Discovery and validation of an NMR-based metabolomic profile in urine as TB biomarker

Abstract

Despite efforts to improve tuberculosis (TB) detection, limitations in access, quality and timeliness of diagnostic services in low- and middle-income countries are challenging for current TB diagnostics. This study aimed to identify and characterise a metabolic profile of TB in urine by high-field nuclear magnetic resonance (NMR) spectrometry and assess whether the TB metabolic profile is also detected by a low-field benchtop NMR spectrometer. We included 189 patients with tuberculosis, 42 patients with pneumococcal pneumonia, 61 individuals infected with latent tuberculosis and 40 uninfected individuals. We acquired the urine spectra from high and low-field NMR. We characterised a TB metabolic fingerprint from the Principal Component Analysis. We developed a classification model from the Partial Least Squares-Discriminant Analysis and evaluated its performance. We identified a metabolic fingerprint of 31 chemical shift regions assigned to eight metabolites (aminoadipic acid, citrate, creatine, creatinine, glucose, mannitol, phenylalanine, and hippurate). The model developed using low-field NMR urine spectra correctly classified 87.32%, 85.21% and 100% of the TB patients compared to pneumococcal pneumonia patients, LTBI and uninfected individuals, respectively. The model validation correctly classified 84.10% of the TB patients. We have identified and characterised a metabolic profile of TB in urine from a high-field NMR spectrometer and have also detected it using a low-field benchtop NMR spectrometer. The models developed from the metabolic profile of TB identified by both NMR technologies were able to discriminate TB patients from the rest of the study groups and the results were not influenced by anti-TB treatment or TB location. This provides a new approach in the search for possible biomarkers for the diagnosis of TB.

Figure 1

Description of the procedure followed to analyse the urine samples of the 332 participants by high and/or low NMR. NMR, Nuclear Magnetic Resonance; TB, tuberculosis; PnP, pneumococcal pneumonia; LTBI, latent TB infection; uninfected, individuals without infection.

Figure 2

Principal Component Analysis (PCA) score plots of urine spectra analyzed by high-field Nuclear Magnetic Resonance of (a) untreated TB patients (n = 19), uninfected individuals (n = 28), pneumococcal pneumonia patients (n = 25) and LTBI individuals (n = 17); (b) untreated TB and uninfected individuals; (c) untreated TB and pneumococcal pneumonia patients; (d) untreated TB and LTBI individuals. TB, tuberculosis; PnP, pneumococcal pneumonia; LTBI: latent TB infection; PC, Principal Component.

Figure 3

Principal Component Analysis (PCA) loading plots of 89 urine spectra analyzed by high-field Nuclear Magnetic Resonance reveals the metabolomic fingerprint of TB corresponding to 31 chemical shift regions assigned to eight metabolites. (a) PCA loading PC1-PC2 biplot and PC1 loading plot between TB patients and uninfected individuals; (b) PCA loading PC1-PC2 biplot and PC2 plot between TB patients and patients with pneumococcal pneumonia (PnP); (c) PCA loading PC2-PC3 biplot and PC2 loading plot between TB patients and individuals with LTBI. Multiple regions for the discrimination between groups were pointed outside the boundaries of a Hotelling's T2 statistics ellipse (pointed red line) in PCA loading biplots. TB, tuberculosis; PnP, pneumococcal pneumonia; LTBI: latent TB infection; PC, Principal Component.

Figure 4

Principal Component Analysis (PCA) score plots of urine spectra analyzed by low-field Nuclear Magnetic Resonance between 39 untreated TB (x) and: (a) 29 uninfected individuals (circle), (b) 31 pneumococcal pneumonia patients (cross), and (c) 53 LTBI individuals (triangle). TB, tuberculosis; PnP, pneumococcal pneumonia; LTBI: latent TB infection; PC, Principal Component.

Figure 5

Comparison of the metabolomic fingerprints of tuberculosis (TB) identified in urine spectra analyzed by high-field Nuclear Magnetic Resonance (green) and low-field Nuclear Magnetic Resonance (red) showing the identification of the TB metabolite biomarkers. PC: Principal Component.