Tuberculosis is the infectious disease with the worldwide largest disease burden and there remains a great need for better diagnostic biomarkers to detect latent and active M. tuberculosis infection. RNA molecules hold great promise in this regard, as their levels of expression may differ considerably between infected and uninfected subjects. We have measured expression changes in the four major classes of small noncoding RNAs in blood samples from patients with different stages of TB infection. We found that, in addition to miRNAs (which are known to be highly regulated in blood cells from TB patients), expression of piRNA and snoRNA is greatly altered in both latent and active TB, yielding promising biomarkers. Even though the functions of many sncRNA other than miRNA are still poorly understood, our results strongly suggest that at least piRNA and snoRNA populations may represent hitherto underappreciated players in the different stages of TB infection.

BackgroundPleural tuberculosis (PlTB) is the most common extrapulmonary manifestation of this infectious disease which still presents high mortality rates worldwide. Conventional diagnostic tests for PlTB register multiple limitations, including the lack of sensitivity of microbiological methods on pleural specimens and the need of invasive procedures such as pleural biopsy performance. In this scenario, the search for biological markers on pleural fluid (PF) has been the target of several studies as a strategy to overcome the limitations of PlTB diagnosis. This study aims to evaluate the use either isolated or in combination with adenosine deaminase (ADA), interferon-gamma (IFN-γ), interferon-gamma inducible protein of 10-kD (IP-10) levels on PF in order to guide an accurate anti-TB treatment in microbiologically non-confirmed cases.Methods and findingsEighty patients presenting pleural effusion under investigation were enrolled in a cross-sectional study conducted at Pedro Ernesto University Hospital, Rio de Janeiro, RJ, Brazil. Peripheral blood (PB) and PF samples collected from all patients were applied to the commercial IFN-γ release assay, QuantiFERON-TB Gold In-Tube, and samples were analyzed for IFN-γ and IP-10 by immunoassays. ADA activity was determined on PF by the colorimetric method. Based on microbiological and histological criteria, patients were categorized as follow: confirmed PlTB (n = 16), non-confirmed PlTB (n = 17) and non-PlTB (n = 47). The Mycobacterium tuberculosis antigen-specific production of IFN-γ and IP-10 on PB or PF did not show significant differences. However, the basal levels of these biomarkers, as well as the ADA activity on PF, were significantly increased in confirmed PlTB in comparison to non-PlTB group. Receiver operating characteristics curves were performed and the best cut-off points of these three biomarkers were estimated. Their either isolated or combined performances (sensitivity [Se], specificity [Sp], positive predictive value [PPV], negative predictive value [NPV] and accuracy [Acc]) were determined and applied to Venn's diagrams among the groups. Based on the confirmed PlTB cases, IFN-γ showed the best performance of them at a cut-off point of 2.33 IU/mL (Se = 93.8% and Sp = 97.9%) followed by ADA at a cut-off of 25.80 IU/L (Se = 100% and Sp = 84.8%) and IP-10 (Cut-point = 4,361.90 pg/mL, Se = 75% and Sp = 82.6%). IFN-γ plus ADA (cut-point: 25.80 IU/L) represent the most accurate biomarker combination (98.4%), showing Se = 93.7%, Sp = 100%, PPV = 100% and NPV = 97.9%. When this analysis was applied in non-confirmed PlTB, 15/17 (88.2%) presented at least two positive biomarkers in combination.ConclusionIFN-γ, IP-10, and ADA in PlTB effusions are significantly higher than in non-PlTB cases. IFN-γ is an excellent rule-in and rule-out test compared to IP-10 and ADA. The combination of IFN-γ and ADA, in a reviewed cut-off point, showed to be particularly useful to clinicians as their positive results combined prompts immediate treatment for TB while both negative resu...

The PstS1 antigen is highly immunogenic, principally when combined with CFP10 during both latent and active TB infection. In the present study, a selected pstS1 gene fragment was cloned, fused with CFP10, and expressed in Escherichia coli. The product M ycobacterium tuberculosis, a highly successful parasite, is the cause of tuberculosis (TB). A major health care concern, the disease results in approximately 1.4 million annual deaths (990,000 among HIV-negative individuals), infecting roughly one-third of the world's population. Among the infected, 5% to 10% will develop active disease in their lifetimes and 90% will harbor the latent form. According to a recent mathematical projection of TB eradication, the treatment of latent TB infection (LTBI) and active TB is urgently required in order to lower and ultimately prevent the further spread of the disease at its present rate (1, 2).Two principal approaches based on the adaptive immune responses elicited by M. tuberculosis infection are currently used to identify TBI (3): the in vivo tuberculin skin test (TST) and the ex vivo gamma interferon (IFN-␥) release assay (IGRA).Developed in 1908, TST became the standard means of assessing the presence of TB infection. Via TST, prior TB exposure is measured by a type 4 delayed-type hypersensitivity reaction when a purified protein derivative (PPD) of M. tuberculosis is injected intradermally. Although TST has some biological limitations such as the occurrence of anergies in one-third of active TB cases, crossreactivity with M. bovis bacillus Calmette-Guérin (BCG) and non-TB mycobacteria does not, according to some authors, result in major sensibility differences in IGRAs (4).The IGRA was recently developed using antigens that are expressed in M. tuberculosis but not in BCG or M. bovis strains as stimuli. These antigens measure the production of IFN-␥ in peripheral blood mononuclear cells (3,5). According to a recent World Health Organization (WHO) report (1), the two currently commercially available IGRAs have yet to generate sufficient data or enough high-quality evidence regarding their performance in the low-and middle-income countries that typically have a high TB and/or HIV burden and where the coverage of BCG vaccination may cause some interference (5, 6).Both IGRAs are based on Mycobacterium tuberculosis-specific antigens, namely, early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP10) (5). These antigens must be validated in different scenarios, but other antigen combinations also need to be evaluated to improve IGRA coverage.The 38-kDa M. tuberculosis antigen, also known as phosphatespecific transporter-1 (PstS-1), coded by a pstS1 gene that composes one of the 3 putative pst operons, is a lipoprotein phosphate transport receptor on the cell surface. These operons probably constitute a subtle biochemical adaptation of M. tuberculosis, enabling it to grow and survive under different phosphate-limiting conditions during its infectious cycle. The PstS-1 protein is ac-

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