Blood levels of Mycobacterium tuberculosis (Mtb)antigen-triggered immune markers in people exposed to tuberculosis with regard to Mtb infection status and receipt of tuberculosis preventive therapy
Description
Abstract Background: Interferon-γ release assays (IGRAs) for tuberculosis infection (TBI) cannot distinguish different stages of the TBI spectrum (including spontaneously cleared infection). Here, we investigated patterns of Mtb-specific blood mediators in people with and without TBI during tuberculosis preventive therapy (TPT). Methods: Individuals with likelihood of recent Mtb exposure, aged 15-25 years, with valid IGRA results, in whom tuberculosis (TB) had been excluded, were included. Persons with TBI were sampled prior to TPT (IGRA+ pre-treatment, n=15) or after completion of TPT (IGRA+ post-treatment, n=15). Five persons without TBI were included as controls (IGRA-). Levels of 40 mediators (GM-CSF, IFN-γ, IL-10, IL-17A, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, eotaxin, I-TAC, IL-12, IL-13, IP-10, MCP-1, MIP-1α, MIP-1β, TNF-α, VEGF, G-CSF, GRO, IFN-α2a, IL-12p70, IL-15, IL-18, IL-1RA, IL-1α, IL-7, IL-9, Galectin-9, I-309, IL-22, IL-27, MCP-2, OXO-40, TRAIL, MIG, MIF, RANTES) related to TB immune control in blood incubated with Mtb antigens in the QuantiFERON-TB Plus® kit were assessed with electrochemiluminescence assay and compared between participant categories. Results: The concentration of 10 mediators (GM-CSF, interferon-γ, IL-2, I-TAC, IL-12, IP-10, I-309, MCP-2, MIG, and VEGF) significantly differed between IGRA+ pre-treatment and IGRA-. A non-significant trend in levels of these markers was observed between IGRA+ pre-treatment, IGRA+ post-treatment and IGRA-. Based on these mediators two clusters were identified: A (n=16), including 5 IGRA-, 4 IGRA+ pre-treatment, 7 IGRA+ post-treatment and B (n=19), including 11 IGRA+ pre-treatment and 8 IGRA+ post-treatment. Conclusion: Plasma levels of several Mtb-triggered mediators differed with regard to TBI status among persons recently exposed to TB, suggesting the potential for alternative markers to assess TBI status. Longitudinal analysis of these mediators during TPT is warranted to explore whether these markers can be used to assess the likelihood of persistence of viable bacilli in Mtb-exposed individuals. ClinicalTrials.gov ID: NCT05621343
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Research methodology 2.1 Inclusion and exclusion criteria This study was conducted at the Department of Infectious diseases, Skane University Hospital, Malmö and Lund, Sweden. Individuals who had undergone QuantiFERON-TB Plus (QFT) testing in routine health care either because of recent and close contact to a person with confirmed pulmonary TB or because of recent immigration to Sweden from a TB endemic country were eligible for inclusion. Persons with positive QFT results (IFN-γ >0.70 IU/ml) aged 15-25 years, with origin in a TB-endemic country were further categorized with regard to tuberculosis preventive therapy (TPT) status (either before initiation of TPT [IGRA+ pre-treatment] or within 0-6 months after TPT completion [IGRA+ post-treatment]). TPT was prescribed as rifampicin (4 months) or isoniazid (6-9 months). Persons without TBI (IGRA-; defined as QFT result <0.35 IU/ml >3 months after exposure, age 15-25 years and with no clinical sign of active TB) were identified from routine contact tracing after exposure to pulmonary TB. The following exclusion criteria were applied: symptoms or signs compatible with TB, underlying chronic disease, immunosuppressive treatment, pregnancy (including 6 months post-partum), previous treatment for TB, or TPT. For this study, we included 15 IGRA+ pre-treatment, 15 IGRA+ post-treatment, and 5 IGRA-. 2.3 Sample collection and processing Venous blood was collected in QFT tubes and directly transported to the study laboratory. Following incubation for 16 to 24 hours at 37 °C, samples were centrifuged for 15 min at 2,000g. Supernatants were aliquoted and stored at -80 °C. After completion of sample collection, levels of selected mediators were measured using Meso Scale Diagnostics LLC (MSD, Rockville, MD, USA) electrochemiluminescence assay. After the selection process (Suppl. Fig. 1) 40 mediators were included: GM-CSF, IFN-γ, IL-10, IL-17A, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, eotaxin, I-TAC, IL-12, IL-13, IP-10, MCP-1, MIP-1α, MIP-1β, TNF-α, VEGF, G-CSF, GRO, IFN-α2a, IL-12p70, IL-15, IL-18, IL-1RA, IL-1α, IL-7, IL-9, Galectin-9, I-309, IL-22, IL-27, MCP-2, OXO-40, TRAIL, MIG, MIF, RANTES). 2.5 Mediator analysis Supernatants were diluted 4-fold according to the manufacturer’s instructions. Supernatants of 10 of the 43 selected mediators were diluted further to obtain quantifiable results. IL-1RA, IP-10, MCP-1, MCP-2, MIF, MIP-1α and RANTES were diluted x100 and GRO-α, IL-8 and MIP-1β were diluted x400. Mediator levels were determined using electrochemiluminescence assay according to the manufacturer’s instructions. Data was processed and stored with the use of DISCOVERY WORKBENCH 4.0 MSD®. All mediator levels were corrected by subtracting background values. These calculations were carried out using Microsoft Excel® version 16.78.3.
Institutions
- Lunds universitet Institutionen for translationell medicin
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Funders
- Region SkåneSweden