Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Comparative miRNA Expression Profiles in Individuals with Latent and Active Tuberculosis

ABSTRACT: To study the role of miRNAs in the transition from latent to active TB and to discover candidate biomarkers that may help predict TB progression, we have employed miRNA microarray expression profiling as a discovery platform to probe the transcriptome of peripheral blood mononuclear cells (PBMCs) with active TB, latent TB infection (LTBI), and healthy donors.Patients were recruited at the Shanghai Public Health Clinical Centre (Shanghai, China) from December, 2008 to May, 2009. The diagnosis of active TB was based on clinical presentation, chest radiography, and acid-fast stain of sputum smear.All the patients were HIV negative, as diagnosed by the Livzon Anti-HIV1/2 EIA Kit (Livzon Pharmaceutical Group Inc., Guangdong, China). Additional tests were also performed to detect hepatitis B virus (HBV) and hepatitis C virus (HCV) by using the Abbott AxSYM anti-HBsAg and HCV 3.0 antibody assay kit (Abbott Laboratories, Illinois) to exclude HBV- and HCV-positive patients (these 2 diseases are highly prevalent in China). Patients with a diabetes history were also excluded because diabetes could increase the risk of TB. Peripheral venous blood was drawn before treatment. Subjects with LTBI and healthy donors both without a history of clinical TB or other infectious diseases were recruited from the staff at the Shanghai Public Health Clinical Centre. TST and IGRA (T-SPOTM-BM-..TB, Oxford Immunuotec, Oxfordshire, U.K) results were used to distinguish the two groups. The LTBI group was TST-positive (TST>10 mm) and IGRA-positive while the healthy donors were TST-negative (TST<5 mm) and IGRA-negative. RNA of PBMC from 6 patients with active TB, 6 donors with Latent TB, and 3 healthy controls (total of 15 biologically independent samples) were used to perform Agilent Human miRNA (version 3) microarray , No replicates were included

ORGANISM(S): Homo sapiens

SUBMITTER: qian gao

PROVIDER: E-GEOD-29190 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Comparative miRNA expression profiles in individuals with latent and active tuberculosis.

Wang Chuan C Yang Shunyao S Sun Gang G Tang Xuying X Lu Shuihua S Neyrolles Olivier O Gao Qian Q

PloS one 20111007 10

The mechanism of latent tuberculosis (TB) infection remains elusive. Several host factors that are involved in this complex process were previously identified. Micro RNAs (miRNAs) are endogenous ∼22 nt RNAs that play important regulatory roles in a wide range of biological processes. Several studies demonstrated the clinical usefulness of miRNAs as diagnostic or prognostic biomarkers in various malignancies and in a few nonmalignant diseases. To study the role of miRNAs in the transition from la ...[more]

PMID: 22003408

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Project description: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/).

Project description:While blood transcriptional profiling has improved diagnosis and understanding of disease pathogenesis of adult tuberculosis (TB), no studies applying gene expression profiling of children with TB have been described so far. In this study, we have compared whole blood gene expression in childhood TB patients, as well as in healthy latently infected (LTBI) and uninfected (HC) children in a cohort of Warao Amerindians in the Delta Amacuro in Venezuela. We identified a 116-gene signature set by means of random forest analysis that showed an average prediction error of 11% for TB vs. LTBI and for TB vs. LTBI vs. HC in our dataset. Furthermore, a minimal set of only 9 genes showed a significant predictive value for all previously published adult studies using whole blood gene expression, with average prediction errors between 17% and 23%. Additionally, a minimal gene set of 42 genes with a comparable predictive value to the 116-gene set in both our dataset and the previously published literature cohorts for the comparsion of TB vs. LTBI vs. HC was identified. In order to identify a robust representative gene set that would hold stand among different ethnic populations, we selected ten genes that were highly discriminative between TB, LTBI and HC in all literature datasets as well as in our dataset. Functional annotation of these ten genes highlights a possible role for genes involved in calcium signaling and calcium metabolism as biomarkers for active TB. These ten genes were validated by quantitative real-time polymerase chain reaction in an additional cohort of 54 Warao Amerindian children with LTBI, HC and non-TB pneumonia. Decision tree analysis indicated that five of the ten genes were sufficient to diagnose 78% of the TB cases correctly with 100% specificity. We conclude that our data justify the further exploration of our signature set as biomarkers to diagnose childhood TB. Furthermore, as the identification of different biomarkers in ethnically distinct cohorts is apparent, it is important to cross-validate newly identified markers in all available cohorts. In this study, 27 children 1 to 15 years of age with TB (n=9), LTBI (n=9) and HC (n=9) were recruited between May 2010 and December 2010. Tuberculin skin test (TST) and QuantiFERON-TB Gold In-Tube assay (QFT-GIT) were performed on all children. A sputum sample was collected from all children with expectoration and a gastric aspirate was taken from all children under 6 years of age. Children with active TB were diagnosed based on culture of M. tuberculosis (n=2) or on the basis of clinical, epidemiological and radiological features (n=7). The latter group were children with a TST = 10 mm or a positive QFT-GIT result who presented all of: persistent fever >38M-BM-0C objectively recorded daily for at least two weeks, persistent cough for more than three weeks, weight loss (>5% reduction in weight compared with the highest weight recorded in last three months) or failure to thrive (documented crossing of percentile lines in the preceding three months), persistent lethargy or decrease in playfulness/activity reported by the parent and absence of clinical response on broad-spectrum antibiotics. Standard antero-posterior and lateral chest radiographs (CXRs) were taken from all children. Two independent experts, blinded to all clinical information, evaluated the CXRs and documented their findings on a standard report form. Where the two objective experts disagreed, a third expert was consulted and final consensus was achieved. A diagnosis of TB was only made when the CXR was consistent with TB9 and the child showed a positive clinical response to anti-TB treatment. Children were followed up clinically, radiologically and, in case of a negative TST at inclusion, by means of TST at six and 12 months after inclusion. LTBI was defined as a TST = 10mm and a positive QFT-GIT with a negative culture result on inclusion in the absence of radiological and clinical evidence of TB disease on inclusion as well as on t=6 and t=12 months. HC were children with a TST = 0 mm at inclusion and at t=6 and t=12 months. The HC had a negative QFT-GIT and a negative culture result at inclusion without radiological or clinical evidence of TB disease on inclusion nor on t=6 and t=12 months. TB patients were sampled before initiation of anti-TB treatment. Of three of the nine TB patients, a follow-up sample was taken when the patient was in anti-TB treatment for five months. All children were HIV-negative. 27 samples in total where analyzed, active TB infection (TB, n=9), Latent TB infection (LTBI, n=9) and healthy controls (HC, n=9) . Gene expression values were log2-transformed and differentially expressed genes were identified based on log2 fold changes (M-values). P values were calculated with a Bayes-regularized one-way ANOVA. Random Forest recursive feature elimination was used to find a signature geneset capable of discrimination active TB from latent TB and from non-infected individuals.

