BackgroundPleural tuberculosis (TB) diagnosis often requires invasive procedures such as pleural biopsy. The aim of this study was to evaluate the role of real-time polymerase chain reaction (PCR) for the IS6110 sequence of M. tuberculosis in pleural fluid specimens as a rapid and non-invasive test for pleural TB diagnosis.FindingsFor this cross-sectional study, 150 consecutive patients with pleural effusion diagnosed by chest radiography, who were referred for diagnostic thoracocentesis and pleural biopsy and met eligibility criteria, had a pleural fluid specimen submitted for real-time PCR testing. Overall, 98 patients had pleural TB and 52 had pleural effusion secondary to other disease. TB diagnosis was obtained using acid-fast bacilli (AFB) smear or culture for mycobacteria and/or histopathologic examination in 94 cases and by clinical findings in 4 cases. Sensitivity, specificity, positive and negative predictive values of PCR testing for pleural TB diagnosis were 42.8% (95% CI 38.4 - 44.8), 94.2% (95% CI 85.8 - 98.0), 93.3% (95% CI 83.6 - 97.7), and 48.5% (95% CI 44.2 - 50.4), respectively. The real-time PCR test improved TB detection from 30.6% to 42.9% when compared to AFB smear and culture methods performed on pleural fluid specimens, although the best sensitivity was achieved by combining the results of culture and histopathology of pleural tissue specimens.ConclusionThe real-time PCR test of pleural fluid specimens is a useful and non-invasive additional assay for fast diagnosis of pleural TB.

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We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3% and 98.1%, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6% and 100%, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100% consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis.

Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC-->ACC (Ser-->Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100% of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.

Mycobacterium tuberculosis (M.tb), the pathogen responsible for tuberculosis (TB) poses as the major cause of death among infectious diseases. The knowledge about the molecular diversity of M.tb enables the implementation of more effective surveillance and control measures and, nowadays, Whole Genome Sequencing (WGS) holds the potential to produce high-resolution epidemiological data in a high-throughput manner. Florianópolis, the state capital of Santa Catarina (SC) in south Brazil, shows a high TB incidence (46.0/100,000). Here we carried out a WGS-based evaluation of the M.tb strain diversity, drug-resistance and ongoing transmission in the capital metropolitan region. Resistance to isoniazid, rifampicin, streptomycin was identified respectively in 4.0% (n = 6), 2.0% (n = 3) and 1.3% (n = 2) of the 151 studied strains by WGS. Besides, resistance to pyrazinamide and ethambutol was detected in 0.7% (n = 1) and reistance to ethionamide and fluoroquinolone (FQ) in 1.3% (n = 2), while a single (0.7%) multidrug-resistant (MDR) strain was identified. SNP-based typing classified all isolates into M.tb Lineage 4, with high proportion of sublineages LAM (60.3%), T (16.4%) and Haarlem (7.9%). The average core-genome distance between isolates was 420.3 SNPs, with 43.7% of all isolates grouped across 22 genomic clusters thereby showing the presence of important ongoing TB transmission events. Most clusters were geographically distributed across the study setting which highlights the need for an urgent interruption of these large transmission chains. The data conveyed by this study shows the presence of important and uncontrolled TB transmission in the metropolitan area and provides precise data to support TB control measures in this region.

BACKGROUND In high tuberculosis (TB) burden countries, there are few data on the performance of new molecular commercialised assays developed locally.OBJECTIVE To evaluate the performance of a new molecular commercialised assay for TB diagnosis (Detect-TB) in three laboratories.METHODS A total of 302 sputum samples from an equal number of patients with presumptive diagnosis of pulmonary tuberculosis (PTB) were submitted for routine smear microscopy, culture, and Detect-TB assay at three different sites in Brazil (the cities of Caxias do Sul, São Paulo and Canoas).FINDINGS Seventy four (24.7%) TB cases were diagnosed (65 bacteriologically confirmed). When compared to smear microscopy/culture results, the overall sensitivity and specificity of Detect-TB assay was 84.6% (CI 95%; 73.7-91.6) and 93.1% (CI 95%; 89.1-95.8), respectively. When compared to bacteriological and clinical diagnostic criteria, the sensitivity and specificity of Detect-TB assay was 74.3% (CI 95%; 63.3-82.9) and 92.9% (CI 95%; 88.7-95.6), respectively. Among the three sites - Caxias do Sul, São Paulo and Canoas - the sensitivity and specificity were respectively 94.7% and 97.8%; 71.4% and 93.9%, 82.1% and 88.9%.MAIN CONCLUSIONS These findings suggest that the Detect-TB assay could be applied routinely in reference laboratories across different regions in Brazil.

The present study analysed the concordance among four different molecular diagnostic methods for tuberculosis (TB) in pulmonary and blood samples from immunocompromised patients. A total of 165 blood and 194 sputum samples were collected from 181 human immunodeficiency virus (HIV)-infected patients with upper respiratory complaints, regardless of suspicious for TB. The samples were submitted for smear microscopy, culture and molecular tests: a laboratory-developed conventional polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) and the Gen-Probe and Detect-TB Ampligenix kits. The samples were handled blindly by all the technicians involved, from sample processing to results analysis. For sputum, the sensitivity and specificity were 100% and 96.7% for qPCR, 81.8% and 94.5% for Gen-Probe and 100% and 66.3% for Detect-TB, respectively. qPCR presented the best concordance with sputum culture [kappa (k) = 0.864)], followed by Gen-Probe (k = 0.682). For blood samples, qPCR showed 100% sensitivity and 92.3% specificity, with a substantial correlation with sputum culture (k = 0.754) and with the qPCR results obtained from sputum of the corresponding patient (k = 0.630). Conventional PCR demonstrated the worst results for sputa and blood, with a sensitivity of 100% vs. 88.9% and a specificity of 46.3% vs. 32%, respectively. Commercial or laboratory-developed molecular assays can overcome the difficulties in the diagnosis of TB in paucibacillary patients using conventional methods available in most laboratories.