br Methods br Results br Discussion A main health

Methods
Results
Discussion A main health obstacle to control TB is that latent bacilli may remain viable for decades and reactivate later to cause active TB. Thus, there is a need to identify LTBI individuals and treat them to eliminate the risk of developing TB. Since there is no diagnostic gold standard for LTBI and several reports have demonstrated the potential use of read what he said employed in current IGRAs as protective vaccines, it urges to find new M. tuberculosis antigens to detect latently infected individuals (Davila et al., 2012; Cervantes-Villagrana et al., 2013; Reither et al., 2014; van Dissel et al., 2011; Sester et al., 2011). Currently, both TST and IGRAs are used for LTBI diagnosis worldwide. Still, in BCG-vaccinated populations IGRAs may be preferable to TST since they are more specific for M. tuberculosis infection (Pai et al., 2014). In fact, in our study 13% of the individuals assessed had discordant results between TST and IGRA tests (Supplementary Fig. 2). To develop T cell-based assays that discriminate active TB and LTBI, improving the current available tests, it is necessary to identify new host markers expressed in response to M. tuberculosis antigens that are uniquely recognized by LTBI individuals (Chegou et al., 2012). Thus, present IGRAs might be complemented by using antigens to which patients with active TB do not respond. Here, we describe an immune-stimulatory function of the 16-kDa conserved protein coded by the M. tuberculosis open reading frame Rv2626c, one of the most abundant proteins under low-oxygen conditions in M. tuberculosis lysates (Rosenkrands et al., 2002). Variable results using Rv2628 and Rv2626c latency antigens were reported by others, although the disparities could be related to differences in stimulation schemes, antigen concentrations, ethnic backgrounds, and BCG vaccination history (Schuck et al., 2009; Chegou et al., 2012; Lin et al., 2007; Black et al., 2009; Riano et al., 2012; Hozumi et al., 2013; Mensah et al., 2014). Bashir et al. reported that Rv2626c induced higher IFN-γ levels in TB patients than in HD, but LTBI individuals were not analyzed in this study (Bashir et al., 2010). Leyten et al. demonstrated that TST+ individuals strongly recognized more latency antigens (e.g.,: Rv2628 and Rv2626c) as compared to cured patients or patients undergoing anti-TB treatment (Leyten et al., 2006). The study by Goletti et al. evaluated T-cell IFN-γ responses to M. tuberculosis latency antigens in a mainly European population composed by subjects at different stages of TB infection, concluding that remote LTBI individuals showed significantly higher IFN-γ responses to Rv2628 antigen than recent LTBI subjects, active TB and controls (Goletti et al., 2010). However, we studied a very different population: 20% of QFT-GIT+ and 12% of QFT-GIT− subjects were from other Latin American countries (Table 1), while the rest were Argentinean. In addition, the genetic mixture of Argentinean people is comprised by 79.9% European, 15.8% Native American, and 4.3% African contributions, which would imply that there was a fairly diverse genetic background in the population studied (Corach et al., 2010; Avena et al., 2006). Since some HD subjects produced low levels of IFN-γ against Rv2626c (Fig. 2), we searched for Rv2626c immunodominant epitopes to which only LTBI individuals responded. None of the mentioned studies that tested latency antigens investigated the immunodominance of peptide pools to discriminate LTBI individuals (Schuck et al., 2009; Leyten et al., 2006; Chegou et al., 2012; Lin et al., 2007; Black et al., 2009; Riano et al., 2012; Hozumi et al., 2013; Mensah et al., 2014; Bashir et al., 2010; Goletti et al., 2010). We observed that pools C, B, D, E and F all allowed improving the discernment between LTBI and HD (Table 3), but pools B, D and F resulted the most specific and immunogenic ones. These findings reinforced our data showing that the response to Rv2626c might allow discriminating between LTBI and HD. Additionally, a significantly higher cumulative IFN-γ response to the immunogenic peptide pools B, D and F was detected in LTBI individuals as compared to HD (Table 3). Together, we demonstrated that specific immunogenic peptide pools from Rv2626c might improve the discrimination of LTBI individuals from HD in a BCG-vaccinated population. Interestingly, when we analyzed the response to Rv2626c both in PBMCs and in whole blood from LTBI, HD and TB patients, we observed that only LTBI individuals secreted significant amounts of IFN-γ against this antigen (Fig. 2A and B), indicating that the use of Rv2626c or its immunogenic peptides might comprise a new tool to differentiate LTBI individuals from both HD and patients with active disease. Thus, the use of Rv2626c in combination with ESAT-6 and CFP-10 could improve current kits to allow the differential diagnosis of LTBI and active TB subjects. Additionally, Rv2626c or its immunogenic peptide pools might be candidates to replace potential vaccine antigens present in current IGRAs. In conclusion, the vast genetic mixture of Argentinean people makes the use of Rv2626c or its immunogenic peptide pools promising to discriminate individuals with active and latent M. tuberculosis infections in BCG-vaccinated people. However, future studies should determine whether Rv2626c might be also useful discriminating LTBI from TB patients and healthy donors in non-BCG-vaccinated populations.