 2%

IPT effectiveness: 50%

Petruccioli et al, ERJ 2016

Take home message: novelties

Available as routine tests: QuantiFERON Plus

Reasonably available soon as routine tests: C-Tb skin test

Potentially available soon as routine tests: COR (likely modified)

Many thanks to…

CD8 studies

Barcellini, Borroni, Cirillo, Emerging Bacterial Pathogen Unit San Raffaele Scientific Institute(MI)

Ruffo Codecasa, Ferrarese, CastellottiO.N. Niguarda (MI)

Tadolini, Ospedale Sant’Orsola (BO)

Brunetti, IRCCS San Matteo (PV)

Brown, Lipman, Free Royal Hospital, London

QIAGEN

We thank QIAGEN (Hilden, Germany) for providing QuantiFERON-TB Glod Plus kits free of charge

Assays for TB progression

Scriba and Heatherhill, South African Tuberculosis Vaccine Initiative, Institute of Infectious Disease and Molecular Medicine, and Division of Immunology, Department of Pathology, University of Cape Town, Cape Town, South Africa

Ottenhoff and Joosten, Leiden University Medical Center, Leiden, Netherlands

Cirillo, Emerging Bacterial Pathogen Unit San Raffaele Scientific Institute, Italy

Denkinger and Shumaker, Tuberculosis and Hepatitis Programme, FIND, Geneva, Switzerland

Petruccioli and Petrone, Translational Research Unit, National Institute for Infectious diseases, Italy

My My thanks and conclusion…

A lot to do still……..