Case\u2013control diagnostic accuracy study of a non-sputum CD38-based TAM-TB test from a single milliliter of blood

Hellen Hiza,Damien Portevin,Sebastien Gagneux,Jerry Hella,Ainhoa Arbués,Mohamed Sasamalo,Klaus Reither,Beatrice Magani

doi:10.1038/s41598-021-92596-z

Hellen Hiza, Damien Portevin + Show 6 more

Open Access

https://doi.org/10.1038/s41598-021-92596-z

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Abstract

CD4 T cell phenotyping-based blood assays have the potential to meet WHO target product profiles (TPP) of non-sputum-biomarker-based tests to diagnose tuberculosis (TB). Yet, substantial refinements are required to allow their implementation in clinical settings. This study assessed the real time performance of a simplified T cell activation marker (TAM)-TB assay to detect TB in adults from one millilitre of blood with a 24 h turnaround time. We recruited 479 GeneXpert positive cases and 108 symptomatic but GeneXpert negative controls from presumptive adult TB patients in the Temeke District of Dar-es-Salaam, Tanzania. TAM-TB assay accuracy was assessed by comparison with a composite reference standard comprising GeneXpert and solid culture. A single millilitre of fresh blood was processed to measure expression of CD38 or CD27 by CD4 T cells producing IFN-γ and/or TNF-α in response to a synthetic peptide pool covering the sequences of Mycobacterium tuberculosis (Mtb) ESAT-6, CFP-10 and TB10.4 antigens on a 4-color FACSCalibur apparatus. Significantly superior to CD27 in accurately diagnosing TB, the CD38-based TAM-TB assay specificity reached 93.4% for a sensitivity of 82.2% with an area under the receiver operating characteristics curve of 0.87 (95% CI 0.84–0.91). The assay performance was not significantly affected by HIV status. To conclude, we successfully implemented TAM-TB immunoassay routine testing with a 24 h turnaround time at district level in a resource limited setting. Starting from one millilitre of fresh blood and being not influenced by HIV status, TAM-TB assay format and performance appears closely compatible with the optimal TPP accuracy criteria defined by WHO for a non-sputum confirmatory TB test.

Highlights

Abbreviations ART Antiretroviral therapy AUC Area under the curve body mass index (BMI) Body mass index cycle threshold (Ct) Cycle threshold NTLP National Tuberculosis and Leprosy Program peripheral blood mononuclear cells (PBMC) Peripheral Blood Mononuclear Cells RIF Rifampicin T cell activation markers (TAM) T cell activation marker TB Tuberculosis target product profiles (TPP) Target product profiles
Diagnostic accuracy studies should rather be conducted in real time and only on patients showing signs of TB including people living with HIV
The implementation of TAM-based assays beyond research settings calls for a simplified protocol fulfilling the following conditions: (i) being compatible with the most basic flow cytometry apparatus; (ii) excluding the need of PBMC isolation; and (iii) requiring a minimal amount of blood to be compatible with TB diagnosis in children

Summary

Introduction

Abbreviations ART Antiretroviral therapy AUC Area under the curve BMI Body mass index Ct Cycle threshold NTLP National Tuberculosis and Leprosy Program PBMC Peripheral Blood Mononuclear Cells RIF Rifampicin TAM T cell activation marker TB Tuberculosis TPP Target product profiles. Previous studies were mostly conducted retrospectively relying on small sample sets of cryopreserved peripheral blood mononuclear cells (PBMC) or whole-blood samples fixed and cryopreserved post-stimulation. These studies would often use as comparator healthy controls or asymptomatic but possibly latently infected individuals[10,11,12,13,15,16,17,18,19,20,21]. The implementation of TAM-based assays beyond research settings calls for a simplified protocol fulfilling the following conditions: (i) being compatible with the most basic flow cytometry apparatus; (ii) excluding the need of PBMC isolation; and (iii) requiring a minimal amount of blood to be compatible with TB diagnosis in children. Following the STARD guidelines[23], we aimed to report the diagnostic performance of this assay during clinical care of a large prospective cohort of adult TB patients and controls conveniently recruited at a district-level diagnostic centre in a resource-limited setting

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