Serum based metabolomics analysis revealed highly sensitive and specific panel of metabolic markers for differential diagnosis of Pulmonary Sarcoidosis and Tuberculosis
Creators: name: Kumar, Dinesh
name: Raj, Ritu
name: Jain, Avinash
name: Guleria, Anupam
name: Kumar, Umesh
name: Mohit K. Rai
name: Harshit Singh
name: Saurabh Chaturvedi
name: Nath, Alok
name: Durga Prasanna Misra
name: Agarwal, Vikas
Publisher: name: F1000 Research Limited
Year: 2019

description: Tuberculosis (TB) and Sarcoidosis (SAR) are two inflammatory granulomatous diseases and often pose a diagnostic challenge for treating physicians due to their clinic-radiological resemblance [1]. Both these diseases mainly involve lungs and lymph nodes and often share similar clinical symptoms (such as fever, fatigue, weight loss and cough) [1]. TB, being infectious disease, requires demonstration of <i>Mycobacterium tuberculosis</i>. However, demonstration of pathogen is not always possible; therefore diagnosis is made based on clinical, pathological and radiological findings. On the other hand, SAR is diagnosed based on exclusion of tuberculosis and other granulomatous disorders; thus often requires repeated biopsies, confirmation of non-caseating granuloma, negative Genexpert test and cultures, and negative Mantoux-tuberculin-skin test (TST). However, histopathological examination of the involved tissue and culture, is not only time taking and painful (invasive biopsies); it results in delayed treatment as well [2]. Therefore, new non-invasive markers are urgently required to improve the diagnosis of sarcoid and guide the treatment. This is particularly very relevant for developing countries like India where prevalence of tuberculosis is very high (~97%) [3] and sarcoid patients (~3%) are often misdiagnosed and mistreated with anti-tubercular therapy (ATT) and therefore, lung damage continues to progress. Metabolomics analysis of serum holds great potential to provide distinctive patterns of metabolic profiles relevant to underlying pathophysiology of the diseases and thus to improve clinical diagnosis and disease management strategies. The present study aims to compare the serum metabolic profiles of SAR and TB patients so that to identify diagnostic panel of biomarkers. For this, the serum samples were collected from 40 SAR, 28 TB and 76 normal control (NC) subjects and their metabolic profiles were measured using 1D <sup>1</sup>H CPMG NMR spectra recorded on 800 MHz NMR spectrometer. The recorded NMR spectra were first corrected for their baseline using PROCESSOR module of commercial software CHENOMX and then these were calibrated w.r.t. formate (used here as an internal reference and concentration set to 10 micromolar i.e. nearly close to the detection limit of 800 MHz NMR spectrometer). The resulted spectra were then analyzed using PROFILER-Module of CHENOMX and concentrations of following seventeen metabolites were explicitly measured: acetate, 3-hydroxybutyrate, alanine, citrate, creatine, creatinine, glucose, glutamate, glutamine, glycine, isoleucine, N-alpha acetyllysine (NAAL), phenylalanine, pyruvate, tyrosine, valine and methyl-histidine. Additionally, we also involved ratios and combinations of specific metabolites to improve the performance of used multivariate discriminatory models i.e. partial-least-squares-discriminant-analysis (PLS-DA) and PLS-DA with orthogonal-signal-correction (OPLS-DA).<br>Compared to SAR, the sera of TB patients were characterized by (a) elevated levels of acetate, 3HB, and glutamate and (b) decreased levels of glucose, citrate, pyruvate, creatinine, glutamine, NAAL, phenylalanine and tyrosine. Compared to TB, however, the serum levels of isoleucine, glycine, histidine, creatine and alanine were decreased in SAR (changes were variable, but not statistically significant) suggesting relatively more aberrant amino acid catabolism in SAR [4]; valine on the other hand was found to be increased in SAR (but not statistically significant). The decreased glucose, glutamine and citrate levels and concomitant increase in glutamate suggested that TB infection involves (a) augmented utilization of glucose through aerobic glycolysis (also known as Warburg effect) [5] and (b) activated glutaminolysis to replenish energy demand for regulating complex inflammatory and immunity responses [6]. The elevated acetate (the end product of lipid-metabolism) and 3HB levels in TB patients were also well consistent to previous reports showing elevated host lipid metabolism in TB patients [7]. As purposed, the aim of current study is to test the utility of NMR based serum metabolic profiles in the diagnostic screening of SAR from TB patients. Therefore, ROC curve analysis was performed further to evaluate the diagnostic potential of discriminatory metabolites between SAR and TB groups. Considering AUC value&gt;0.72 and p-value&lt;0.001 as the criterion for diagnostic potential; fifteen key metabolite entities have been identified as biomarkers for discriminating SAR from TB. Of them, seven metabolite entities (i.e. glutamate-to-glutamine ratio, citrate-to-tyrosine ratio, acetate, glutamine, citrate, (3HB+Acetate) and Glutamate) were found with AUROC value more than 0.9 suggesting their high diagnostic potential in differentating SAR from TB. However, the use of these biomarkers in clinical settings will require future studies on large patient cohorts and procedural optimization as well to improve accuracy. To continue our efforts in this direction so that the patients can be benefited both in terms of accurate diagnosis and appropriate treatment, further longitudinal studies have been started so that these biomarkers can be rigorously validated on follow-up patients showing clinical improvement and response to therapy. We hope these lab findings will be validated soon and translated into diagnostic tool for distinguishing SAR from TB patients.<br><br>References:<br> <br>[1]       F. Badar, S.F. Azfar, I. Ahmad, S. Yasmeen, S. Kirmani, Diagnostic difficulties in differentiating sarcoidosis from tuberculosis, Oman medical journal 26 (2011) 210-211.<br><br>[2]       A. Jain, A. Kumar, H. Singh, M.K. Rai, S. Chaturvedi, A. Guleria, A. Nath, D. Kumar, D.P. Misra, V. Agarwal, 114 Nuclear magnetic resonance (NMR) based serum metabolomics in sarcoidosis and tuberculosis: search for a biomarker, Rheumatology 57 (2018) key075-key338.<br><br>[3]       K. Thulasidoss, L. Asokan, P. Chandra, P. Rejliwal, The clinical conundrum of diagnosing and treating systemic sarcoidosis in a high TB burden area, BMJ case reports 2017 (2017) bcr-2016.<br><br>[4]       T.L. McGaha, L. Huang, H. Lemos, R. Metz, M. Mautino, G.C. Prendergast, A.L. 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