New real-time PCR able to detect in a single tube multiple rifampin resistance mutations and high-level isoniazid resistance mutations in Mycobacterium tuberculosis

Abstract

The emergence of resistance to antituberculosis drugs is a relevant matter worldwide, but the retrieval of antibiograms for Mycobacterium tuberculosis is severely delayed when phenotypic methods are used. Genotypic methods allow earlier detection of resistance, although conventional approaches are cumbersome or lack sensitivity or specificity. We aimed to design a new real-time PCR method to detect rifampin (RIF)- and isoniazid (INH)-resistant M. tuberculosis strains in a single reaction tube. First, we characterized the resistant isolates in our area of Spain by DNA sequencing. Some mutation was found within the rpoB core region in all the RIF-resistant (RIF(r)) strains. Forty-six percent of the INH-resistant (INH(r)) strains showed a mutation in katG codon 315, and most of these were associated with high MICs. Eighteen of the RIF(r), INH(r), and multidrug-resistant strains sequenced were tested by our real-time PCR assay; and full concordance of the results of the PCR with the sequencing data was obtained. In addition, a blind test was performed with a panel of 15 different susceptible and resistant strains from throughout Spain, and our results were also in 100% agreement with the sequencing data. Ours is the first assay based on rapid-cycle PCR able to simultaneously detect in a single reaction tube a large variety of mutations associated with RIF resistance (12 different mutations affecting 8 independent codons, including the most prevalent mutations at positions 526 and 531) and the most frequent INH resistance mutations. Our design could be a model for new, rapid genotypic methods able to simultaneously detect a wide variety of antibiotic resistance mutations.

FIG. 1.

Schematic representation of the rpoB (rpoβ) core (a) and katG (b) regions that include the mutations for resistance to RIF and INH. The FRET probes used in the real-time PCR assay are indicated by the boxes above the corresponding complementary sequence. Codon coordinates are indicated as a reference. The mutations studied and the corresponding nucleotide substitutions are indicated below the affected codons. The fluorescein and Red labels are indicated. For the katG region the probe designed to be complementary with ACA in codon 315 is indicated.

FIG. 2.

Representative experimental melting patterns for wt, RIF^r, INH^r, and MDR isolates, as measured in fluorimetric channels F2 (a) and F3 (b). The graphs show the result of taking the first negative derivative of the melting curve for the probes to obtain the point at which the slope changes (T_m). The T_m regions for wt and mutant isolates are indicated above the curves. (a) Measurement of the fluorescent signals from RPO1 and katG-specific probes. For the wt strain two peaks corresponding to each of the two probes are shown. A, representative result for an INH^r isolate (the T_m of the katG-specific probes deviates with respect to that for the wt; the T_m of the RPO1 probes does not deviate with respect to that for the wt); B, representative result for an RIF^r isolate with a mutation in RPO1 (the T_m for the RPO1 probes is deviated compared to the reference wt value; the T_m for the katG-specific probes is not deviated with respect to the wt value); C, representative result for an MDR isolate (simultaneous deviations both for the T_m of the RPO1 probes and for the T_m of the katG-specific probes). (b) Measurement of the fluorescent signal from the RPO2 probes. The results for three representative RIF^r strains with mutations in RPO2 (peaks D, E, and F) are shown. In all of the strains, deviations in the T_m with respect to that for the wt were observed.