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Transcriptome analysis of mycobacteria in sputum samples of pulmonary tuberculosis patients

ABSTRACT: Sputum expectorated by the pulmonary tuberculosis patients still remains a primary diagnostic specimen. The expression pattern of mycobacteria in sputum will lead to an insight of bacterial adaptation at the most highly transmissible stage of infection and can also help in identifying newer diagnostic as well as drug targets. Further, the identification of crossreactive gene targets from the sputum samples of other lung diseases will help to find the diagnostic candidates which will be highly specific to pulmonary tuberculosis.

OTHER RELATED OMICS DATASETS IN: PRJNA360604

ORGANISM(S): uncultured bacterium Mycobacterium tuberculosis

PROVIDER: GSE93316 | GEO | 2017/04/18

SECONDARY ACCESSION(S): PRJNA360604

REPOSITORIES: GEO

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Project description:Background: Assessments of airways inflammation in patients with chronic obstructive pulmonary diseases (COPD) require semi-invasive procedures and specialized sample processing know-how. In this study we aimed to set up and validate a novel non-invasive processing-free method for RNA sequencing (RNAseq) of spontaneous sputum samples collected from COPD patients. Methods: Spontaneous sputum samples were collected and stabilized, with or without selection of plugs and with or without the use of a stabilizer specifically formulated for downstream diagnostic testing (PrimeStore® Molecular Transport Medium). After 8-day storage at ambient temperature RNA was isolated according to an optimized RNAzol® method. An average percentage of fragments longer than 200 nucleotides (DV200) >30% and an individual yield >50ng were required for progression of samples to sequencing. Finally, to assess if the transcriptome generated would reflect a true endotype of COPD inflammation, the outcome of single-sample gene-set enrichment analysis (ssGSEA) was validated using an independent set of processed induced sputum samples Results: RNA extracted from spontaneous sputum using a stabilizer showed an average DV200 higher than 30%. 70% of the samples had a yield >50ng and were submitted to downstream analysis. There was a straightforward correlation in terms of gene expression between samples handled with or without separation of plugs. This was also confirmed by principal component analysis and ssGSEA. The top ten enriched pathways resulting from spontaneous sputum ssGSEA were associated to features of COPD, namely, inflammation, immune responses and oxidative stress; up to 70% of these were in common within the top ten enriched pathways resulting from induced sputum ssGSEA. Conclusion: This analysis confirmed that the typical COPD endotype was represented within spontaneous sputum and supported the current method as a non-invasive processing-free procedure to assess the level of sputum cell inflammation in COPD patients by RNAseq analysis.

Project description:Purpose: Accurate diagnosis of complete inactivation of tuberculosis lesions is still a challenge with respect to sputum-negative tuberculosis. RNA-sequencing was conducted to uncover potential lncRNA indicators of metabolic activity in tuberculosis lesions. Methods: Lung tissues with high metabolic activity (PET-high) and low metabolic activity (PET-low) demonstrated by fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) were collected from five sputum-negative tuberculosis patients for RNA-sequencing. Differentially expressed mRNAs and lncRNAs were identified, and their correlations were evaluated to constructed lncRNA-mRNA co-expression network. Afterwards, cis- or trans-targets of differentially expressed lncRNAs were predicted, followed by establishing lncRNA-target-pathway network, in which lncRNAs and mRNAs with high degrees were measured by quantitative real-time PCR using samples collected from 11 patients. Prediction efficiencies of lncRNA indicators were assessed by receiver operating characteristic curve analysis, Bioinformatics analysis was performed for potential lncRNAs. Results: In total, 386 mRNAs and 44 lncRNAs were identified to be differentially expressed. Differentially expressed mRNAs in lncRNA-mRNA co-expression network were significantly associated with the fibrillar collagen, platelet-derived growth factor binding, and leukocyte migration involved in inflammatory response. One cis-target gene and 440 trans-target genes were predicted for differentially expressed lncRNAs. Seven genes (C1QB, CD68, CCL5, CCL19, MMP7, HLA-DMB, and CYBB) and two lncRNAs (ENST00000429730.1 and MSTRG.93125.4) were validated to be significantly up-regulated. The area under the curve of ENST00000429730.1 and MSTRG.93125.4 was 0.750 and 0.813, respectively. Conclusion: Two lncRNAs ENST00000429730.1 and MSTRG.93125.4 might be considered as potential indicators of metabolic activity in tuberculosis lesions for sputum-negative tuberculosis.

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Transcriptome analysis of mycobacteria in sputum samples of pulmonary tuberculosis patients

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