Project description:The aim of the project was to define transcriptional signatures in whole blood of TB patients (before drug treatment) and healthy controls to distinguish the signature of Latent and Active TB patients from each other and from healthy controls. This will help in diagnosis of active tuberculosis which normally relies on culture of the bacilli, which can take up to 6 wks, and sometimes the bacilli cannot be obtained from sputum thus requiring invasive techniques obtaining bronchoalveolar lavage (BAL). In some cases the bacill cannot be grown from sputum or BAL. Secondly the aim was to determine whether Latent patients have a homogeneous or heterogeneous signature, one may expect the latter since it is not possible to determine by the present tests (Tuberculin skin test - TST - or MTb antigen responsiveness of blood cells to produce IFN-gamma - IGRA assay) whether the mycobacteria has been cleared, is still present but is controlled, or if patients are recently infected or reactivated and will develop active TB. The latter situation may be determined if Latent patients have a blood transcriptional signature similar to that in Active patients. The transcriptional signature in Active TB patients may also provide information as to the factors leading to immunopathogenesis, thus possibly identifying therapeutic targets. The transcriptional profile in Latent TB may give information as to the protective factors controlling the infection, thus important for monitoring vaccine development. A further aim was to examine the transcriptional blood profile of active TB patients at the time of recruitment (before drug treatment) and then subsequently at specific time points after drug treatment to determine whether the signature is extingsuihed with treatment and when. London is a site of intermediate burden of TB - the study was initiated in London, across a broad range of ethnicities to obtain a robust signature that could be used in different developing countries where there is a high burden TB disease. Experimental design : Whole blood collected in tempus tubes from patients with different spectra of TB disease and healthy controls. All patients were sampled prior to the initiation of any antimycobacterial therapy. Active Pulmonary TB: PTB - All patients confirmed by isolation of Mycobacterium Tuberculosis on culture of sputum or bronchoalvelolar lavage fluid. Latent TB: LTB - All patients were screened at a tuberculosis clinic, being either new entrants to the UK from endemic countries or being household contacts of infectious cases. All were positive by tuberculin skin test (>14mm if BCG vaccinated, >5mm if not vaccinated) and were also positive by Interferon-Gamma Release assay(IGRA); specifically Quantiferon Gold In-Tube Assay (Cellestis, Australia). Latent patients had no clinical, radiological or microbiological evidence of active infection and were asymptomatic. Healthy controls - these were volunteers without exposure to TB who were negative by both tuberculin skin test (<15mm if BCG vaccinated, <6mm if unvaccinated); who were also negative by IGRA (as described above). In this dataset: PTB n= 13; LTB n = 17; BCG+ n = 6, BCG-, n =6. Experimental variables : Patient group: Active PTB; Latent TB, Healthy controls (BCG vaccinated and unvaccinated). ethnicity - a wide range of ethnic groups is represented. However all controls in this dataset are broadly Caucasian/White. The active PTB group incorporates a range of smear positive and smear negative disease and a spectrum of disease extent/severity.

Dataset Information

Comparative miRNA Expression Profiles in Individuals with Latent and Active Tuberculosis

Publications

Comparative miRNA expression profiles in individuals with latent and active tuberculosis.

Similar Datasets

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