Rapid detection of multidrug-resistant Mycobacterium tuberculosis in Cotonou (Benin) using two low-cost colorimetric methods: resazurin and nitrate reductase assays

Abstract

We have evaluated two simple, rapid and low-cost colorimetric methods for the detection of multidrug-resistant Mycobacterium tuberculosis. A total of 151 M. tuberculosis strains were tested for resistance to rifampicin (RMP) and isoniazid by resazurin microplate assay (REMA) and nitrate reductase assay (NRA) in comparison with the conventional proportion method (PM) on Lö wenstein-Jensen medium. A complete agreement was found between NRA and PM, while one false RMP-susceptible result was found by REMA. REMA and NRA tests are rapid and inexpensive, and could be good alternatives to the conventional PM in low-resource countries. INTRODUCTIONTuberculosis (TB) is still a major public-health problem all over the world, particularly in developing countries. According to the latest World Health Organization (WHO) report in 2005, there were 8.8 million new TB cases and 1.6 million deaths were attributed to the disease worldwide (WHO, 2007). The situation becomes more complicated due to the rising human immunodeficiency virus/AIDS pandemic, the emergence of multidrug-resistant (MDR) TB and the recently described extensively drugresistant TB (WHO, 2004;Aziz et al., 2006;Shah et al., 2007).Current standard methods for the detection of MDR Mycobacterium tuberculosis include the proportion method (PM) performed on Löwenstein-Jensen (LJ) medium or agar, absolute concentration and resistance ratio methods (Canetti et al., 1963(Canetti et al., , 1969Kent & Kubica, 1985) and the radiometric method in the BACTEC-460 system (Roberts et al., 1983). However, these methods either are lengthy or produce radioactive waste that is difficult to manage in low-resource countries.Commercial methods, such as the mycobacteria growth indicator tube (MGIT) and molecular methods, have been introduced (Rossau et al., 1997;Palomino et al., 1999); however, though rapid, these methods are expensive and could not be easily implemented in developing countries. Recently, several rapid and inexpensive tests to detect drug resistance in M. tuberculosis have been developed (Wilson et al., 1997;Abate et al., 2004;Caviedes et al., 2000;Angeby et al., 2002;Palomino et al., 2002); unfortunately, such tests have been evaluated only in a few low-resource countries (Nateche et al., 2006;Rivoire et al., 2007) but not so far in a TB reference laboratory in West Africa.In this study we have evaluated two rapid and low-cost colorimetric tests, the nitrate reductase assay (NRA) and the resazurin microplate assay (REMA), for the detection of resistance to rifampicin (RMP) and isoniazid (INH) in strains of M. tuberculosis in a TB reference laboratory in Cotonou, Benin. The results were compared to those obtained by the standard PM performed on LJ medium. METHODSSetting. The study was performed in the National Reference Mycobacteriology Laboratory (Laboratoire de Référence des Mycobactéries) in Cotonou, Benin, a West African country. External quality control of the laboratory is performed by the Supranational Mycobacteriology Laboratory of the Institute of Tropical Medi...

Detection of a streptomycin-resistant Mycobacterium bovis strain through antitubercular drug susceptibility testing of Tunisian Mycobacterium tuberculosis complex isolates from cattle

“…Among these, REMA, an indirect method based on the reduction of the coloured dye resazurin added to liquid culture medium on a microtitre plate after exposure of mycobacterial strains to antituberculosis drugs in vitro, was successfully tested on human isolates of M. tuberculosis [ 35 , 36 , 39 , 40 ]. The REMA plate method proposed by Palomino and colleagues [ 35 ] has recently been simplified by Marianelli and colleagues by testing only two concentrations per drug per isolate, the R and S cut-off values, the d-REMA assay [ 27 ].…”

Comparative evaluation of Anyplex II MTB/MDR/XDR and resazurin microtiter assay for detection of drug resistant Mycobacterium tuberculosis

“…Also, REMA has the benefit of simply determining the MIC; MIC results in this study were in accordance with Nateche et al (Nateche et al, 2006) who reported that the MIC by REMA among INH resistant isolates (n =17) was >1 µg/ml, while among RIF resistant isolates (n =12) MIC was >2 µg/ml (10/17), 1 µg/ml (1/17) and 0.5µg/ml (1/17). Similarly, Affolabi et al (2008) reported that 82.6% (19/23) of the isolates resistant to INH had MIC ≥1 µg/ml and 93.9% (14/15) of the isolates resistant to RIF had MIC ≥ two µg/ml. In Egypt, the MICs of OFX and KAN reported by Khalifa et al (2013) were quite different than that in our study.…”

Resazurin assay for rapid drug susceptibility testing of Mycobacterium tuberculosis

“…Similarly 65 (97.01%) and 67(100%) of the results were available on 7 th and 10 th day respectively for Rifamycin. Affolabi D et al 14 observed that 100% results by 8 th day. Franzblau SG et al 7 has also reported 100% results on 8 th day.…”