Resazurin Microtiter Assay Plate: Simple and Inexpensive Method for Detection of Drug Resistance in Mycobacterium tuberculosis

Abstract

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...

Manganese(II) complexes with thiosemicarbazones as potential anti-Mycobacterium tuberculosis agents

“…The anti-MTB activity of the compounds was determined by the REMA (Resazurin Microtiter Assay) method according to Palomino et al [47]. Stock solutions of the tested compounds were prepared in DMSO and diluted in Middlebrook 7H9 broth (Difco) supplemented with oleic acid, albumin, dextrose and catalase (OADC enrichment -BBL/Becton-Dickinson), to obtain final drug concentration ranges of 0.09-25 μg/mL.…”

Synthesis and SAR evaluation of novel thioridazine derivatives active against drug-resistant tuberculosis

“…The anti-M. tuberculosis activity of the compounds against M. tuberculosis H37Rv (ATTC27294), M. tuberculosis CF73 and two MDR-TB clinical isolates (CF104 and CF81) was determined using the Resazurin Microtiter Assay (REMA) method according to Palomino et al, [30]. Stock solutions of the tested compounds were prepared in dimethyl sulfoxide (DMSO), then diluted in Middlebrook 7H9 broth (Difco, Detroit, MI, USA) supplemented with oleic acid, albumin, dextrose and catalase (OADC enrichment) to obtain a final drug concentration range of 0.09-100 µg/mL.…”

Avaliação da atividade antimicobacteriana da lignana diidrocubebina extraída da Piper cubeba e de seus derivados semissintéticos

“…A atividade antimicobacteriana dos compostos 1-6 foi avaliada in vitro por meio da técnica de microdiluição em microplaca utilizando resazurina como indicador de atividade microbiana (REMA-Resazurin Microtiter Assay), (Palomino et al, 2002) …”

“…Embora existam inúmeros programas governamentais e estratégias para o controle da TB, a descoberta de novas drogas pode ser uma das alternativas para auxiliar no controle da doença (Raviglione, 2003;Okunade & Elvin-Lewis, 2004;Koul et al, 2011;Riccardi & Pasca, 2014).…”

“…Em nossos estudos anteriores sobre as lignano lactonas e lactóis várias propriedades biológicas foram relatadas (Saraiva et al, 2007;2009, Silva et al, 2009, Silva et al, 2005Silva et al, 2008;Souza et al, 2005;Silva et al, 2011;Pistolozzi et al, 2013), incluindo a atividade antimicobacteriana (Silva et al, 2009 O extrato etanólico (EE) foi suspenso em solução hidrometanólica (9:1), para cada 20 g de EE foram utilizados aproximadamente 100 mL de solução hidrometanólica. Esta solução foi submetida a partição com hexano (7 x 250 mL), e em seguida separada e concentrada.…”