Cell proliferation and interferon-gamma response to recombinant MBP-3, NarL, MT-10.3, and 16kDa Mycobacterium tuberculosis antigens in Brazilian tuberculosis patients

Human pulmonary tuberculosis (TB) is a worldwide public health problem. In resistant individuals, control of the infection mainly requires development of a Th1 cell immune response with production of cytokines, of which interferon-gamma (IFN-gamma)plays an important role. Several antigens from Mycobacterium tuberculosis complex has been described for use in vaccine development or for diagnostic purposes, however little evaluation has been done in endemic area for TB. The proliferative and IFN-gamma human T cell immune responses, to four recombinant proteins (MBP-3, NarL, MT-10.3, 16 kDa) and PPD, of 38 Brazilian TB patients (6 untreated and 32 treated) and 67 controls (38 positive and 29 negative tuberculin skin test - TST) were compared. The highest reactivity mean rate was obtained with PPD followed by 16 kDa in TB patients. While most of the patients (87%) and controls (> 64%) respond to the PPD, 16kDa was more specifically recognized (> 21%) although less sensitive (54%). When TB patients were divided according to treatment status, opposite to PPD, higher average level of IFN-gamma was induced by 16kDa in untreated (505 pg/ml) compared to treated TB patients and TST+ (269.8 pg/ml x 221.6pg/ml, respectively), although the difference was not significant. These data show that in contrast with the other recombinant proteins, the stimulatory potency of 16kDa to induce proliferative and INF-gamma response was more effective and is more recognized by active TB untreated patients, eliciting in control individuals a more selective immune response than PPD

Application of Venn's diagram in the diagnosis of pleural tuberculosis using IFN-γ, IP-10 and adenosine deaminase.

BACKGROUND:Pleural 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 FINDINGS:Eighty 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. CONCLUSION:IFN-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 results suggest further investigation

Reprogramming of small noncoding RNA populations in peripheral blood reveals host biomarkers for latent and active Mycobacterium tuberculosis infection

