Resazurin Microtiter Assay Plate Testing of Mycobacterium tuberculosis Susceptibilities to Second-Line Drugs: Rapid, Simple, and Inexpensive Method

Abstract

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...

Evaluation of rapid MTT tube method for detection of drug susceptibility of <i> mycobacterium tuberculosis</i> to rifampicin and isoniazid

“…[7,11,12,14] However, since M. tuberculosis is a conventional pathogen and many of the isolates could also be MDR, performing the test in microtitre plates could pose a grave laboratory hazard because of the generation of aerosols. Abate et al in 1998 and Foongladda et al, in 2002 had therefore used screw cap test tubes for their experiments.…”

“…The working concentrations were prepared subsequently by diluting with 7H9 broth supplemented with OADC to achieve the concentrations of 2 μg/mL and 0.4 μg/mL respectively for RIF and INH. [12] …”

“…[4] The reduction of the dye by the bacteria when incubated in the presence of the drug indicates that the bacteria are viable which in turn indicates that the isolate is resistant to the given drug and vice versa. This principle has been utilized by many authors [5][6][7][8][9][10][11][12][13][14][15] to assess the susceptibility of the mycobacterial cells in the presence of anti-TB drugs, as well as for susceptibility testing of certain fungi and bacteria. This kind of study has not been reported so far from India where it is indeed necessary as India has a large population of MDR strains.…”

“…This kind of study has not been reported so far from India where it is indeed necessary as India has a large population of MDR strains. [16] In some of the previous studies, [7,11,12,14] this assay was performed in the microtitre plates posing risk to the laboratory personnel as the open wells of the plate used lead to easy spillage and generation of aerosols. [8,9] The present study has performed the assay in tubes which not only decreased the hazard but is most appropriate for routine use in the simpler laboratory settings.…”

Evaluación de métodos fenotípicos y genotípicos para la detección de farmacorresistencia de Mycobacterium tuberculosis.

“…Para detectar esta susceptibilidad de M. tuberculosis a los medicamentos se emplean microplacas con medio líquido, y de acuerdo con el indicador utilizado se conocen estas pruebas como Rema (resazurin microtiter assay) (11,12) y Maba (microdilution alamar blue assay) (13,14). Otro colorante usado es la sal de tetrazolio MTT (bromuro de 3(4,5-dimetiltizol-2-i)-2,5-difenil tetrazolio) útil en ensayos de proliferación y citotoxicidad de células eucariotas (15,16).…”