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
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