Application of the resazurin microtitre assay for detection of multidrug resistance in Mycobacterium tuberculosis in Algiers

Abstract

This study assessed the performance of a rapid, low-cost, colorimetric method, the resazurin microtitre assay (REMA) plate method, for the detection of resistance to isoniazid and rifampicin in 136 clinical isolates of Mycobacterium tuberculosis from two hospitals in Algiers. MICs were determined and the results were compared with those obtained with the conventional proportion method on Lö wenstein-Jensen medium. Excellent results were obtained for the REMA plate method, with a sensitivity of 100 % for both isoniazid and rifampicin and a specificity of 98?3 and 99?2 %, respectively. The REMA plate method appears to be a reliable method for the rapid determination of multidrug-resistant tuberculosis and is a good alternative for use in resource-limited countries such as Algeria. INTRODUCTIONTuberculosis (TB) is a public health problem worldwide. According to the World Health Organization, 8?8 million new cases of TB were reported in 2003 and 1?7 million deaths were attributed to the disease (WHO, 2005). The human immunodeficiency virus pandemic and multidrugresistant TB (MDRTB) have emerged as major obstacles in the treatment and efficient control of the disease (Barnes et al., 2002;Corbett et al., 2003;Frieden et al., 2003). MDRTB is defined as TB that is resistant to at least rifampicin (RIF) and isoniazid (INH), the two most important drugs in the treatment of TB.In Algeria, the incidence of TB in 2003 was notified as 53 cases per 100 000 inhabitants (WHO, 2005). For the year 2001, the prevalence of drug resistance was 6?2 % and MDRTB in untreated patients amounted to 1?1 % (WHO, 2004).Effective treatment and prevention of MDRTB rely upon the prompt availability of drug-susceptibility testing (DST) results. For this reason, alternative, inexpensive and rapid methods of DST are needed urgently. The conventional agar and Löwenstein-Jensen (LJ) proportion method is laborious, with results only available after 3-6 weeks. The BACTEC radiometric system (Becton Dickinson) had the advantage of being more rapid than the proportion method, but required the use of radioisotopes, which was a disadvantage with respect to the disposal of waste material and was expensive to perform (Siddiqi et al., 1981;Roberts et al., 1983). The Mycobacteria Growth Indicator Tube or MGIT (Palomino et al., 1999; Goloubeva et al., 2001) and the Etest (Wanger & Mills, 1996), both commercial methods, are simple and rapid to perform, but are still expensive, making them impractical for routine use in developing countries. Molecular methods for detection of drug resistance have also been described, such as the line probe assay INNO-LiPA (Innogenetics), but need substantial investment in equipment, which makes them impractical for routine use (De Beenhouwer et al., 1995;Nachamkin et al., 1997). In recent years, several new methods have been proposed for the rapid performance of DST of Mycobacterium tuberculosis, including phage assays (Símboli et al., 2005) and cytofluorometry (Norden et al., 1995;Moore et al., 1999).Recently, a new method using...

Evaluation of antimicrobial activity of five Vincetoxicum taxa growing in Turkey

“…The broth dilution method assay was used for antibacterial [33] and antifungal [34][35][36] activities and the resazurin microtitre assay (REMA) plate method was used for antimycobacterial activity [37].…”

“…The REMA plate method: This method was performed in duplicate according to the method mentioned at Nateche et al [37] with minor changes. As the standard strain M. tuberculosis H37Rv and as reference antimycobacterial agents Ethambutol and Isoniazid were used.…”

“…There is no investigation on antibacterial activities of Turkish Vincetoxicum species in the literature. Therefore, the aim of the present study was to evaluate antibacterial, antifungal and antimycobacterial effects of ethanolic extracts obtained from aerial parts and roots of five [33] and antifungal [34][35][36] activities were performed using the broth dilution method assay and antimycobacterial activity [37] was performed using the REMA plate method assay. Ampicillin, Ethambutol, Isoniazid and Fluconazole were used as reference antimicrobial agents.…”

Green synthesis of highly functionalized octahydropyrrolo[3,4-c]pyrrole derivatives using subcritical water, and their anti(myco)bacterial and antifungal activity

“…Antimycobacterial properties of 4a-j were determined using the resazurin microtitre assay (REMA) plate method. 48 Culture medium: 7H9-S medium was used for the REMA plate method, consisting of Middlebrook 7H9 broth (BBL, Becton Dickinson and Company, Sparks, MD, USA) containing 0.1% casitone, 0.5% glycerol and 10% oleic acid-albumin-dextrose-catalase (OADC, BBL, Becton Dickinson and Company, Sparks, MD, USA). Resazurin reagent: Resazurin sodium salt powder (Sigma R7017) was dissolved in distilled water at a concentration of 0.01% (w/v) and sterilized by filtration through a 0.22 μm membrane filter (Ministar, Sartorius Stedim Biotech GmbH, Goettingen, Germany) to obtain a working solution which was stored at 4 °C for up to 1 week.…”

“…Resazurin reagent: Resazurin sodium salt powder (Sigma R7017) was dissolved in distilled water at a concentration of 0.01% (w/v) and sterilized by filtration through a 0.22 μm membrane filter (Ministar, Sartorius Stedim Biotech GmbH, Goettingen, Germany) to obtain a working solution which was stored at 4 °C for up to 1 week. The REMA plate method was used in duplicate as described by Nateche et al 48 with minor changes.…”