Department for Tropical Medicine & Infectious Diseases

Improving diagnostic possibilities for childhood TB – the TAM TB assay

Christof Geldmacher7th FIND Session, Barcelona

29.10.2014

Childhood tuberculosis

• non-specific symptoms

• difficult sputum collection in children

• unsatisfactory sensitivity of AFB smear microscopy & Xpert MTB/RIF

Assay

• MTB culture can be time consuming

• Current Immunodiagnostic approaches lack specificity for active disease

Novel diagnostic approaches are urgently needed !

Diagnosis of paediatric TB diseaseremains a major challenge

TAM-TB Background

Phenotype and Function of MTB-specific T cells reflect TB disease status

Central MemoryT cell:

Lymph-nodeHoming

„TAMs“

Cell surface proteins

onT cells

TAM-TB Background

persistent antigen-stimulation in vivo

Phenotype and Function of MTB-specific T cells reflect TB disease status

Effector Memory T cell:Homing toSITES OF INFECTION

Materials and Methods

Blood

Ficoll

Serum

RBC/PMN

PBMC CryopreservionSampletreatment

Sample thawed, rested and viability check

Antigen incubation (Unstimulated / E6C10 / PPD / SEB)

Antibody staining and flow data acquisition and analyses

TAM‐TB Assay results

Sampleanalysis

TAM‐TB assay Assays were done independently at two sites

The TAM TB assay procedure

Materials & Methods

No IFN+ MTB‐specific CD4 T cells response detected=

No active Tuberculosis disease

Definition of TAM-TB assay results

IFN+ MTB‐specific CD4 T cells response detected(≥0.05% of CD4 T cells and ≥2x unstimulated control )

Determine CD27 phenotype of MTB-specific T cells

CD27 MFI total CD4 T cells

CD27 MFI of IFN+ CD4 T cells

CD27 staining flourescence Intensity

0 10 3 10 4 10 50 10 3 10 4 10 50 10 10 100 10 10 10

Active TB

MTB-specific CD4 T cells

Total CD4 T cells

0 10 3 10 4 10 50 10 3 10 4 10 50 10 3 10 4 10 50 10 3 10 4 10 5

latent TB

MTB-specific CD4 T cells

Total CD4 T cells

rela

tive

Cel

l cou

nt

Intensity of CD27 staining on CD4 T cell surface

Median Flourescence Intensity

Determination of the CD27 Median flourescence intensity ratio

CD27 MFI total CD4 T cells

CD27 MFI of IFNg+ CD4 T cells

high value in active TB diseaselow value latent MTB infection

TB child study ‐ Evaluation of new and emerging diagnostics for childhood Tuberculosis disease in high burden countries 

Inclusion criteria• Eight weeks‐15 years of age• Suspicion of active Tuberculosis disease: 

a) persistent, non‐remitting cough (14 days), not responding to antibiotics 

b) repeated episodes of fever (14 days) not responding to antibiotics, and after malaria has been excluded

c) weight loss or failure to thrive within the previous 3 months d) signs and symptoms suggestive of  extrapulmonary TB

Strategy: Test novel diagnostic approaches in comparison to 1) reference standard MTB culture and 2.) clinical classification for culture negative “clinical” TB cases

Childhood tuberculosis

Classification system for paediatric TB suspects

Culture confirmed MTB-culture confirmed Tuberculosis

Culture confirmed MTB-culture negative “clinical TB”

Culture confirmed No TB

Childhood tuberculosis

Flow Diagram of children enrolled

^̂

Portevin et al 2014 Lancet ID

Performance of the TAM TB assay in comparison to MTB‐culture

Portevin et al 2014 Lancet ID

Performance of the TAM TB assay

0

25

50

75

100

MTB culture+ TB highly probable TB

probable TB definite no TB

MTB culture

Xpert

TAM‐TB

IGRA + TST

Comparison of TAM TB assay results with other assays

% o

f sub

ject

s w

ith

Conclusions

Conclusions and Outlook

• Proof of principle that novel immunodiagnostic strategies can improve rapid diagnosis of TB in pediatric suspects from endemic countries

• Major Limitation: TAM TB Assay needs to be simplified

• Our Vision: • should be as simple as a “CD4 T cell count”

• Whole blood assay

• Automatize where possible

• End-product could be used at District level hospitals, where CD4 T cell counts are peformed

Andrea RachowElmar Saathoff

Michael HoelscherUniversity of Munich

Felicien MoukambiAsli Bauer

Mkunde ChachagePetra Clowes

Andrea RachowAnke Kohlenberg

Nyanda EliasOnesmo Mgaya

Epiphania MichaelNIMR-MMRC

Damien PortevinClaudia Daubenberger

Anneth TumboTedson LukindoKlaus Reither

IHI/STPH

Scientific AdvisorBen Marais

University of Sydney