Rapid Detection of Cell-Free Mycobacterium tuberculosis DNA in Tuberculous Pleural Effusion

Abstract

Tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, but its diagnosis remains difficult. In this study, we report for the first time on the detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and an evaluation of a newly developed molecular assay for the detection of cell-free Mycobacterium tuberculosis DNA. A total of 78 patients with pleural effusion, 60 patients with tuberculous pleurisy, and 18 patients with alternative diseases were included in this study. Mycobacterial culture, the Xpert MTB/RIF assay, the adenosine deaminase assay, the T-SPOT.TB assay, and the cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. The cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities of 75.0% and 68.3%, respectively, than mycobacterial culture and the Xpert MTB/RIF assay, which had sensitivities of 26.7% and 20.0%, respectively (P < 0.01). All four of these tests showed good specificities: 88.9% for the adenosine deaminase assay and 100% for the remaining three assays. The T-SPOT.TB assay with pleural effusion showed the highest sensitivity of 95.0% but the lowest specificity of 38.9%. The cell-free Mycobacterium tuberculosis DNA assay detected as few as 1.25 copies of IS6110 per ml of pleural effusion and showed good accordance of the results between repeated tests (r = 0.978, P = 2.84 × 10^-10). These data suggest that the cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology.

Keywords: Mycobacterium tuberculosis; cell-free DNA; pleural effusion.

FIG 1

Validation of cell-free M. tuberculosis DNA test with tuberculous pleural effusion. (A) Copy number of IS6110 in 45 cell-free M. tuberculosis DNA test-positive pleural effusion samples; (B) copy number of IS6110 in 15 pleural effusion samples from tuberculous pleurisy patients by two repeated cell-free M. tuberculosis DNA tests.

FIG 2

Comparison of five diagnostic tests with samples from different patient groups. Pearson's chi-square test was used to determine the significance among categorical variables, except that Fisher's exact test was used to determine the significance between cf-TB and T-SPOT in the group with definite TP. NS, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001; Δ, analysis was by Fisher's exact test.