PCR-Restriction Fragment Length Polymorphism for Rapid, Low-Cost Identification of Isoniazid-Resistant Mycobacterium tuberculosis

Abstract

PCR-restriction fragment length poymorphism (PCR-RFLP) is a simple, robust technique for the rapid identification of isoniazid-resistant Mycobacterium tuberculosis. One hundred consecutive isolates from aVietnamese tuberculosis hospital were tested by MspA1I PCR-RFLP for the detection of isoniazid-resistant katG_315 mutants. The test had a sensitivity of 80% and a specificity of 100% against conventional phenotypic drug susceptibility testing. The positive and negative predictive values were 1 and 0.86, respectively. None of the discrepant isolates had mutant katG_315 codons by sequencing. The test is cheap (less than $1.50 per test), specific, and suitable for the rapid identification of isoniazid resistance in regions with a high prevalence of katG_315 mutants among isoniazid-resistant M. tuberculosis isolates.Tuberculosis (TB) control is threatened by increasing resistance to first-line antituberculous agents. The WHO estimates that in the year 2004, 4.3% of new TB cases globally were multidrug-resistant TB (MDR-TB) (29), defined as resistance to at least isoniazid and rifampin. Treatment regimens for MDR-TB are more complex, less potent, more toxic, and more expensive than first-line regimens, resulting in poorer tolerability and adherence and higher morbidity and mortality rates. Early identification of drug-resistant and particularly MDR strains is routine in developed low-prevalence countries, but the methods are currently unavailable to and too costly for resource-poor nations, where the burden of disease is greatest. Early identification of MDR-TB is crucial in order to permit the timely administration of appropriate drug regimens and could potentially improve the efficacy of MDR treatment schemes, such as directly observed treatment, short course-plus.Isoniazid is a prodrug that is activated through cleavage by catalase peroxidase, the product of the katG gene. The active drug has a complex mode of action, which remains to be fully elucidated but principally targets mycolic acid biosynthesis pathways. Upregulation of the inhA gene product, an enoyl-ACP reductase, can overwhelm the impact of isoniazid.Resistance to isoniazid is achieved through mutations in the katG gene, predominantly at codon 315, and in the inhA promoter for the majority of isolates. Mutations in other genes, such as ahpC and ndh, have been implicated in isoniazid resistance, but their roles are not yet proven. A role for kasA gene mutation in isoniazid resistance has recently been discredited (7). Many isolates are isoniazid resistant without any mutations in known target genes, and the mechanism of resistance for these isolates remains obscure.Phenotypic sensitivity testing of Mycobacterium tuberculosis is time-consuming, requiring 2 to 4 weeks from primary specimen in settings with rapid liquid culture systems, such as BACTEC MGIT 960, and 6 to 8 weeks in settings with solidmedium culture (14).Several approaches to the rapid identification of isoniazid resistance have been reported in the literature, including realtime PCR (22), s...

Detection of katG Ser315Thr substitution in Mycobacterium tuberculosis infected patients using PCR -RFLP method for screening Isoniazid-Resistance

“…After confi rmation the katG amplicons were further used for RFLP analysis using The Mutation in katG Ser315Thr observed in isoniazid resistant isolates. The drug susceptibility profi le of these isolates were showed resistant to in isoniazid and rifampicin whereas 3 isolates were resistant to Ethambutol drug and Streptomycin respectively alternatively other Seven ( 7) and multiplex-allele-specifi c PCR but the RFLP approach has several advantages over others methods because it is very cheap, robust, and simple to both perform and interpret, requires PCR equipment [8]. Early detection of resistance to drugs will helpful for treatment modalities and effective control measures to reduce the risk of further disease progression and transmission.…”

Analysis of GSTM1 Deletion on Adult Pulmonary Tuberculosis Patients

“…Acetylhydrazine also metabolized by CYP2E1 into toxic metabolitethat will subsequently be excreted after detoxification by glutathione S-transferase (GST). 11,13,14 Several studies have investigated the relationship between genetic variation on drug-metabolizing enzyme and hepatotoxicity incidence. Most have reported that GSTM1 deletion significantly related to hepatotoxicity incidence.…”

Detección de la mutación S315T en el gen katg como estrategia para identificación de Mycobacterium tuberculosis resistente a isoniacida en un laboratorio de referencia

“…Para la detección de la mutación en el codón 315 se utilizó la estrategia RPC-PLFR, utilizando los partidores katG2F (5'-GGTCGACATTCGCGAGACGTT-3) y katG2R (5'-CGGTGGATCAGCTTGTACCAG-3') para amplificar un fragmento de ADN de 377 pb contentivo del codón 315 correspondiente a una secuencia del gen katG de M. tuberculosis, de acuerdo al protocolo descrito previamente 26 .…”