Mycobacterial antigens in pleural fluid mononuclear cells to diagnose pleural tuberculosis in HIV co-infected patients.

BACKGROUND: Extra pulmonary manifestation of tuberculosis (TB) accounts for approximately one-half of TB cases in HIV-infected individuals with pleural TB as the second most common location. Even though mycobacteria are cleared, mycobacterial antigens may persist in infected tissues, causing sustained inflammation and chronicity of the disease. The aim of this study was to explore various mycobacterial antigens in pleural effusions, the impact of HIV infection and CD4+ T-cell depletion on the presence of antigens, and the diagnostic potential of antigens for improved and rapid diagnosis of pleural TB. METHODS: Pleural fluid specimens were collected from patients presenting with clinically suspected pleural TB, and processed routinely for culture, cytology, and adenosine deaminase activity analysis. HIV status and CD4+ T-cell counts were recorded. Pleural fluid mononuclear cells (PFMC) were isolated, and cell smears were stained with acid-fast staining and immunocytochemistry for various mycobacterial antigens. Real-time and nested-PCR were performed. Patients were categorized as pleural TB or non-TB cases using a composite reference standard. Performance of the mycobacterial antigens as diagnostic test was assessed. RESULTS: A total of 41 patients were enrolled, of which 32 were classified as pleural TB and 9 as non-TB. Thirteen patients had culture confirmed pleural TB, 26 (81%) were HIV-TB co-infected, and 64% had < 100 CD4+ T-cells/microL. Both secreted and cell-wall mycobacterial antigens were detected in PFMC. Lipoarabinomannan (LAM) was the most frequently detected antigen. There was no direct correlation between positive culture and antigens. Cases with low CD4+ T-cell counts had higher bacterial and antigen burden. By combining detection of secreted antigen or LAM, the sensitivity and specificity to diagnose pleural TB was 56 and 78%, respectively, as compared to 41 and 100% for culture, 53 and 89% for nested PCR, and 6 and 100% for real-time PCR. CONCLUSION: Mycobacterial antigens were detectable in PFMC from tuberculous pleural effusions, even in cases where viable mycobacteria or bacterial DNA were not always detected. Thus, a combination of secreted antigen and LAM detection by immunocytochemistry may be a complement to acid-fast staining and contribute to rapid and accurate diagnosis of pleural TB.

The COX- inhibitor indomethacin reduces Th1 effector and T regulatory cells in vitro in Mycobacterium tuberculosis infection.

BACKGROUND: Tuberculosis (TB) causes a major burden on global health with long and cumbersome TB treatment regimens. Host-directed immune modulating therapies have been suggested as adjunctive treatment to TB antibiotics. Upregulated cyclooxygenase-2 (COX-2)-prostaglandin E2 (PGE2) signaling pathway may cause a dysfunctional immune response that favors survival and replication of Mycobacterium tuberculosis (Mtb). METHODS: Blood samples were obtained from patients with latent TB (n = 9) and active TB (n = 33) before initiation of anti-TB chemotherapy. COX-2 expression in monocytes and ESAT-6 and Ag85 specific T cell cytokine responses (TNF-α, IFN-γ, IL-2), proliferation (carboxyfluorescein succinimidyl ester staining) and regulation (FOXP3+ T regulatory cells) were analysed by flow cytometry and the in vitro effects of the COX-1/2 inhibitor indomethacin were measured. RESULTS: We demonstrate that indomethacin significantly down-regulates the fraction of Mtb specific FOXP3+ T regulatory cells (ESAT-6; p = 0.004 and Ag85; p < 0.001) with a concomitant reduction of Mtb specific cytokine responses and T cell proliferation in active TB. Although active TB tend to have higher levels, there are no significant differences in COX-2 expression between unstimulated monocytes from patients with active TB compared to latent infection. Monocytes in both TB groups respond with a significant upregulation of COX-2 after in vitro stimulation. CONCLUSIONS: Taken together, our in vitro data indicate a modulation of the Th1 effector and T regulatory cells in Mtb infection in response to the COX-1/2 inhibitor indomethacin. The potential role as adjunctive host-directed therapy in TB disease should be further evaluated in both animal studies and in human clinical trials.

Cytokine Patterns in Tuberculosis Infection; IL-1ra, IL-2 and IP-10 Differentiate Borderline QuantiFERON-TB Samples from Uninfected Controls.

BACKGROUND: Interferon gamma release assays (IGRAs) do not discriminate between active tuberculosis (TB) and latent TB infection (LTBI), which limit their use in TB endemic areas. Subjects with QuantiFERON-TB (QFT) results around the diagnostic cut-off more likely show inconsistent results on serial testing which makes the interpretation of the assay difficult. We have studied potential biomarkers in patients with various stages of TB infection and with borderline QFT tests compared to those with higher values. METHODS: 27 soluble biomarkers were analysed in QFT supernatants from patients with active TB (n = 18), individuals with LTBI (n = 48) and from QFT negative controls (n = 16) by the Multiplex bead assay. The LTBI group was classified into two groups according to QFT IFN-γ levels; QFT borderline (0.35-0.70 IU/mL, n = 11) or QFT high (>0.70 IU/mL, n = 36). RESULTS: The levels of IL-1ra, IL-2, IL-13, IL-15, IFN-γ, IP-10 and MCP-1 in background corrected TB antigen stimulated supernatants (TBAg-Nil) significantly distinguished both active TB and LTBI QFT high groups from the QFT negative controls (p≤0.004). In addition, IL-1ra, IL-2 and IP-10 significantly differentiated the QFT borderline group from the controls (p≤0.001). Still, in the QFT borderline group the IL-1ra and IP-10 levels were not significant different from neither the QFT high nor the active TB group, whereas the IL-2 levels were lower (p≤0.003). The level of IP-10 showed the best separation between the QFT borderline group and the QFT negative controls (AUC 0.92) and offered 100% sensitivity for active TB. CONCLUSION: IL-1ra, IL-2 and IP-10 differentiate QFT borderline samples from uninfected controls and the majority of QFT borderline subjects were classified as LTBI by these markers. Still, inconsistency was seen, and further studies are needed to examine the performance of alternative markers before concluded if they could be used as diagnostics tools.