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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.

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Transcriptome analysis of mycobacteria in sputum samples of pulmonary tuberculosis patients

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