Diagnosis of paediatric tuberculosis by optically detecting two virulence factors on extracellular vesicles in blood samples

Sensitive and specific blood-based assays for the detection of pulmonary and extrapulmonary tuberculosis would reduce mortality associated with missed diagnoses, particularly in children. A team led by researchers at the Tulane University School of Medicine have developed a nanoparticle-enhanced immunoassay read by dark-field microscopy that detects two Mycobacterium tuberculosis virulence factors (the glycolipid lipoarabinomannan and its carrier protein) on the surface of circulating extracellular vesicles. In a cohort study of 147 hospitalized and severely immunosuppressed children living with HIV, the assay detected 58 of the 78 (74%) cases of paediatric tuberculosis, 48 of the 66 (73%) cases that were missed by microbiological assays, and 8 out of 10 (80%) cases undiagnosed during the study. It also distinguished tuberculosis from latent-tuberculosis infections in non-human primates. The researchers adapted the assay to make it portable and operable by a smartphone. With further development, the assay may facilitate the detection of tuberculosis at the point of care, particularly in resource-limited settings.

NEI optimization for sensitive detection of EV-associated TB biomarkers

Fig. 2

a, Schematic of the NEI image capture workflow and signal. Created with BioRender.com. b,c, NEI images (left) and 3D heat maps (right) before (b) and after (c) image processing to remove artefacts (blue and white signal) to improve detection of AuNRs (red signal) bound to Mtb EVs (top) and negative control EVs (bottom). d, Standard curves for AuNR dilutions quantified by counting positive pixels versus mean pixel intensity. e, AuNR titration to maximize signal-to-noise (highlighted in blue shadow) in samples near the EV-ELISA limit of detection (150 ng EV protein per ml). f, NEI signal increase versus background (signal-to-noise) when using the indicated capture and detection antibody pairs to detect NEI signal from a low-concentration EV sample (150 ng protein per ml) and a negative control (blank) sample. g, EV LAM and LprG NEI signal linearity with an Mtb EV concentration curve generated using EVs from Mtb-infected macrophages. h, Linear regression line and correlation coefficient of NEI EV LAM signal without and after noise reduction in background-normalized serum samples spiked with EVs isolated from Mtb-infected macrophages. For dh: mean ± s.d.; N = 3. i, Receiver operating characteristic (ROC) analysis for the ability of single and integrated (combined) EV LAM and LprG NEI signals to distinguish children with and without TB, indicating areas under the ROC curve. jl, NEI signal for LAM (j), LprG (k) and integrated LAM and LprG (l) expression on serum EVs of children with TB (N = 15) and with no evidence of TB (N = 5), N.I., NEI signal intensity (arbitrary units; a.u.). Solid lines indicate mean ± s.d.; dashed lines indicate the threshold for positive signal determined in corresponding ROC analysis in i. P values were determined by two-sided Mann-Whitney U test.

Zheng W, LaCourse SM, Song B, Singh DK, Khanna M, Olivo J, Stern J, Escudero JN, Vergara C, Zhang F, Li S, Wang S, Cranmer LM, Huang Z, Bojanowski CM, Bao D, Njuguna I, Xiao Y, Wamalwa DC, Nguyen DT, Yang L, Maleche-Obimbo E, Nguyen N, Zhang L, Phan H, Fan J, Ning B, Li C, Lyon CJ, Graviss EA, John-Stewart G, Mitchell CD, Ramsay AJ, Kaushal D, Liang R, Pérez-Then E, Hu TY. (2022) Diagnosis of paediatric tuberculosis by optically detecting two virulence factors on extracellular vesicles in blood samples. Nat Biomed Eng 6(8):979-991. [article]

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