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

Abstract

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.

Evaluation of FluoroType MTB for direct detection of Mycobacterium tuberculosis complex and GenoType MTBDRplus for determining rifampicin and isoniazid resistance

“…Felkel et al [18] reported four false positive results and a false negative one when comparing them with cultures of patients without a history of MTBC treatment. Ferreira Junior et al [27] reported that the GenoType MTBDRplus identified two out of 40 (5%) INH resistant and three out of 40 (7.5%) RIF resistant strains as sensitive. In the study reported by Gu et al [20], with this assay, one out of six (16.6%) RIF resistant strains and one out of seven (14.2%) INH resistant strains were falsely detected as sensitive.…”

Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus

“…Genotype® MTBDRplus has been compared to phenotypic and other molecular assays with very impressive performance and was endorsed by WHO for MDR-TB molecular detection in 2008 ( Nikolayevsky et al 2009 , Ferreira Jr et al 2014 , Asante-Poku et al 2015 ). Since the first report using MLPA as a promising alternative for TB genetic characterisation ( Bergval et al 2008 ), few studies evaluating MLPA in comparison to other assays have been described ( Bergval et al 2012 , Sengstake et al 2014 , Chaidir et al 2016 ).…”

Multicenter evaluation of TB-SPRINT 59-Plex Beamedex®: accuracy and cost analysis

“…Target genes of both drugs were amplified as described elsewhere by Junior et al (2014) [16] and submitted to DNA sequencing using Big Dye® Kit Terminator Cycle Sequencing (Applied Biosystems, Foster City, CA, USA). Capillary electrophoresis was performed with an automated genetic analyzer ABI Prism 3130xl (Applied Biosystems), following the manufacturer’s instructions.…”