New study enables differentiation between active and latent tuberculosis using diagnostic "TB flow assay"
Labor Berlin - Charité Vivantes GmbH, together with the Institute of Immunology at the University Medical Center Schleswig-Holstein in Kiel, has developed a flow cytometric immunoassay that uses phenotyping of tuberculosis-reactive T cells and a diagnostic score to differentiate between latent tuberculosis infection (LTBI) and tuberculosis disease (TB). With the help of this "TB flow assay", the usually lengthy and sometimes difficult diagnostic differentiation between LTBI and TB could be significantly simplified and the associated therapy decisions accelerated. The results of the clinical-experimental study were published in May[ERJ publication on the TB flow assay].
In the prospective study in collaboration with Charité - Universitätsmedizin and the Heckeshorn Lung Clinic at Helios Klinikum Emil von Behring, the blood samples of 81 HIV-negative patients with culture-confirmed TB (n=47) or LTBI (n=34) were examined for the activity pattern of their TB-specific T lymphocytes before or at the start of antituberculous therapy. The participants were assigned to the respective cohort according to the applicable diagnostic criteria. A positive interferon-gamma release assay (IGRA) with a simultaneous absence of clinical and microbiological signs of TB in combination with a corresponding medical assessment led to inclusion in the LTBI cohort. TB disease was diagnosed on the basis of positive microbiological tests (culture, PCR, histology) or a clear clinical diagnosis. However, the decisive inclusion criterion for the TB cohort used to establish the assay was the cultural detection of the pathogen in temporal proximity to the sample collection. The TB cohort included patients with pulmonary (n=30), extrapulmonary (n=15) and mixed (n=2) involvement ranging in age from 19 to 84 years. About two-thirds (64%) of the participants were male (n=52).
As part of the study, the blood samples from both cohorts were stimulated with two different antigen pools (ESAT-6/CFP-10 peptide pools and purified protein derivative, PPD) and the number of reactive CD4+ T cells was then determined using flow cytometry. As the pure quantity of reactive T cells did not yet allow reliable discrimination between active and latent tuberculosis, the specific expression pattern of activity markers of the cells was used. Previous studies had already identified a number of markers (CD38, HLA-DR and KI-67) that can be used to diagnose tuberculosis. On their own, however, these markers did not allow reliable differentiation of a latent tuberculosis infection. However, the combined evaluation of the expression of several markers in the form of a scoring system comprising 12 parameters made it possible to reliably differentiate between TB and LTBI in the study.
With a sensitivity of at least 91% and a specificity of at least 93%, the TB flow assay score was highly diagnostic. The differentiation was reliable for both pulmonary and extrapulmonary involvement. The assay was applicable regardless of the gender and age of the patients and, thanks to laboratory standardization measures, can be used independently of the examiner and, with the same technical equipment, independently of the laboratory.
Patients who did not meet the inclusion criteria of the TB cohort were also examined. Among the culture-negative patients who, according to medical assessment, had TB disease, the TB flow assay identified all cases with positive TB PCR. There was less agreement in cases without culture and PCR confirmation, which were diagnosed solely on the basis of clinical, radiological or histological findings (6/9 matches). It remained unclear at the time of publication of the data whether these cases are not detected by the TB flow assay or whether the assay is better able to differentiate TB from LTBI than is possible using clinical, radiologic and histologic data alone.
Repeat measurements in the further course of TB therapy showed on average decreasing score values in the TB flow assay as the therapy progressed. This correlated with a lower activation of TB-specific T cells and could give the TB flow assay a role in therapy monitoring and early assessment of treatment success in the future.
The individual results of the study provide interesting insights into the pathogenesis of tuberculosis and the specific immune response of the human body. However, further prospective and longitudinal studies are needed to confirm these findings. The next step will be to evaluate the validity of the score in particularly vulnerable patients. In addition to HIV co-infected and otherwise immunocompromised people, this also includes children and adolescents, in whom TB diagnosis remains a challenge due to immunological characteristics. The applicability of the score in regions of the world with fewer resources also requires a separate evaluation.
The method is characterized by the simple sample collection and technically standardized feasibility and could contribute to the diagnosis of TB in industrialized countries. In Germany, the direct detection of tuberculosis pathogens using microscopic, cultural or molecular biological methods remains the gold standard for the diagnosis of TB.
Literature:
Mantei A, Meyer T, Schürmann M, Beßler C, Bias H, Krieger D, Bauer T, Bacher P, Helmuth J, Volk H-D, Schürmann D, Scheffold A, Meisel C. Mycobacterium tuberculosis-specific CD4 T-cell scoring discriminates tuberculosis infection from disease. European Respiratory Journal. 2022:2101780.
