Quantification of circulating Mycobacterium tuberculosis antigen peptides allows rapid diagnosis of active disease and treatment monitoring

Abstract

Tuberculosis (TB) is a major global health threat, resulting in an urgent unmet need for a rapid, non-sputum-based quantitative test to detect active Mycobacterium tuberculosis (Mtb) infections in clinically diverse populations and quickly assess Mtb treatment responses for emerging drug-resistant strains. We have identified Mtb-specific peptide fragments and developed a method to rapidly quantify their serum concentrations, using antibody-labeled and energy-focusing porous discoidal silicon nanoparticles (nanodisks) and high-throughput mass spectrometry (MS) to enhance sensitivity and specificity. NanoDisk-MS diagnosed active Mtb cases with high sensitivity and specificity in a case-control study with cohorts reflecting the complexity of clinical practice. Similar robust sensitivities were obtained for cases of culture-positive pulmonary TB (PTB; 91.3%) and extrapulmonary TB (EPTB; 92.3%), and the sensitivities obtained for culture-negative PTB (82.4%) and EPTB (75.0%) in HIV-positive patients significantly outperformed those reported for other available assays. NanoDisk-MS also exhibited high specificity (87.1-100%) in both healthy and high-risk groups. Absolute quantification of serum Mtb antigen concentration was informative in assessing responses to antimycobacterial treatment. Thus, a NanoDisk-MS assay approach could significantly improve the diagnosis and management of active TB cases, and perhaps other infectious diseases as well.

Keywords: blood test; nanodisk; rapid diagnosis; treatment monitoring; tuberculosis.

Fig. 1.

Schematic illustration of the NanoDisk-MS platform. (A) CFP-10 and ESAT-6 are secreted into the circulation from active Mtb infections. (B) Serum samples are subjected to microwave-assisted tryptic digestion and mixed with functionalized nanodisks and stable isotope-labeled internal standard peptides. (C) Peptide quantification. Step 1: Recognition and enrichment of target peptides and stable isotope-labeled internal standard peptides by antibody-conjugated nanodisks. Step 2: A nanodisk effect to enhance MALDI signal allows quantification of target peptide at low concentrations, as determined by MS intensity ratio of target and isotope-labeled internal standard peptides.

Fig. 2.

Method optimization and multiplex quantification of Mtb target peptides. (A) MS signal intensity of target peptides analyzed alone (no NPs) or with graphene, silver (Ag), gold (Au), silicon (Si), silica nanoparticles (NPs), or nanodisks. (B) Scanning electron microscopy image of nanodisk structure. (C and D) Transmission electron microscopy images of cross-sectional structure (C) and silica modification (D) of nanodisk inner pore surfaces. (E) CFP-10 (m/z 1,593.75; Left) and ESAT-6 (m/z 1,900.95; Right) MS intensity in an antigen-spiked healthy serum sample that was trypsin-digested overnight (12 h) or by rapid microwave-irradiation (20 min) and then analyzed without IP, after IP and elution from target-specific Dynabeads or nanodisks, or by NanoDisk-MS. (F) Calibration curves for CFP-10 and ESAT-6 quantitation in serum (n = 3; R² >0.98). (G) Representative MS spectra of CFP-10 and ESAT-6 peptides (m/z 1,593.75 and 1,900.95, respectively) and their internal standards (m/z 1,603.60 and 1,910.80, respectively) in serum of a healthy control (blue) and a TB case (red) analyzed by NanoDisk-MS. (H) CFP-10 (Left) and ESAT-6 (Right) MS intensity ratios of 1× (undiluted), 2×, 4×, 8×, 16×, and 32× diluted serum of a TB case analyzed by MALDI-TOF/TOF MS without IP, MALDI-TOF/TOF MS with Dynabead IP enrichment, and NanoDisk-MS. Data represent mean ± SEM. n = 3. ***P < 0.001.

Fig. 3.

Identification of active TB in adult patients. (A and B) Serum CFP-10 and ESAT-6 concentrations in adult HIV-negative (A) and HIV-positive (B) groups. Each column represents CFP-10 (upper cell, in red) and ESAT-6 (lower cell, in blue) results from a subject, ranked by high to low CFP-10 concentration. Antigen levels are indicated by the color intensity in the matching gradient bars. (C) Combined Mtb antigen (CFP-10 + ESAT-6) concentrations and means (black line) for the indicated groups. Data represent mean. n = 3. **P < 0.01.

Fig. 4.

Evaluation of anti-TB treatment efficacy. CFP-10 and ESAT-6 quantitation in archived serum samples of HIV-negative TB patients during anti-TB therapy (2–11 mo). Treatment start and end dates are indicated by blue and red dotted lines, respectively. Data represent mean ± SEM. n = 3.