Who
Study population: Adults aged 18 years or older with new-onset unilateral pleural effusion requiring thoracentesis.
Setting: Consecutive patients screened at the Department of Medicine & Therapeutics, Prince of Wales Hospital, Hong Kong SAR.
Eligible and analyzed cohort: 329 patients with established causes of pleural effusion.
TB pleuritis group (TBP): 34 patients (10.3% of the cohort).
16/34 had positive pleural fluid M. tuberculosis culture.
13/34 did not undergo pleural biopsy because of physician preference or patient refusal.
Non-TBP group: 295 patients, including:
134 malignant pleural effusion
93 fluid overload
55 parapneumonic effusion
13 other causes
Excluded patients:
39/403 eligible patients had uncertain diagnosis
19 lacked pleural fluid adenosine deaminase results
16 lacked M. tuberculosis culture results
Exclusion criteria: Prior TBP or bacterial pleural infection, prior instrumentation in the ipsilateral pleural space, or >14 consecutive days of anti-TB treatment in the prior 3 months.
What
Study focus: To assess the diagnostic utility of targeted Mycobacterium tuberculosis sequencing in pleural fluid and plasma for diagnosing tuberculous pleuritis (TBP), compared with conventional diagnostic methods.
Key innovation: A bioinformatics masking strategy was developed to exclude regions of high genomic similarity between M. tuberculosis and nontuberculous mycobacteria (NTM), aiming to improve taxonomic specificity.
Primary findings in pleural fluid:
M. tuberculosis DNA fragments were detected in all TBP cases, with median 267.6 RP10M (IQR 30.8–2644.3).
In non-TBP, fragments were absent in 288/295 (97.6%); 7/295 (2.4%) had low-level signal (0.31–4.34 RP10M).
Diagnostic discrimination was excellent:
AUC 0.9996 (95% CI 0.9988–1.0000)
At a cutoff of 2 RP10M:
Sensitivity: 97.1% (33/34; 95% CI 84.7–99.9%)
Specificity: 99.7% (95% CI 98.1–100.0%)
Comparison with conventional pleural fluid tests:
M. tuberculosis culture sensitivity: 47.1% (95% CI 29.8–64.9%)
Xpert MTB/RIF Ultra sensitivity: 26.5% (95% CI 12.9–44.4%)
Sequencing sensitivity was significantly higher than culture (P<0.001).
Performance by culture subgroup:
In culture-positive TBP, sequencing sensitivity was 93.8% (15/16; 95% CI 69.8–99.8%)
In culture-negative TBP, sequencing sensitivity was 100.0% (18/18)
Xpert Ultra sensitivities:
37.5% for culture-positive TBP
16.7% for culture-negative TBP
Plasma findings:
Paired plasma available for 31 TBP and 257 non-TBP patients.
M. tuberculosis DNA detected in 28/31 TBP plasma samples, median 6.2 RP10M (IQR 1.5–27.9)
Low-level signal found in 9/257 non-TBP plasma samples
AUC 0.9475 (95% CI 0.8929–1.000)
Plasma Xpert Ultra sensitivity was 0%
Drug resistance analysis:
16 TBP samples had reads covering at least 1 of 15 WHO-relevant resistance-associated regions.
No resistance-associated mutations were detected.
Sequencing findings agreed with phenotypic susceptibility in culture-positive cases, all susceptible to first-line agents.
Additional biological findings:
M. tuberculosis cell-free DNA fragments in pleural fluid and plasma were mostly short fragments.
Pleural fluid human cell-free DNA end motif profiles clustered by disease subgroup, suggesting association with pleural pathology.
Authors’ conclusion from provided text: Targeted M. tuberculosis sequencing of pleural fluid showed very high sensitivity and specificity for TBP and outperformed pleural fluid culture and Xpert Ultra, including in culture-negative TBP cases.
