Fast test for assessing the susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by real-time PCR

Abstract

Mycobacterium tuberculosis is the bacterium that causes tuberculosis (TB), a leading cause of death from infectious disease worldwide. Rapid diagnosis of resistant strains is important for the control of TB. Real-time polymerase chain reaction (RT-PCR) assays may detect all of the mutations that occur in the M. tuberculosis 81-bp core region of the rpoB gene, which is responsible for resistance to rifampin (RIF) and codon 315 of the katG gene and the inhA ribosomal binding site, which are responsible for isoniazid (INH). The goal of this study was to assess the performance of RT-PCR compared to traditional culture-based methods for determining the drug susceptibility of M. tuberculosis. BACTEC™ MGIT™ 960 was used as the gold standard method for phenotypic drug susceptibility testing. Susceptibilities to INH and RIF were also determined by genotyping of katG, inhA and rpoB genes. RT-PCR based on molecular beacons probes was used to detect specific point mutations associated with resistance. The sensitivities of RT-PCR in detecting INH resistance using katG and inhA targets individually were 55% and 25%, respectively and 73% when combined. The sensitivity of the RT-PCR assay in detecting RIF resistance was 99%. The median time to complete the RT-PCR assay was three-four hours. The specificities for tests were both 100%. Our results confirm that RT-PCR can detect INH and RIF resistance in less than four hours with high sensitivity.

Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides

“…(9) Thus, we showed that DNA extracts from ZN-stained slides may be used as a template for the amplification of M. tuberculosis DNA by qPCR technique. Similar results were also obtained by Gonçalves et al, (25) who used the same DNA extraction technique for detection by qPCR assays. De Almeida et al, (26) evaluated six different techniques for M. tuberculosis DNA extraction, including DNA extraction by Chelex from cultures of microorganism and FN-stained slides.…”

Performance of an in-house real-time polymerase chain reaction for identification of Mycobacterium tuberculosis isolates in laboratory routine diagnosis from a high burden setting

“…Detection of the mpt64 gene was 100% sensitive and specific for MTC when real-time PCR was tested against previously identified mycobacterial and non-mycobacterial species. Under routine conditions, this assay also showed high sensitivity and specificity in the detection of MTC, as found by other authors ( Gonçalves et al 2012 , Pinhata et al 2015 ). Gonçalves et al (2012) obtained high sensitivity and specificity using molecular beacons in an in-house real-time PCR assay to detect resistance to isoniazid and RIF in 988 MTC primary isolates received at the IAL.…”

“…Under routine conditions, this assay also showed high sensitivity and specificity in the detection of MTC, as found by other authors ( Gonçalves et al 2012 , Pinhata et al 2015 ). Gonçalves et al (2012) obtained high sensitivity and specificity using molecular beacons in an in-house real-time PCR assay to detect resistance to isoniazid and RIF in 988 MTC primary isolates received at the IAL. In this study, we decided to design a real-time PCR assay to identify MTC isolates in our TB diagnosis routine.…”

Cutaneous tuberculosis: diagnosis, histopathology and treatment - Part II

“…With the amplified DNA, it is also possible to separate atypical mycobacteria from Mtb, genotype mycobacteria and detect mutations that induce resistance to antibiotics. 17 , 18 The main molecular typing methods are:…”