PLoS One. 2013 Nov 21;8(11):e80149.

miRNA signatures in Sera of patients with active pulmonary tuberculosis.

Miotto P, Mwangoka G, Valente IC, Norbis L, Sotgiu G, Bosu R, Ambrosi A, Codecasa LR, Goletti D, Matteelli A, Ntinginya EN, Aloi F, Heinrich N, Reither K, Cirillo DM.

Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.

ABSTRACT:

Several studies showed that assessing levels of specific circulating microRNAs (miRNAs) is a non-invasive, rapid, and accurate method for diagnosing diseases or detecting alterations in physiological conditions. We aimed to identify a serum miRNA signature to be used for the diagnosis of tuberculosis (TB). To account for variations due to the genetic makeup, we enrolled adults from two study settings in Europe and Africa. The following categories of subjects were considered: healthy (H), active pulmonary TB (PTB), active pulmonary TB, HIV co-infected (PTB/HIV), latent TB infection (LTBI), other pulmonary infections (OPI), and active extra-pulmonary TB (EPTB). Sera from 10 subjects of the same category were pooled and, after total RNA extraction, screened for miRNA levels by TaqMan low-density arrays. After identification of “relevant miRNAs”, we refined the serum miRNA signature discriminating between H and PTB on individual subjects. Signatures were analyzed for their diagnostic performances using a multivariate logistic model and a Relevance Vector Machine (RVM) model. A leave-one-out-cross-validation (LOOCV) approach was adopted for assessing how both models could perform in practice. The analysis on pooled specimens identified selected miRNAs as discriminatory for the categories analyzed. On individual serum samples, we showed that 15 miRNAs serve as signature for H and PTB categories with a diagnostic accuracy of 82% (CI 70.2-90.0), and 77% (CI 64.2-85.9) in a RVM and a logistic classification model, respectively. Considering the different ethnicity, by selecting the specific signature for the European group (10 miRNAs) the diagnostic accuracy increased up to 83% (CI 68.1-92.1), and 81% (65.0-90.3), respectively. The African-specific signature (12 miRNAs) increased the diagnostic accuracy up to 95% (CI 76.4-99.1), and 100% (83.9-100.0), respectively. Serum miRNA signatures represent an interesting source of biomarkers for TB disease with the potential to discriminate between PTB and LTBI, but also among the other categories.

PMID: 24278252

 

SUPPLEMENT:

New diagnostic tests and therapeutic monitoring tools are needed for many poverty-related and neglected diseases, but in particular for tuberculosis. Disease-specific biomarkers (defined as measurable markers related to biological processes, pathogenic features, or pharmacological responses to a therapeutic intervention) are of great value not only as diagnostic or a prognostic tools, but also in providing novel insights into the pathogenesis of the disease.

New research has revealed that circulating miRNAs could serve as non-invasive biomarkers for several physiological and pathological conditions, offering the prospect for earlier diagnosis, monitoring disease progression and therapeutic outcomes. miRNAs are short noncoding RNAs that post-transcriptionally regulate gene expression by targeting coding and untranslated regions of messenger RNA (mRNA). Nevertheless, miRNA networks modulate many important aspects of cellular physiology and thus have been found to be dysregulated in multiple disease states.

Several studies have demonstrated stable and detectable miRNAs in different body fluids including serum and plasma; among the various miRNA-carriers that have been described there are exosomes, membrane-derived vesicles, lipoproteins, and ribonucleoprotein complexes. Whereas almost all cell types are thought to release extracellular vesicles that are found in the plasma and other bodily fluids, including breast milk, saliva, urine, and sputum, less is known about the mechanism of cellular uptake of miRNAs. Once secreted, extracellular vesicles seem to act on the microenvironment, or passively traffic through the bloodstream or through other bodily fluids to provide long-range communication. miRNA profiles of extracellular carriers show specific sets of miRNAs that differ from parental cell-type profiles, thus suggesting that some miRNAs might be transcribed only to be exported. The selective packaging of miRNAs for exporting is probably related to the specific biological functions of the secreted miRNAs. Several in vitro studies have shown that cell-to-cell miRNA-transfer by the above-mentioned types of carriers are functional and can regulate gene expression in recipient cells. Although the complete mechanism of gene regulation mediated by specifically selected extracellular circulating miRNAs has yet to be clearly demonstrated in vivo, these studies suggest a plausible form of cell-to-cell communication in which donor cells send their miRNAs to distant recipient cells where they exert their regulatory functions.

Characterization of circulating miRNAs as biomarkers for viral/bacterial or parasitic infections is still in its infancy. We recently described a serum miRNA signature discriminating healthy subjects and patients with active pulmonary tuberculosis. In particular, we have been looking at serum miRNA-patterns not only related to a disease status and its differential diagnoses, but also to an infection status: we aimed at identifying miRNA profiles associated with the different phases of M. tuberculosis infection (active pulmonary disease, active extra-pulmonary disease, latent infection), and non-tubercular lung infections as well as in healthy condition. Other aspects of novelty are represented by:

(i) taking into account subjects from different nationalities and ethnicities: previous studies did not consider the impact of genetic background, whereas considering subjects from African and European ethnicity should increase the likelihood of detecting more miRNAs selectively expressed during active tuberculosis. On the other side, this approach allowed to define “population specific” signatures, showing higher diagnostic performances on specific genetic backgrounds;

(ii) applying the concept of biomarker’s “signature”: while much research is still focused on assessing the quality of single biomarkers, there is an emerging interest in panels of biomarkers composed of multiple candidate targets which are neither specific nor sensitive when used as single tests, but highly increasing the performance when used in combination;

(iii) considering only miRNAs detectable in at least the 80% of cases (thus providing higher diagnostic index).

Several challenges in pre-analytical and analytical phases remain in the analysis of circulating miRNAs. The extraction and quantification of circulating miRNAs is a challenging task, due to the low amount of total miRNAs in serum samples and the lack of reliable endogenous normalizers specific for circulating miRNAs. In our study we performed qRT-PCR quantification from various tuberculosis – and non tuberculosis- categories using TaqMan® Low Density Array (TLDA) Human MicroRNA Panels A and B, investigating an overall of 671 different miRNAs (Life Technologies). Sample quality has been widely considered at various levels, including the assessment of free haemoglobin levels to exclude haemolysis. After normalization procedure, the Ct values of four miRNAs detected by both array A and array B were found to be consistent across samples.

Overall, circulating miRNAs offer great hope and opportunity as non-invasive biomarkers for detecting different infection statuses. The future combinatory usage of newly established miRNA markers with other conventional markers might show even more advantages in early diagnosis/disease progression monitoring. Understanding the mechanism of transition from latent tuberculosis infection to active disease remains challenging and incomplete: multiple host and pathogen factors are involved in this complex process. miRNAs signatures could help in discriminating between active disease and latent infection, providing novel insight into the pathophysiology of the infection. Interestingly, circulating miRNAs have been recently described also as markers of immune activation (1). Thus, miRNA profiling may provide not only new tools for a better diagnosis and prognostic stratification, but may even provide new useful insights for understanding M. tuberculosis-specific immune response and eventually for the development of new vaccines against tuberculosis.

References

1. de Candia P, Torri A, Pagani M, Abrignani S. Serum microRNAs as Biomarkers of Human Lymphocyte Activation in Health and Disease. Front Immunol. 2014 Feb 10;5:43. eCollection 2014.

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann