Clinical Evaluation of a Blood Assay to Diagnose Paucibacillary Tuberculosis via Bacterial Antigens

Abstract

Background: The diagnosis of active tuberculosis (TB) cases primarily relies on methods that detect Mycobacterium tuberculosis (Mtb) bacilli or their DNA in patient samples (e.g., mycobacterial culture and Xpert MTB/RIF assays), but these tests have low clinical sensitivity for patients with paucibacillary TB disease. Our goal was to evaluate the clinical performance of a newly developed assay that can rapidly diagnose active TB cases by direct detection of Mtb-derived antigens in patients' blood samples.

Methods: Nanoparticle (NanoDisk)-enriched peptides derived from the Mtb virulence factors CFP-10 (10-kDa culture factor protein) and ESAT-6 (6-kDa early secretory antigenic target) were analyzed by high-throughput mass spectrometry (MS). Serum from 294 prospectively enrolled Chinese adults were analyzed with this NanoDisk-MS method to evaluate the performance of direct serum Mtb antigen measurement as a means for rapid diagnosis of active TB cases.

Results: NanoDisk-MS diagnosed 174 (88.3%) of the study's TB cases, with 95.8% clinical specificity, and with 91.6% and 85.3% clinical sensitivity for culture-positive and culture-negative TB cases, respectively. NanoDisk-MS also exhibited 88% clinical sensitivity for pulmonary and 90% for extrapulmonary TB, exceeding the diagnostic performance of mycobacterial culture for these cases.

Conclusions: Direct detection and quantification of serum Mtb antigens by NanoDisk-MS can rapidly and accurately diagnose active TB in adults, independent of disease site or culture status, and outperform Mycobacterium-based TB diagnostics.

Conflict of interest statement

Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

© 2017 American Association for Clinical Chemistry.

Figures

Fig. 1. Participant disposition flow chart

Fig. 2. NanoDisk-MS assay procedure

Serum samples of suspected TB cases are trypsin digested and incubated with antibody-functionalized NanoDisk particles that bind Mtb-specific peptides (A). Serum Mtb CFP-10 and ESAT-6 concentration are determined by the mass spectra intensity ratios of the Mtb target peptides (1593.75 and 1900.95 m/z) and their isotope-labeled internal standards, respectively. Representative NanoDisk-MS results for the peaks of interest from a clinically confirmed active TB case (upper) and a control subject with a negative TB diagnosis (lower) (B).

Fig. 3. Serum CFP-10 (red) and ESAT-6 (green) concentrations (CFP-10 + ESAT-6) in each clinically confirmed TB case, with patients grouped according to their Mtb culture status and disease site

Results indicate an average of 3 sample replicates per subject.

Fig. 4. NanoDisk-MS results in different TB subgroups

Mtb culture-positive vs culture-negative cases (A). AFB smear-positive vs smear-negative cases (B). Mtb culture-positive and culture-negative pulmonary and extrapulmonary TB cases and non-TB cases (C). Data points indicate the NanoDisk-MS readout for each study participant, while black lines indicate the median and interquartile range. NanoDisk-MS readout is the combined CFP-10 and ESAT-6 concentration.