Clinical Research Directory

Diagnostic Utility of Mycobacterium Tuberculosis Cell-free DNA

Tuberculosis (TB) is one of the major global health threats and is the second leading infectious cause of death after COVID-19 in 2022. Extrapulmonary TB (EPTB), amongst which tuberculous pleuritis (TBP) is one of the most common subtypes, poses additional obstacles to global TB control due to its difficulty in diagnosis. The diagnosis of TBP is challenging. The ideal way of confirming TBP is by direct detection of TB bacteria or its specific component in the pleural space. However, the performance of available diagnostic tests is far from satisfactory, and no single test can achieve multiple diagnostic goals simultaneously, including high detection sensitivity, high specificity to exclude other diseases, low invasiveness and detection of drug resistance. The inability to diagnose TBP early leads to unnecessary invasive pleural procedures and delayed curative treatment. There is a pressing need for a better diagnostic test to diagnose TBP confidently. When TB bacteria die or break down, the DNA materials shed into the pleural space, forming Mycobacterium tuberculosis cell-free DNA (MTB cfDNA), which may aid in diagnosing TBP. However, only limited literature explored this aspect, and the sensitivity rates reported were still suboptimal due to the scarcity of DNA materials in the pleural fluid. Based on a small patient cohort, our group has recently developed a new laboratory assay measuring MTB cfDNA to overcome this problem, with a superior diagnostic performance to conventional tests. This assay can potentially capture the genes harbouring drug resistance towards anti-TB medications. There are three aims in this research proposal. First, the diagnostic accuracy of the new MTB cfDNA assay in diagnosing TBP will be determined using a large cohort containing pleural fluid samples of various causes from countries with different TB burdens. Second, the clinical and laboratory factors determining the pleural fluid MTB cfDNA level will be identified. Third, the ability of the assay to capture different anti-TB drug-resistance genes will be explored. This new diagnostic method will significantly enhance the pickup rate of TBP, benefit patients with less invasive procedures, shorter hospital stays and timely treatment.

Diagnostic Utility of Mycobacterium Tuberculosis Cell-free DNA in Hong Kong

Tuberculosis (TB) is an endemic infectious disease in Hong Kong and a global health threat. Tuberculous pleuritis (TBP) is the most common form of extrapulmonary tuberculosis in China. Its presentation is frequently non-specific and the diagnosis is challenging due to its paucibacillary nature. Various studies have shown that there are limitations for conventional diagnostic modalities, including low sensitivity for pleural fluid microbiological tests (acid-fast bacilli \[AFB\] stain, Mycobacterium tuberculosis \[MTB\] culture and MTB polymerase chain reaction \[PCR\]), lack of specificity for pleural fluid biomarkers (adenosine deaminase) especially in low TB prevalence regions, invasiveness for pleural biopsy (AFB stain, MTB culture and MTB PCR by bedside or pleuroscopy biopsy). The diagnostic journey can be lengthy due to the long turnaround time for microbiological tests and the need for multiple invasive diagnostic procedures. Therefore, the diagnosis of TBP is frequently based on a composite of clinical, radiological and laboratory endpoints to maximise the diagnostic yield and limit the invasiveness. The application of pleural fluid MTB cell-free DNA (cfDNA) as a liquid biopsy to diagnose tuberculous pleuritis has been explored. Previous study groups focused on TB-specific single gene fragments revealed diagnostic sensitivity between 41.4% and 79.5%. Due to the suboptimal sensitivity, the clinical utility based on the previous technique was limited. Our study group has developed a novel MTB cfDNA assay based on different cfDNA processing techniques and interpretation algorithms, with sensitivity and specificity greater than 95% in diagnosing TBP. Since the performance of this novel MTB cfDNA assay was developed based on a limited number of selected cases, its clinical utility should be examined in a non-selective cohort of new-onset unilateral pleural effusion. Successful validation of MTB cfDNA in pleural fluid, as a liquid biopsy obtained through thoracentesis, can obviate the need to perform a pleural biopsy.