Practical implication: This approach may be a promising diagnostic method for paucibacillary TB pleuritis, where conventional microbiologic tests often have limited sensitivity.
When
Recruitment period: September 1, 2022, to March 31, 2024
Follow-up information for non-TBP diagnoses: reviewed through July 2025
Study design timeframe: Prospective enrollment during the above study period.
Where
Location: Prince of Wales Hospital, Hong Kong SAR
Institutional context: Department of Medicine & Therapeutics
Ethics and registration: Approved by the Chinese University of Hong Kong / New Territories East Cluster Ethics Committee; registered at ClinicalTrials.gov (NCT05397730) as the MYDNITE study.
Why
Rationale: TB pleuritis is difficult to diagnose because conventional tests such as pleural fluid culture and PCR have limited sensitivity, especially in paucibacillary disease.
Knowledge gap: Whether targeted sequencing of pleural fluid and plasma, coupled with a method to distinguish M. tuberculosis from NTM background DNA, can improve diagnostic yield.
Objective: To evaluate the diagnostic performance of targeted M. tuberculosis sequencing for TBP against conventional diagnostic methodologies.
How
Study design: Prospective cohort study
This is an observational diagnostic accuracy study, which provides moderate evidence for test performance in a real-world consecutive cohort.
Sampling approach: Consecutive recruitment of eligible patients with unilateral pleural effusion.
Reference standard for TBP diagnosis: Positive microbiology, histologic evidence, or a composite reference standard.
Specimens collected:
Pleural fluid for targeted sequencing, Xpert Ultra, acid-fast bacilli stain, culture, cell counts, biochemistry, and cytology
Paired plasma when available
Index test: Targeted M. tuberculosis sequencing using a custom-designed Roche Diagnostics capture-probe system
Bioinformatics method:
Compared the M. tuberculosis genome with 2543 reference genomes from 1075 microbial species in the Actinobacteria phylum
Regions shared with NTM were masked
Only reads aligning to the masked M. tuberculosis genome were counted as M. tuberculosis DNA
DNA abundance reported as reads per 10 million (RP10M)
Sequencing depth: Pleural fluid sequencing had median 41.9 million raw reads (IQR 31.7–52.5 million)
Statistical/analytic methods mentioned:
McNemar’s test for sample size planning
AUC for diagnostic performance
Bonferroni correction for multiple comparisons in end motif analysis
Unsupervised hierarchical clustering for motif profile analysis
Sample size calculation:
Based on assumed sensitivity difference between culture (27%) and cell-free DNA analysis (80%)
Required 28 TBP patients, inflated to 32 allowing for 10% attrition
Blinding: Investigators performing molecular analyses were masked to the clinical diagnosis
Limitations noted or evident from the text:
13/34 TBP patients lacked pleural biopsy and additional histology/microbiology
Some diagnostic classification relied on a composite reference standard
There was at least one apparent false-positive at the selected cutoff
Plasma analysis was limited to samples “if available”
Single-center study, which may limit generalizability
Funding/conflicts of interest: Not specified in the provided text.
Bottom line
In this prospective Hong Kong cohort of 329 patients with pleural effusion, targeted M. tuberculosis sequencing of pleural fluid using a genome-masking alignment strategy showed 97.1% sensitivity and 99.7% specificity for diagnosing TB pleuritis at a cutoff of 2 RP10M, clearly outperforming pleural fluid culture and Xpert Ultra. Performance remained strong even in culture-negative TBP, suggesting this method may be particularly valuable for diagnosing paucibacillary pleural TB.
Source: Lam WJ, Chan KK, Wang G, Lai CK, Kang G, Chan C, Leung AC, Wong NH, Tso CS, Chow KM, Ramakrishnan S. Sequencing of Pleural Fluid and Plasma for Tuberculous Pleuritis. NEJM evidence. 2026 Mar 24;5(4):EVIDoa2500237. https://benangmerah.net/record/46/sequencing-of-pleural
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