BackgroundThe clinical application of IFN-γ release assays (IGRAs) has recently improved the diagnosis of latent tuberculosis infection. In a multicenter study of the Tuberculosis Network European Trialsgroup (TBNET) we aimed to ascertain in routine clinical practice the accuracy of a novel assay using selected peptides encoded in the mycobacterial genomic region of difference (RD) 1 for the diagnosis of active tuberculosis in comparison with tuberculin skin test (TST), QuantiFERON-TB GOLD In-Tube (Cellestis Ltd., Carnegie, Australia) and T-SPOT.TB (Oxfordimmunotec, Abingdon, UK).Principal Findings425 individuals from 6 different European centres were prospectively enrolled. We found that sensitivity of the novel test, TST, QuantiFERON-TB GOLD In-Tube and T-SPOT.TB was respectively 73.1%, 85.3%, 78.1%, and 85.2%; specificity was respectively 70.6%, 48.0%, 61.9% and 44.3%; positive likelihood ratios were respectively 2.48, 1.64, 2.05, and 1.53; negative likelihood ratios were respectively 0.38, 0.31, 0.35, 0.33. Sensitivity of TST combined with the novel test, QuantiFERON-TB GOLD In-Tube and T-SPOT.TB increased up to 92.4%, 97.7% and 97.1%, respectively. The likelihood ratios of combined negative results of TST with, respectively, the novel test, QuantiFERON-TB GOLD In-Tube and T-SPOT.TB were 0.19, 0.07 and 0.10.ConclusionsThe assay based on RD1 selected peptides has similar accuracy for active tuberculosis compared with TST and commercial IGRAs. Then, independently of the spectrum of antigens used in the assays to elicit mycobacterial specific immune responses, the novel test, IGRAs, and the TST do not allow an accurate identification of active tuberculosis in clinical practice. However, the combined use of the novel assay or commercial IGRAs with TST may allow exclusion of tuberculosis.

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A reliable approach differentiating between active and latent TB infection (LTBI) is yet unavailable. Recent data suggest the involvement of CD8 T cells in protective response to mycobacterium tuberculosis (MTB). We compared the antigen-specific responses and subset composition of peripheral blood CD8 T cells in patients with active infection (ATB), recent household contacts (RC), highly-exposed health-care workers (HW), and BCG-vaccinated healthy controls (HC). Study groups included 20 ATB, 21 RH, 21 HW and 15 HC. Active TB was diagnosed according to microbiological and clinical criteria. RD1-specific CD4 and CD8 T cells were determined by intracellular cytokine staining (ICS). CD8 T subsets were defined as naïve (CD45RA+CD27+), memory (M, CD45RA-CD27+), effector (E, CD27-CD45RA-), and terminal effector (TE, CD27-CD45RA+), and analyzed by flow cytometry. The levels of RD-1-specific CD4 T cells were highest in ATB but not significantly different from LTBI groups. A significant RD-1-specific CD8 T cell IFNγ response (0.289%) differentiated RC from ATB, HE and HC subjects (0.053, 0.027 and 0.011, p < 0.05) and was associated with decreased M/TE ratio. Finally, an increased M/E CD8 T ratio distinguished HW from RC and ATB subjects (7.6 vs. 2.4 and 2.6, p < 0.05). The study of MTB-specific CD8 T-cell response in combination with CD8 T subset composition adds to the differentiation between active and latent TB.Key words: CD8 T-cell response, RD-1-specific T cells, ICS, MTB infection 587 Introduction. Mycobacterium tuberculosis (MTB) is one of the most frequent causes of infectious morbidity worldwide [ 1 ]. A large majority of M. tuberculosis-infected individuals remains asymptomatic while unable to clear the bacterium, with a lifelong probability to develop active TB of about 5-10% [ 2 ]. This probability is highest within the first two years after infection, and in subjects with recently-established LTBI preventive therapy can be effective. Thus the distinction between the different forms and stages of MTB infection is fundamental for the effective control of TB transmission.The correct diagnosis of LTBI remains a challenge. Until recently, tuberculin skin test (TST) has been the most frequently used screening method, leading to a high rate of false positive results in M. bovis BCG-vaccinated individuals, and false negative tests in immunosuppressed persons. The new interferon gamma release assays (IGRAs) employing RD-1 antigens specific for virulent mycobacteria have excellent specificity unaffected by BCG vaccination and increased sensitivity While CD4 Th1 cells are the well-known effectors of MTB-specific immune response, a lot of evidence from humans and from animal models indicate a critical role of CD8 T cells for MTB control as well [ 6-8 ]. However, data about the characteristics of CD8 T cell responses in individuals with LTBI and in patients with active tuberculosis are still scarce.In this study, using intracellular cytokine staining (ICS) and flow cytometry, we compared the CD8 T cell responses ...