Two-Hit in vitro T-Cell Stimulation Detects Mycobacterium tuberculosis Infection in QuantiFERON Negative Tuberculosis Patients and Healthy Contacts From Ghana

Abstract

IFN-γ release assays [e.g., QuantiFERON (QFT)] are widely used for diagnosis of Mycobacterium tuberculosis ( Mtb ) infection. T-cell responses against QFT antigens ESAT6 and CFP10 are highly Mtb specific but previous studies indicated suboptimal assay sensitivity. Especially for potentially infected healthy contacts (HCs) of tuberculosis patients, alternative antigen usage and more sensitive tests may contribute to improved detection of latent Mtb infection. In a pilot case-control study of tuberculosis patients ( n = 22) and HCs ( n = 20) from Ghana, we performed multifaceted in vitro assays to identify optimal assay conditions. This included a two-hit stimulation assay, which is based on initial and second re-stimulation with the same antigen on d6 and intracellular IFN-γ analysis, to compare T-cell responses against ESAT6/CFP10 (E6/C10) and selected latency antigens (i.e. Rv2628, Rv1733, Rv2031, Rv3407) of Mtb . Considerable subgroups of tuberculosis patients (64%) and HCs (75%) had negative or indeterminate QFT results partially accompanied by moderate PHA induced responses and high IFN-γ background values. Intracellular IFN-γ analysis of E6/C10 specific CD4 + T-cell subpopulations and evaluation of responder frequencies had only moderate effects on assay sensitivity. However, two-hit in vitro stimulation significantly enhanced E6/C10 specific IFN-γ positive T-cell proportions especially in QFT non-responders, and in both study groups. Mtb latency antigen-specific T cells against Rv1733 and Rv2628 were especially detected in HCs after two-hit stimulation and T-cell responses against Rv2628 were highly capable to discriminate tuberculosis patients and HCs. Two-hit in vitro stimulation may improve moderate sensitivity of short term IFN-γ based assays, like QFT, to detect Mtb infection. Latency stage-specific antigens added significantly to detection of Mtb infection in HCs and tuberculosis patients with negative QFT test results.

Plasma cytokine levels characterize disease pathogenesis and treatment response in tuberculosis patients

“…There is increasing evidence that tuberculosis pathology affects innate and adaptive immune cell phenotype and functions [2]. As a consequence, effector T-cell and antigen-presenting cell functions in tuberculosis patients are impaired with potential implications on anti-mycobacterial host response [3][4][5]. Importantly, several pathognomonic factors are detectable in blood and also affect immune cells, which had not been in direct contact with the pathogen itself or affected tissues [3][4][5][6].…”

Aberrant cytokine milieu and signaling affect immune cell phenotypes and functions in tuberculosis pathology: What can we learn from this phenomenon for application to inflammatory syndromes?

“…However, we and others detected lower mitogen-induced IFNγ secretion in tuberculosis patients from Ghana than in healthy controls. 3 , 7 Mitogen-induced IFNγ levels were below the detection limit for a subgroup of tuberculosis patients, which led to false-negative or ‘indeterminate’ IGRA results. 3 These results suggested generally impaired T-cell cytokine expression as an immunopathognomonic feature in tuberculosis patients.…”

“… 3 , 7 Mitogen-induced IFNγ levels were below the detection limit for a subgroup of tuberculosis patients, which led to false-negative or ‘indeterminate’ IGRA results. 3 These results suggested generally impaired T-cell cytokine expression as an immunopathognomonic feature in tuberculosis patients. This assumption was strengthened by T-cell phenotype analyses.…”

The Biological and Clinical Aspects of a Latent Tuberculosis Infection

“…QFT-GIT-positive people with a remote infection had a fivefold higher response to Rv2628, which could be explained by the immune-mediated protection against TB, and, thus, can be used for distinguishing between an LTBI and a recently acquired infection [ 103 ]. A two-hit assay, combining the use of ESAT-6/CFP-10 with additional antigens (Rv2628, Rv1733, Rv2031, and Rv3407), allowed for the identification of the LTBI possessors who were not responsive in the QFT assay [ 104 ].…”