In house colorimetric reverse hybridisation assay for detection of the mutation most frequently associated with resistance to isoniazid in Mycobacterium tuberculosis

Verza, Mirela; Maschmann, Raquel de Abreu; Silva, Márcia Susana Nunes; Costa, Elis Regina Dalla; Ribeiro, Marta Osório; Rosso, Franciele; Suffys, Philip Noël; Tortoli, Enrico; Marcelli, Fiorella; Zaha, Arnaldo; Rossetti, Maria Lúcia Rosa

doi:10.1590/s0074-02762009000500008

Cited by 7 publications

(6 citation statements)

References 32 publications

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“…The sample was defined as positive when it had a detectable cycle threshold (Ct) and the melting temperature (Tm) was the same as for the positive control (88.8°C). Our group has obtained successful results using the IS 6110 region to detect M. tuberculosis complex DNA, and thus this region was chosen for the study [15,16]. …”

Section: Methodsmentioning

confidence: 99%

Evaluation of real-time PCR of patient pleural effusion for diagnosis of tuberculosis

et al. 2011

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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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Section: Methodsmentioning

confidence: 99%

Evaluation of real-time PCR of patient pleural effusion for diagnosis of tuberculosis

et al. 2011

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“…The assay showed high analytical sensitivity and specificity. This technique had already been proved very promising in previous study in which the katG315 mutation was successfully characterized in 100% samples compared to the DNA sequencing (Verza et al 2009).…”

Section: Discussionmentioning

confidence: 99%

“…In this study, we tested a molecular assay for the detection of M. tuberculosis RIF resistance based on colorimetric reverse dot blot hybridization (CRDH). Previously, a similar strategy was developed by Verza et al (2009) in our laboratory. The results from the CRDH were compared with the data obtained by DNA sequencing and antimicrobial susceptibility test (AST) and some DNAs were also analyzed by the GenoType MTBDR commercial test.…”

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confidence: 99%

Detection of rifampin-resistant genotypes in Mycobacterium tuberculosis by reverse hybridization assay

Maschmann

Verza

Silva

et al. 2011

Mem. Inst. Oswaldo Cruz

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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.

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“…The genes encoding rpo B, kat G and the inh A promoter, which are related to RMP and INH, respectively, had been sequenced as part of an earlier study (Verza et al 2009, Maschmann et al 2011). The H37Rv (ATCC27294) reference strain was used as a control for both DST and genotyping.…”

Section: Methodsmentioning

confidence: 99%

“…To determine the specificity of LINE-TB/MDR, 100 ng of genomic DNA from each of the following bacteria were submitted to the entire procedure: Neisseria meningitidis, Streptococcus pneumoniae, Salmonella enterica, Haemophilus influenzae, Escherichia coli (obtained from IPB/LACEN-RS), Mycobacterium marinum, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium gordonae, Mycobacterium avium, Mycobacterium smegmatis, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium fortuitum-peregrinum and Mycobacterium phlei (obtained from Oswaldo Cruz Foundation, Brazil) (Verza et al 2009, Maschmann et al 2011). …”

Section: Methodsmentioning

confidence: 99%

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

Ferreira

Costa

Santos

et al. 2014

Mem. Inst. Oswaldo Cruz

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Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

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In house colorimetric reverse hybridisation assay for detection of the mutation most frequently associated with resistance to isoniazid in Mycobacterium tuberculosis

Cited by 7 publications

References 32 publications

Evaluation of real-time PCR of patient pleural effusion for diagnosis of tuberculosis

Evaluation of real-time PCR of patient pleural effusion for diagnosis of tuberculosis

Detection of rifampin-resistant genotypes in Mycobacterium tuberculosis by reverse hybridization assay

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

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