A method for detecting multidrug-resistant Mycobacterium tuberculosis by using a reduction of resazurin is described. Eighty clinical isolates were evaluated against isoniazid and rifampin; results at 7 days were compared with those of the proportion method. Specificity and sensitivity were excellent. The method is simple, inexpensive, and rapid and might be used with other antituberculosis drugs.Multidrug-resistant (MDR) tuberculosis (TB), defined as resistance to isoniazid (INH) and rifampin (RIF), is a severe problem for TB control. Recent reports document the global emergence of highly resistant Mycobacterium tuberculosis strains, particularly in countries of Eastern Europe (7, 36). The emergence of MDR TB highlights the need for drug susceptibility testing (DST), patient management, and drug resistance surveillance. Early diagnosis is essential for starting an effective treatment regimen and reducing its transmission in the population.In countries with low resources, DST involves conventional culture methods with Löwenstein-Jensen (L-J) and Middlebrook agars and requires 3 to 6 weeks to yield results (1, 2, 16). Faster methods which use liquid media, such as the BACTEC radiometric method and the mycobacteria growth indicator tube method, require radioisotopes (27, 28), expensive equipment and media, or commercial products not always available in most developing countries (34). Recently, molecular methods for rapid detection of drug resistance have appeared (6,30,31,35). However, their costs and the requirement for equipment and skilled personnel have precluded their routine implementation in countries with low resources.Colorimetric assays employing oxidation-reduction indicators for DST have been previously used with mycobacteria (11,21,38). A simple method employing Alamar blue for DST of M. tuberculosis was recently described (23). Resazurin, an oxidation-reduction indicator, has been used to assess viability and bacterial contamination and to test for antimicrobial activity (3,17,29). Since Alamar blue has been recently identified as resazurin in cell cytotoxicity studies (22), we have standardized and evaluated a microplate method which uses the reduction of resazurin for DST to INH and RIF in clinical isolates of M. tuberculosis from low-income countries. The results were compared to those of the proportion method (PM) on L-J medium.Eighty clinical isolates were studied: 65 from TB patients from a region of Peru with a high prevalence of MDR TB and 15 from the Tuberculosis Reference Laboratory at the Instituto Nacional de Laboratorios de Salud, La Paz, Bolivia. American Type Culture Collection (Manassas, Va.) reference strains were used as controls. INH and RIF (Sigma-Aldrich NV/SA, Bornem, Belgium) solutions were prepared at concentrations of 1 mg/ml in distilled water and 10 mg/ml in methanol, respectively, filter sterilized, and frozen until used. Resazurin sodium salt powder (Acros Organic N.V., Geel, Belgium) was prepared at 0.01% (wt/vol) in distilled water and filter sterilized; it can be store...
The emergence of multidrug-resistant tuberculosis calls for new, rapid drug susceptibility tests. We have tested 150 Mycobacterium tuberculosis isolates against the second-line drugs ethionamide, kanamycin, capreomycin, ofloxacin, and para-aminosalicylic acid by the colorimetric resazurin microtiter assay and the proportion method. By visual reading, MICs were obtained after 8 days. A very good correlation between results by the colorimetric resazurin microtiter assay and the proportion method was obtained. The colorimetric resazurin microtiter assay is inexpensive, rapid, and simple to perform, and implementation of the assay is feasible for low-resource countries.The global situation with respect to tuberculosis (TB) has worsened with the emergence of multidrug-resistant (MDR) TB worldwide (5, 36). MDR TB, defined by resistance to at least isoniazid and rifampin, is ubiquitous (12) and is most prevalent in some countries of Eastern Europe as well as China and India (14,47). Immigration from areas where TB is endemic, human immunodeficiency virus infection (2,16,23,28), homelessness, poor socioeconomic situations (6, 21), and prison populations (10,39,41) are among the responsible factors. Some patients with MDR TB do not respond to treatment with first-line drugs (isoniazid, rifampin, ethambutol, pyrazinamide, and streptomycin) (17). Several studies have shown that MDR TB can be cured by a combination of second-line drugs under DOTS-Plus, the treatment strategy proposed by the World Health Organization to address the management of MDR TB in settings with good control programs (15,18,22,30,35,48). However, these drugs are expensive, have to be taken for long periods, and can cause adverse reactions (48). Drug susceptibility testing (DST) with Löwenstein-Jensen (LJ) medium or Middlebrook agar requires 3 to 6 weeks to obtain results (7,8). Critical drug concentrations for second-line drugs have not been completely established. The development of new, rapid DST which is easy to use and inexpensive is thus an urgent priority for determining the susceptibility to secondline drugs (29,32). A rapid and inexpensive colorimetric method based on the oxidation-reduction indicators Alamar blue and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] have been successfully used for determining MICs of first-line drugs in DST of Mycobacterium tuberculosis (1,9,20,31,38,49). Since resazurin has been recently identified as the main component of Alamar blue (34, 43), we recently developed a resazurin microtiter assay (REMA) plate (37) for detecting MDR TB and demonstrated a very good correlation between results by this method and the proportion method (PM). In this study we evaluated the second-line drugs ethionamide (ETH), kanamycin monosulfate (KAN), capreomycin sulfate (CAP), ofloxacin (OFX), and para-aminosalicylic acid (PAS) with clinical isolates of M. tuberculosis by the colorimetric method using the REMA plate, and we compared the results with those of the PM.One hundred fifty clinical isolates from Bolivia, Peru...
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