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Antimicrobial Agents and Chemotherapy, January 2005, p. 144-147, Vol. 49, No. 1
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.1.144-147.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

New Multiplex PCR for Rapid Detection of Isoniazid-Resistant Mycobacterium tuberculosis Clinical Isolates

Laura Herrera-León,* Tamara Molina, Pilar Saíz, Juan Antonio Sáez-Nieto, and Maria Soledad Jiménez

Mycobacterial Reference Laboratory, Servicio de Bacteriología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain

Received 4 February 2004/ Returned for modification 26 June 2004/ Accepted 11 September 2004

In this study, we describe a multiplex PCR to detect a AGC->ACC (serine to threonine) mutation in the katG gene and a –15 C-to-T substitution (inhAC–15T) at the 5' end of a presumed ribosome binding site in the promoter of the mabA-inhA operon. These mutations have been reported in the majority of previous studies as the most frequent mutations involved in the resistance to isoniazid (INH) of Mycobacterium tuberculosis clinical strains with high levels of resistance. The method was optimized and validated after an analysis of 30 M. tuberculosis clinical isolates with known sequences of the relevant part of the katG gene and the regulatory region of the mabA-inhA operon. We analyzed 297 INH-resistant M. tuberculosis isolates collected in Spain from 1996 to 2003 by PCR-restriction fragment length polymorphism (using the katG gene), DNA sequencing, and the newly developed multiplex PCR. The results were concordant for all 297 isolates tested. The analysis revealed that 204 (68.7%) of the isolates carried one or both of the mutations. This finding suggests that with further development this multiplex PCR will be able to detect the majority of the INH-resistant M. tuberculosis clinical isolates from Spain and other countries where a high frequency of similar mutations occur.

* Corresponding author. Mailing address: Instituto de Salud Carlos III, Laboratorio de Referencia de Micobacterias, 28820 Majadahonda, Madrid, Spain. Phone: (34) 918223642. Fax: (34) 915097966. E-mail: lherrera{at}isciii.es.

Antimicrobial Agents and Chemotherapy, January 2005, p. 144-147, Vol. 49, No. 1
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.1.144-147.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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