Diagnosis of pleural tuberculosis by multi-targeted loop-mediated isothermal amplification assay based on SYBR Green I reaction: comparison with GeneXpert® MTB/RIF assay.

BACKGROUND: Diagnosis of pleural tuberculosis (TB) is tedious owing to its close resemblance with malignant pleural effusion and sparse bacterial load in clinical specimens. There is an immediate need to design a rapid and dependable diagnostic test to prevent unnecessary morbidity/mortality. RESEARCH DESIGN AND METHODS: A multi-targeted loop-mediated isothermal amplification (MT-LAMP) was deliberated using mpt64 and IS6110 to diagnose pleural TB within pleural fluids/biopsies. MT-LAMP products were analyzed by gel-based and visual detection methods, viz. SYBR Green I, SYBR Green I+deoxyuridine triphosphate uracil-N-glycosylase (dUTP-UNG), and dry methyl green reactions. RESULTS: In a pilot study, while assessing pleural TB/non-TB control subjects (n = 40), both SYBR Green I+dUTP-UNG/gel-based MT-LAMP assays exhibited better sensitivity/specificity than SYBR Green I and dry methyl green MT-LAMP. Since it is facile to work with SYBR Green I+dUTP-UNG than gel-based MT-LAMP, we validated the performance of SYBR Green I+dUTP-UNG in a higher number of specimens (n = 97), which revealed somewhat higher sensitivity (85.2 vs. 81.5%) and specificity (97.7 vs. 90.7%) than SYBR Green I MT-LAMP. Furthermore, the sensitivity attained by SYBR Green I+dUTP-UNG MT-LAMP was significantly higher (p < 0.001) than GeneXpert. CONCLUSIONS: Our SYBR Green I+dUTP-UNG MT-LAMP is a simple and reliable method to diagnose pleural TB, which may translate into a point-of-care test.

Evaluation of in silico designed inhibitors targeting MelF (Rv1936) against Mycobacterium marinum within macrophages.

We recently identified inhibitors targeting Mycobacterium marinum MelF (Rv1936) by in silico analysis, which exhibited bacteriostatic/bactericidal activity against M. marinum and M. tuberculosis in vitro. Herein, we evaluated the effect of best four inhibitors (# 5175552, # 6513745, # 5255829, # 9125618) obtained from the ChemBridge compound libraries, on intracellular replication and persistence of bacteria within IFN-γ activated murine RAW264.7 and human THP-1 macrophages infected with M. marinum. Inhibitors # 5175552 and # 6513745 significantly reduced (p < 0.05) the intracellular replication of bacilli during day 7 post-infection (p.i.) within RAW264.7 and THP-1 macrophages infected at multiplicity of infection (MOI) of ~1.0. These observations were substantiated by electron microscopy, which revealed the protective effect of # 5175552 in clearing the bacilli inside murine macrophages. Strikingly, # 6513745 displayed synergism with isoniazid against M. marinum in murine macrophages, whereas # 5175552 significantly suppressed (p < 0.05) the persistent bacilli during day 10-14 p.i. in infected RAW264.7 and THP-1 macrophages (MOI of ~ 0.1). Moreover, # 5175552 and # 6513745 were non-cytotoxic to host macrophages at both 1X and 5X MIC. Further validation of these inhibitors against M. tuberculosis-infected macrophages and animal models has potential for development as novel anti-tubercular agents.