A pyrosequencing method for molecular monitoring of regions in the inhA, ahpC and rpoB genes of Mycobacterium tuberculosis.
In this study, a pyrosequencing method for monitoring two genes related to isoniazid (INH)-resistance and a region of the rpoB gene linked to rifampin (RMP)-resistance in Mycobacterium tuberculosis was developed and evaluated. Specifically, a 20-base pair (bp) region of inhA (from -24 to -4), a 35-bp region of ahpC (from -39 to -4), and a 57-bp region of rpoB (from codon 515 to 533) were analysed by pyrosequencing. For the development of the method, selected non-consecutive clinical isolates of M. tuberculosis were analysed, including: 25 isolates susceptible to both INH and RMP, 18 RMP-monoresistant isolates, 17 INH-monoresistant isolates, and 15 multidrug-resistant strains. Our pyrosequencing methodology was further evaluated using 96 M. tuberculosis isolates. Mutations in ahpC were found to be associated with INH resistance (p <0.05). By setting any mutation in ahpC as a marker of resistance, the specificity and the positive predictive value (PPV) were 100%. Similarly, any mutation in the rpoB gene was associated with a RMP resistance phenotype (p <0.01). Using any mutation in rpoB as a marker of RMP resistance, the sensitivity of this assay was 73% and the specificity and PPV were 100%. The use of this pyrosequencing method to analyse the ahpC and rpoB genes allowed us to detect INH- and/or RMP-resistant isolates. Furthermore, this method represents an opportunity to expedite the description of novel mutations related to drug resistance.
Garza-González
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Departamento de Microbiología, Facultad de Medicina, Universidad Autónoma de Nuevo León, Mexico. elvira_garza_gzz@yahoo.com
González
G M
GM
Rentería
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A
Cruz-Pulido
W
W
Rivera
G
G
Bocanegra-García
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V
D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology; D008826:Microbial Sensitivity Tests / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D010088:Oxidoreductases / Q000235:genetics*; D011237:Predictive Value of Tests; D012293:Rifampin / Q000494:pharmacology; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D014376:Tuberculosis / Q000382:microbiology*
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C505805:rpoB protein, Mycobacterium tuberculosis; D010088:Oxidoreductases; C085257:InhA protein, Mycobacterium; D012321:DNA-Directed RNA Polymerases; D007538:Isoniazid; D012293:Rifampin