Sequencing of Pleural Fluid and Plasma for Tuberculous Pleuritis.

Abstract

The laboratory diagnosis of tuberculous pleuritis (TBP) is hindered by the paucibacillary nature of <i>Mycobacterium tuberculosis</i> in the pleural space, resulting in low sensitivity of microbiological culture and polymerase chain reaction-based analyses on pleural fluid. The use of metagenomic next-generation sequencing for diagnosing TBP may be limited by the background noise of DNA from nontuberculous mycobacteria. We performed targeted sequencing to analyze <i>M. tuberculosis</i> DNA in paired pleural fluid and plasma from prospectively enrolled consecutive patients with new-onset pleural effusion. We used a bioinformatics alignment algorithm to the <i>M. tuberculosis</i> genome that was masked for regions with high sequence similarity to nontuberculous mycobacteria. Our primary outcome was a comparison of diagnostic sensitivity between <i>M. tuberculosis</i> sequencing as described above and culture using McNemar's test. Among the included 329 patients with pleural effusion, 34 patients with TBP were identified. Targeted sequencing detected <i>M. tuberculosis</i> DNA fragments in the pleural fluid of all TBP cases (median, 267.6 reads per 10 million [RP10M]; interquartile range [IQR], 30.8-2644.3) but absent in 288 out of 295 (97.6%) non-TBP samples (median, 0 RP10M; IQR, 0-0). Targeted sequencing of pleural fluid achieved a sensitivity of 97.1% for TBP detection at a cutoff of 2 RP10M, in contrast to 47.1% by <i>M. tuberculosis</i> culture (P<0.001, McNemar's test). Sequencing yielded an area-under-the-curve value of 0.9996 (95% confidence interval, 0.9988-1.0000) for differentiating TBP and non-TBP. Plasma analysis by targeted sequencing with the same alignment algorithm reported an area-under-the-curve value of 0.9475 (95% confidence interval, 0.8929-1.0000). Targeted sequencing of pleural fluid with selectively masked <i>M. tuberculosis</i> genomic alignment accurately diagnosed TBP and outperformed conventional diagnostic tests. (Supported by InnoHK and the Hong Kong Tuberculosis, Chest and Heart Diseases Association; ClinicalTrials.gov number, NCT05397730.).