Downloaded from www.microbiologyresearch.org by IP: 54.198.134.45 On: Fri, 03 Mar 2017 06:14:25 Rapid and low-cost colorimetric method using 2,3,5-triphenyltetrazolium chloride for detection of multidrug-resistant Mycobacterium tuberculosis Alireza Mohammadzadeh, 1 Parisa Farnia, 2 Kiarash Ghazvini, 1 Mahdi Behdani, 1 3 Tahereh Rashed 1 and Javad Ghanaat 1 Correspondence Mahdi Behdani Behdani73042@yahoo.com 1 Mycobacteriology Department, Gaem University Hospital, Mashhad University of Medical Science, Iran 2 Iranian National Reference TB laboratory, National Research Institute of Tuberculosis & Lung Disease (NRITLD)/WHO Collaborating Centre, Tehran, Iran Received 29 November 2005 Accepted 21 August 2006 A rapid and inexpensive method for the detection of drug resistance in Mycobacterium tuberculosis is essential for the effective control of tuberculosis. The aim of this study was to evaluate a colorimetric method using 2,3,5-triphenyltetrazolium chloride (TTC) for antibiotic susceptibility testing of M. tuberculosis isolates. Eleven multidrug-resistant (MDR) isolates of M. tuberculosis and 12 isolates which were susceptible to rifampicin (RIF) and isoniazid (INH) were used. The test was performed with a critical concentration of 0?2 mg ml ”1 for INH and 2?0 mg ml ”1 for RIF in 7H9GC broth with 0?625 mg TTC ml ”1 . Each isolate was inoculated under these conditions and inspected daily for colour changes; the results were obtained after a mean of 4?9 days. The sensitivity and specificity of this method were 100 % and 92 %, respectively, for both antibiotics. Considering the speed, technical ease and cost-effectiveness of this method, the TTC assay is a good alternative method for drug susceptibility testing of M. tuberculosis isolates. INTRODUCTION The spread of multidrug-resistant (MDR) Mycobacterium tuberculosis is of increasing public health concern in many parts of the world and the rapid detection of MDR isolates is critical for the effective treatment of affected patients (Tenover et al., 1993). The method of proportion, which has been accepted as the gold standard, requires 3 weeks of incubation before an isolate can be determined as susceptible or resistant (Woods, 2000). The Bactec radio- metric susceptibility method produces results in 7–10 days, but requires a heavy technical investment and is costly to perform (Ardito et al., 2001). Molecular methods for the characterization of genes that confer resistance to first-line antimicrobial agents such as isoniazid (INH) (Musser et al., 1996) and rifampicin (RIF) (Mokrousov et al., 2003) are available; however, the equipment and specialized skills required to perform such methods make them an impractical option, especially in developing countries. Colorimetric assays, using reagents such as Alamar blue (Franzblau et al., 1998; Yojko et al., 1995), 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Abate et al., 2004; Martin et al., 2005; Montoro et al., 2005; Mshana et al., 1998), 2,3-bis(2-methoxy- 4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide (XTT) (De Logu et al., 2002, 2003) and 2,3-diphenyl-5- thienyl-(2)-tetrazolium chloride (Lee et al., 2006; Yamane et al., 1996), have been proposed as a method for the detection of drug susceptibility. The yellow dye 2,3,5- triphenyltetrazolium chloride (TTC) is reduced in living cells by dehydrogenase to produce insoluble red TTC formazan crystals (Caviedes et al., 2002; Denizot & Lang, 1986; Thom et al., 1993). The only reported use of TTC has been for the detection of mesophilic anaerobic bacteria in the canning industry (Kvasnikov et al., 1974). In this study, we have evaluated the possibility of using the TTC colorimetric assay for detecting the susceptibility of M. tuberculosis to INH and RIF in liquid medium. METHODS M. tuberculosis isolates. The M. tuberculosis isolates used in this study were recovered from patients with active clinical infections at the Iranian National Research Institute of Tuberculosis & Lung Disease, Tehran, Iran (2005–2006). These included 11 isolates of M. tuberculosis which were resistant to RIF and INH (MDR isolates), and 12 isolates susceptible to RIF and INH (sensitive isolates). Drug susceptibility testing of these isolates was performed using the pro- portion method with INH (0?2 mg ml 21 ) and RIF (40 mg ml 21 ) in 3Present address: Education Department, Pasteur Institute of Iran, Tehran, Iran. Abbreviations: INH, isoniazid; MDR, multidrug-resistant; RIF, rifampicin; TTC, 2,3,5-triphenyltetrazolium chloride. 46442 G 2006 SGM Printed in Great Britain 1657 Journal of Medical Microbiology (2006), 55, 1657–1659 DOI 10.1099/jmm.0.46442-0