Marcelo Ribeiro Alves. Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Documento produzido em parceria ou por autor vinculado à Fiocruz, mas não consta a informação no documento.Submitted by Janaína Nascimento ([email protected]) on 2019-12-16T13:51:37Z No. of bitstreams: 1 ve_Araujo_Leonardo_etal_INI_2019.pdf: 4561523 bytes, checksum: 31fe5ecf90ccb4d45f353481d14207d7 (MD5)Approved for entry into archive by Janaína Nascimento ([email protected]) on 2019-12-16T14:15:20Z (GMT) No. of bitstreams: 1 ve_Araujo_Leonardo_etal_INI_2019.pdf: 4561523 bytes, checksum: 31fe5ecf90ccb4d45f353481d14207d7 (MD5)Made available in DSpace on 2019-12-16T14:15:20Z (GMT). No. of bitstreams: 1 ve_Araujo_Leonardo_etal_INI_2019.pdf: 4561523 bytes, checksum: 31fe5ecf90ccb4d45f353481d14207d7 (MD5) Previous issue date: 2019TWINCORE Centre for Experimental and Clinical Infection Research. Research Group Biomarkers for Infectious Diseases. Hannover, Germany / Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Rio de Janeiro, RJ, Brasil / Helmholtz Centre for Infection Research. Braunschweig, Germany.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Federal University of Rio de Janeiro. Thoracic Diseases Institute. Rio de Janeiro, RJ, Brazil.TWINCORE Centre for Experimental and Clinical Infection Research. Institute for Experimental Infection Research. Hannover, Germany.TWINCORE Centre for Experimental and Clinical Infection Research. Research Group Biomarkers for Infectious Diseases. Hannover, Germany / Zagazig University. Faculty of Veterinary Medicine. Department of Zoonoses. Zagazig, Egypt.TWINCORE Centre for Experimental and Clinical Infection Research. Research Group Biomarkers for Infectious Diseases. Hannover, Germany / Hannover Medical School. Institute for Laboratory Animal Science. Hannover, Germany.TWINCORE Centre for Experimental and Clinical Infection Research. Research Group Biomarkers for Infectious Diseases. Hannover, Germany.Federal University of Rio de Janeiro. Thoracic Diseases Institute. Rio de Janeiro, RJ, Brazil.Helmholtz Centre for Infection Research. Braunschweig, Germany.Fundação Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.TWINCORE Centre for Experimental and Clinical Infection Research. Research Group Biomarkers for Infectious Diseases. Hannover, Germany / Helmholtz Centre for Infection Research. Braunschweig, Germany / Centre for Individualised Infection Medicine, Hannover, Germany.In tuberculosis (TB), as in other infectious diseases, studies of small noncoding RNAs (sncRNA) in peripheral blood have focused on microRNAs (miRNAs) but have neglected the other major sncRNA classes in spite of their potential functions in host gene regulation. Using RNA sequencing of whole blood, we have therefore determined expression of miRNA, PIWI-interacting RNA (piRNA), small nucleolar RNA (snoRNA), and small nuclear RNA (snRNA) in patients with TB (n = 8), latent TB infection (LTBI; n = 21), and treated LTBI (LTBItt; n = 6) and in uninfected exposed controls (ExC; n = 14). As expected, sncRNA reprogramming was greater in TB than in LTBI, with the greatest changes seen in miRNA populations. However, substantial dynamics were also evident in piRNA and snoRNA populations. One miRNA and 2 piRNAs were identified as moderately accurate (area under the curve [AUC] = 0.70 to 0.74) biomarkers for LTBI, as were 1 miRNA, 1 piRNA, and 2 snoRNAs (AUC = 0.79 to 0.91) for accomplished LTBI treatment. Logistic regression identified the combination of 4 sncRNA (let-7a-5p, miR-589-5p, miR-196b-5p, and SNORD104) as a highly sensitive (100%) classifier to discriminate TB from all non-TB groups. Notably, it reclassified 8 presumed LTBI cases as TB cases, 5 of which turned out to have features of Mycobacterium tuberculosis infection on chest radiographs. SNORD104 expression decreased during M. tuberculosis infection of primary human peripheral blood mononuclear cells (PBMC) and M2-like (P = 0.03) but not M1-like (P = 0.31) macrophages, suggesting that its downregulation in peripheral blood in TB is biologically relevant. Taken together, the results demonstrate that snoRNA and piRNA should be considered in addition to miRNA as biomarkers and pathogenesis factors in the various stages of TB.IMPORTANCE 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

Neutrophil CD64 expression levels in IGRA-positive individuals distinguish latent tuberculosis from active disease

BACKGROUND CD64 (FcγR1) is a high-affinity receptor for monomeric IgG1 and IgG3. Circulating neutrophils express very low amounts of CD64 on their surface. OBJECTIVES Our primary aim was to investigate the utility of neutrophil CD64 surface expression as a biomarker of active pulmonary tuberculosis (TB). We hypothesised that elevated neutrophil CD64 expression in TB infection would be associated with interferon gamma (IFN-g) as an inducer of CD64 expression. METHODS The expression level of CD64 per neutrophil (PMN CD64 index) was quantitatively measured with flow cytometry using a Leuko64 kit in samples from patients with TB and latent TB infection (LTBI) as well as healthy controls, as part of a prospective cohort study in Brazil. FINDINGS The PMN CD64 index in patients with TB was higher than that in healthy controls and LTBI. Receiver operating characteristic curve analyses determined that the PMN CD64 index could discriminate patients with TB from those with LTBI and healthy individuals. PMN CD64 index levels returned to baseline levels after treatment. CONCLUSIONS The positive regulation of CD64 expression in circulating neutrophils of patients with active TB could represent an additional biomarker for diagnosis of active TB and could be used for monitoring individuals with LTBI before progression of TB disease