Simultaneous detection of isoniazid, rifampin, and ethambutol resistance of Mycobacterium tuberculosis by a single multiplex allele-specific polymerase chain reaction (PCR) assay Zhenhua Yang a, T , Riza Durmaz b , Dong Yang a , Selami Gunal b , Lixin Zhang a , Betsy Foxman a , Ahmet Sanic c , Carl F. Marrs a a Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI 48109-2029, USA b Medical Microbiology, Medical Faculty, Inonu University, Malatya, Turkey c Ondokuz Mayis University, Medical Faculty, Medical Microbiology, Samsun, Turkey Received 9 February 2005; accepted 8 June 2005 Abstract Prompt detection of drug resistance of Mycobacterium tuberculosis is essential for effective control of tuberculosis (TB). We developed a multiplex allele-specific polymerase chain reaction (MAS-PCR) that detects the most commonly observed isoniazid (INH), rifampin (RIF), and ethambutol resistance-associated mutations in a single assay. The usefulness of the newly developed method was evaluated with 174 clinical isolates of M. tuberculosis obtained from Turkey. Distinct PCR banding patterns were observed for different mutation profiles and the correlation between MAS-PCR results and DNA sequencing findings was 99.4%. With culture-based phenotypic drug susceptibility testing as a reference standard, the sensitivity and specificity of the newly developed MAS-PCR assay for drug resistance-related genetic mutation detection were determined to be 81.1% and 97.5% for INH, 93.0% and 98.9 % for RIF, and 54.5% and 68.0 % for ethambutol. MAS-PCR provides a rapid, potentially more cost-effective, method of detecting multidrug-resistant TB. D 2005 Elsevier Inc. All rights reserved. Keywords: Multidrug resistant; Tuberculosis; Multiplex PCR 1. Introduction Tuberculosis (TB) has a long and continuing history of causing worldwide morbidity and mortality World Health Organization (World Health Organization, 2004). The emergence of multidrug-resistant TB (MDR-TB), defined as resistance to at least isoniazid (INH) and rifampin (RIF), the 2 principal first-line anti-TB drugs, poses an important threat to TB control as MDR-TB reduces response to standard short-course chemotherapy with first-line anti-TB drugs, leads to higher mortality and treatment failure rates, and increases periods of transmissibility of the disease (Schluger, 2000; WHO, 2000; Schneider and Castro, 2003). An estimated 273 000 new cases of MDR-TB occurred worldwide in 2000, accounting for 3.2% of all new TB cases (Dye et al., 2002). Prompt detection of anti-TB drug resistance is essential for controlling the development and spread of MDR-TB as it facilitates the appropriate and timely delivery of anti-TB therapy reducing overall cost of treatment and transmission of resistant cases. Currently, the detection of drug resistance in Mycobacterium tuberculosis is primarily based on phenotypic drug susceptibility testing, which involves time-consuming culture of the slow-growing M. tuberculosis bacilli in the presence of antibiotics (Canetti and Khomenko, 1969; Libonati et al., 1988; Laszlo et al., 1997). With the increased understanding of the genetic mechanisms of M. tuberculosis drug resistance and the advancement of molecular technologies in recent years, a number of more rapid molecular methods to detect mutations in genes implicated in M. tuberculosis drug resistance have been developed providing a rapid alternative (Cockerill, 1999; Victor et al., 2002). These methods include direct DNA sequencing (Kapur et al., 1995), line probe assay (Debeenhouwer et al., 1995; Cooksey et al., 1997), molecular beacon sequence analysis (Piatek et al., 0732-8893/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2005.06.007 T Corresponding author. Tel.: +1-734-763-4296; fax: +1-734-764-3192. E-mail address: zhenhua@umich.edu (Z. Yang). Diagnostic Microbiology and Infectious Disease 53 (2005) 201 – 208 www.elsevier.com/locate/diagmicrobio