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Journal of Clinical Microbiology, May 2001, p. 1813-1818, Vol. 39, No. 5
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.5.1813-1818.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Lucia Garcia,1 Mercedes Alonso-Sanz,2 Maria J. Rebollo,2,dagger Juan C. Tercero,1 and Fernando Chaves2,*

Pharma Gen S.A., Coslada,1 and Servicio de Microbiología, Hospital Universitario Doce de Octubre,2 28041 Madrid, Spain

Received 11 December 2000/Returned for modification 10 February 2001/Accepted 4 March 2001

Genetic alterations in the rpoB gene were characterized in 50 rifampin-resistant (Rifr) 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 Rifr 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 Rifr allele within each strain, 10 of the 11 Rifr 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 Rifr 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.

* Corresponding author. Mailing address: Servicio de Microbiología, Hospital Universitario Doce de Octubre, Carretera de Andalucia Km 5,400, Madrid 28041, Spain. Phone: (34) 91-3908239. Fax: (34) 91-5652765. E-mail: fchaves{at}hdoc.insalud.es.

dagger Present address: Departamento de Medicina Preventiva y Salud Pública, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid 28029, Spain.

Journal of Clinical Microbiology, May 2001, p. 1813-1818, Vol. 39, No. 5
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.5.1813-1818.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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