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Antimicrobial Agents and Chemotherapy, November 2004, p. 4293-4300, Vol. 48, No. 11
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.11.4293-4300.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Rapid Direct Detection of Multiple Rifampin and Isoniazid Resistance Mutations in Mycobacterium tuberculosis in Respiratory Samples by Real-Time PCR

Mercedes Marín, Darío García de Viedma,* María Jesús Ruíz-Serrano, and Emilio Bouza

Servicio de Microbiología y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain

Received 24 May 2004/ Returned for modification 9 July 2004/ Accepted 21 July 2004

Rapid detection of resistance in Mycobacterium tuberculosis can optimize the efficacy of antituberculous therapy and control the transmission of resistant M. tuberculosis strains. Real-time PCR has minimized the time required to obtain the susceptibility pattern of M. tuberculosis strains, but little effort has been made to adapt this rapid technique to the direct detection of resistance from clinical samples. In this study, we adapted and evaluated a real-time PCR design for direct detection of resistance mutations in clinical respiratory samples. The real-time PCR was evaluated with (i) 11 clinical respiratory samples harboring bacilli resistant to isoniazid (INH) and/or rifampin (RIF), (ii) 10 culture-negative sputa spiked with a set of strains encoding 14 different resistance mutations in 10 independent codons, and (iii) 16 sputa harboring susceptible strains. The results obtained with this real-time PCR design completely agreed with DNA sequencing data. In all sputa harboring resistant M. tuberculosis strains, the mutation encoding resistance was successfully detected. No mutation was detected in any of the susceptible sputa. The test was applied only to smear-positive specimens and succeeded in detecting a bacterial load equivalent to 103 CFU/ml in sputum samples (10 acid-fast bacilli/line). The analytical specificity of this method was proved with a set of 14 different non-M. tuberculosis bacteria. This real-time PCR design is an adequate method for the specific and rapid detection of RIF and INH resistance in smear-positive clinical respiratory samples.

* Corresponding author. Mailing address: Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, C/Dr. Esquerdo 46, 28007 Madrid, Spain. Phone: 34 91 4265104. Fax: 34 91 5044906. E-mail: dgviedma{at}microb.net.

Antimicrobial Agents and Chemotherapy, November 2004, p. 4293-4300, Vol. 48, No. 11
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.11.4293-4300.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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