Mutations in the rpoB Gene of Rifampin-Resistant Mycobacterium tuberculosis Isolates in Spain and Their Rapid Detection by PCR–Enzyme-Linked Immunosorbent Assay

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Abstract
Genetic alterations in the rpoB gene were characterized in 50 rifampin-resistant (Rif r ) clinical isolates of Mycobacterium tuberculosis complex from Spain. A rapid PCR–enzyme-linked immunosorbent assay (ELISA) technique for the identification of rpoB mutations was evaluated with isolates of the M. tuberculosis complex and clinical specimens from tuberculosis patients that were positive for acid-fast bacilli (AFB). Sequence analysis demonstrated 11 different rpoB mutations among the Rif r isolates in the study. The most frequent mutations were those associated with codon 531 (24 of 50; 48%) and codon 526 (11 of 50; 22%). Although the PCR-ELISA does not permit characterization of the specific Rif r allele within each strain, 10 of the 11 Rif r genotypes were correctly identified by this method. We used the PCR-ELISA to predict the rifampin susceptibility of M. tuberculosis complex organisms from 30 AFB-positive sputum specimens. For 28 samples, of which 9 contained Rif r organisms and 19 contained susceptible strains, results were concordant with those based on culture-based drug susceptibility testing and sequencing. Results from the remaining two samples could not be interpreted because of low bacillary load (microscopy score of 1+ for 1 to 9 microorganisms/100 fields). Our results suggest that the PCR-ELISA is an easy technique to implement and could be used as a rapid procedure for detecting rifampin resistance to complement conventional culture-based methods.