Profiling of the host small non-coding RNAome in Mycobacterium tuberculosis infection

We report a study about differentially expressed small non-coding RNAs in the blood of humans harboring a latent (LTBI) or active tuberculosis (TB) infection in comparison with exposed controls (ExC) and treated LTBI (LTBItt). All non-TB subjects enrolled in this study were recent close contacts (rCt) of a newly diagnosed contagious TB cases enrolled in Rio de Janeiro, Brazil. The detailed methodology is described below. According to Brazilian Ministry of Health (BMH) guidelines, the screen to detect LTBI among recent contacts comprises a clinical evaluation by a physician specializing in pulmonary diseases, a chest X-ray (CXR), and a tuberculin skin test (TST, cut-off 5mm). Additionally, as part of this study, blood was collected for short- (st) and long-term (lt) IGRA. St-IGRA was performed by stimulating whole blood with the Mtb antigen ESAT6:CFP10 (expressed as a fusion protein) for 22h (cut-off 10pg/mL). Lt-IGRA involved stimulating peripheral blood mononuclear cells (PBMC) with this same antigen for 5 days (cut-off 100 pg/mL). Cases were defined as follows: ExC were recent close contacts of a TB index case and had a negative response to both TST and in house interferon-gamma release assay (IGRA) by stimulating blood-derived specimens with ESAT6:CFP10 indicating absence of Mycobacterium tuberculosis (Mtb) infection. LTBI was defined as (1) a TST induration >5 mm measured 72 h after intradermal injection of Mtb purified protein derivative (PPD) and (2) a positive IGRA response (to either st-IGRA or lt-IGRA, or both) if indicators of active disease were observed on CXR, (3) the absence of acid-fast bacilli (AFB) and negative Lowenstein-Jensen (LJ) culture of clinical specimens were also required. LTBItt consisted of LTBI cases (TST+/IGRA+ at enrollment) who completed a 6-month course of IPT. Their blood samples were collected >2 months after the end of isoniazid (INH) preventive treatment (IPT). Active TB was defined as (1) respiratory symptoms suggestive of TB, and/or (2) detection of AFB and/or positive LJ culture in sputum, bronchoalveolar lavage or biopsy, followed by (3) remission of symptoms upon anti-TB chemotherapy. Their blood samples were obtained before initiation of treatment. Whole blood was collected in PAXgene RNA tubes (PreAnalytiX, SWZ) and was stored at -80°C for <2 years before RNA extraction. sncRNA libraries. Total RNA (including small RNA) was isolated using the PAXgene Blood miRNA Kit (PreAnalytiX, SWZ), which is indicated for the isolation and purification of total RNA longer than 18 nucleotides. The manufacturer’s instructions were followed at both stages. Total RNA was quantified with a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, EUA) and RNA integrity was assessed via agarose gel electrophoresis. One microgram RNA was used for cDNA library preparation (TruSeq Small RNA Sample Preparation® Kit, Illumina, San Diego, CA) following the manufacturer’s protocols. RNAseq was performed on an Illumina HiSeq® 2500 Sequencing System (Illumina, San Diego, CA), generating 50 bp single reads and ≈16 million reads passing filter for each sample. Pre-processing and differential expression. The FASTQ files were preprocessed (FastQC 0.11.2), adaptors trimmed (Cutadapt 1.7.1), aligned to the human genome (STAR 2.4.1d), counted (featureCounts 1.4.6) on the Oasis 2.0 web platform. Transcripts with <5 reads in at least one sample were excluded. Then, normalized and evaluated for differentially expressed (DE) transcripts using DESeq2 (v. 1.16) on the Oasis 2.0 web platform (https://oasis.dzne.de/). Overall design: We collected blood samples from recent close contacts at recruitment and monitored them for 1 year. All TB cases were treatment-naïve. An active TB sncRNA signature was derived from whole blood RNA sequencing data by comparing TB and non-TB groups. Notably, it classified all TB cases correctly and reclassified 8 presumed LTBI cases as TB, 5 of whom turned out to have features of Mycobacterium tuberculosis infection on chest radiographs.

49

de Araujo LS, Ribeiro-Alves M, Leal-Calvo T, Leung J, Durán V, Samir M, Talbot S, Tallam A, Mello FCQ, Geffers R, Saad MHF, Pessler F