Background Diagnosis of active tuberculosis (ATB) currently relies on detection of M. tuberculosis (Mtb). Identifying patients with extrapulmonary TB (EPTB) remains challenging because microbiological confirmation is often not possible. Highly accurate blood-based tests could improve diagnosis of both EPTB and pulmonary TB (PTB), and timely initiation of anti-TB therapy. Methods A case-control study was performed using discriminant analyses to validate an approach using Mtb-specific CD4+T-cell activation markers in blood to discriminate PTB and EPTB from latent TB infection (LTBI) as well as EPTB from PTB in 270 Brazilian individuals. We further tested the effect of HIV co-infection on diagnostic performance. Frequencies of IFNγ+CD4+T-cells expressing CD38, HLADR, and/or Ki67 were assessed by flow cytometry. Results EPTB and PTB were associated with higher frequencies of CD4+T-cells expressing CD38, HLADR or Ki67 compared to LTBI (all p-values < .001). Moreover, frequencies of HLADR+ (p= .03) or Ki67+ (p< .001) cells accurately distinguished EPTB from PTB. HIV infection did not affect the capacity of these markers to distinguish ATB from LTBI or EPTB from PTB. Conclusion Cell activation markers in Mtb-specific CD4+T-cells distinguished ATB from LTBI, and EPTB from PTB, regardless of HIV infection status. These parameters provide an attractive approach for developing blood-based diagnostic tests for both active and latent TB.

Cellular necrosis during Mycobacterium tuberculosis (Mtb) infection promotes both immunopathology and bacterial dissemination. Glutathione peroxidase-4 (Gpx4) is an enzyme that plays a critical role in preventing iron-dependent lipid peroxidation–mediated cell death (ferroptosis), a process previously implicated in the necrotic pathology seen in Mtb-infected mice. Here, we document altered GPX4 expression, glutathione levels, and lipid peroxidation in patients with active tuberculosis and assess the role of this pathway in mice genetically deficient in or overexpressing Gpx4. We found that Gpx4-deficient mice infected with Mtb display substantially increased lung necrosis and bacterial burdens, while transgenic mice overexpressing the enzyme show decreased bacterial loads and necrosis. Moreover, Gpx4-deficient macrophages exhibited enhanced necrosis upon Mtb infection in vitro, an outcome suppressed by the lipid peroxidation inhibitor, ferrostatin-1. These findings provide support for the role of ferroptosis in Mtb-induced necrosis and implicate the Gpx4/GSH axis as a target for host-directed therapy of tuberculosis.