Abstract
Background: Failure to early detect multidrug-resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes, and highlights the need to rapidly detect resistance to rifampicin (RIF) and isoniazid (INH).Methods: In Multi-Fluorescence quantitative Real-Time PCR (MF-qRT-PCR) assay, 10 probes labeled with four kinds of fluorophores were designed to detect the mutations in regions of rpoB, katG, mabA-inhA, oxyR-ahpC, and rrs.The efficiency of MF-qRT-PCR assay was tested using 261 bacterial isolates and 33 clinical sputum specimens. Among these samples, 227 Mycobacterium tuberculosis isolates were analyzed using drug susceptibility testing (DST), DNA sequencing and MF-qRT-PCR assay.Results: Compared with DST, MF-qRT-PCR sensitivity and specificity for RIF-resistance were 94.6 and 100%, respectively. And the detection sensitivity and specificity for INH-resistance were 85.9 and 95.3%, respectively. Compared with DNA sequencing, the sensitivity and specificity of our assay were 97.2 and 100% for RIF-resistance and 97.9 and 96.4% for INH-resistance. Compared with Phenotypic strain identification, MF-qRT-PCR can distinguish 227 M. tuberculosis complexes (MTC) from 34 Non-tuberculous mycobacteria (NTM) isolates with 100% accuracy rate.Conclusions: MF-qRT-PCR assay was an efficient, accurate, reliable, and easy-operated method for detection of RIF and INH-resistance, and distinction of MTC and NTM of clinical isolates.
Highlights
Worldwide prevalence, high misdiagnosis rate, high treatment cost, and serious side-effects of drug made multidrug-resistant tuberculosis (MDR-TB) a critical problem for global public health (WHO, 2012)
Quantitative real-time polymerase chain reaction is considered as one of the most widely applied molecular assays for diagnosis of drug resistant tuberculosis (Cuevas-Córdoba and ZentenoCuevas, 2010). It can accurately discriminate single nucleotide polymorphism (SNP) with low risk of contamination and had the shortest turn-around time (TAT) so far. qRT-PCR assays have been applied to the detection of RIF and INH resistances (García de Viedma et al, 2002; van Doorn et al, 2003; Marín et al, 2004; Helb et al, 2010) using at most two color probes so far
Several commercial Mycobacterium tuberculosis complex (MTC)/drug susceptibility testing (DST) diagnostic kits based on qRT-PCR assay have appeared and obtained regulatory approval (WHO, 2012; Food and Drug Administration, 2015) including Xpert MTB/RIF (Cepheid) (Boehme et al, 2010), Fluorotype MDRTB (Hain Lifescience) (Pierce et al, 2003; Rice et al, 2012), RealTime MDR TB (Abbott Molecular) (Chen et al, 2015), BDProbeTec (Becton Dickinson) (UNITAID, 2015) and MeltPro Drug-Resistant TB RIF, and Drug-Resistant INH kits(Xiamen Zeesan Biotech) (Hu et al, 2014)
Summary
High misdiagnosis rate, high treatment cost, and serious side-effects of drug made MDR-TB a critical problem for global public health (WHO, 2012). Quantitative real-time polymerase chain reaction (qRT-PCR) is considered as one of the most widely applied molecular assays for diagnosis of drug resistant tuberculosis (Cuevas-Córdoba and ZentenoCuevas, 2010) It can accurately discriminate single nucleotide polymorphism (SNP) with low risk of contamination and had the shortest turn-around time (TAT) so far. Several commercial MTC/DST diagnostic kits based on qRT-PCR assay have appeared and obtained regulatory approval (WHO, 2012; Food and Drug Administration, 2015) including Xpert MTB/RIF (Cepheid) (Boehme et al, 2010), Fluorotype MDRTB (Hain Lifescience) (Pierce et al, 2003; Rice et al, 2012), RealTime MDR TB (Abbott Molecular) (Chen et al, 2015), BDProbeTec (Becton Dickinson) (UNITAID, 2015) and MeltPro Drug-Resistant TB RIF, and Drug-Resistant INH kits(Xiamen Zeesan Biotech) (Hu et al, 2014) While most of these kits can be automated and highthroughput detection of mutations, they still have shortcomings such as high cost, limited detection sites. Failure to early detect multidrug-resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes, and highlights the need to rapidly detect resistance to rifampicin (RIF) and isoniazid (INH)
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