1 Laboratorio Nacional de Referencia e Investigaciones en Tuberculosis y Mycobacteria, Instituto de Medicina Tropical Pedro Kouri, Havana, Cuba; 2 Mycobacteriology Unit, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium
Received 6 September 2004; returned 5 November 2004; revised 26 November 2004; accepted 15 December 2004
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Abstract |
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Methods: One hundred M. tuberculosis clinical isolates were tested by the nitrate reductase assay (NRA), the MTT test and the resazurin microtitre assay (REMA), and the results compared with those obtained with the gold standard proportion method (PM) on Löwenstein Jensen medium.
Results: The results using the three methods showed a good sensitivity and specificity between 94% and 100% for the detection of rifampicin and isoniazid resistance. Specificity for ethambutol and streptomycin using MTT and resazurin was low (5889%). In contrast, NRA showed a good agreement for all first-line drugs tested.
Conclusions: This study shows a high level of agreement of these three low-cost methods compared with the PM for rapid detection of rifampicin and isoniazid resistance. However, more standardization is needed for ethambutol and streptomycin using the MTT test and resazurin microtitre assay. The nitrate reductase assay might represent an inexpensive procedure for rapid detection of resistance to first-line drugs in low-resource countries.
Keywords: antibiotic resistance , colorimetric , rapid methods
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Introduction |
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In the last few years, several methods have been proposed for the rapid detection of drug resistance and for DST of M. tuberculosis.58 Among them, the Mycobacterial Growth Indicator Tube (MGIT, Becton Dickinson), and molecular tools such as the INNO-LiPA Rif.TB (Line probe assay, Innogenetics, Ghent, Belgium) have extensively been applied. However, they are expensive and impractical for routine use. Ängeby et al. have described a new nitrate reductase assay (NRA) for rapid and inexpensive DST of M. tuberculosis.9 The method depends on the ability of M. tuberculosis to reduce nitrate to nitrite which can be detected using specific reagents producing a change in colour. Abate and colleagues have also proposed a rapid colorimetric method based on the reduction of the MTT compound for detection of resistance to rifampicin.10,11 More recently, Morcillo et al. have used this same compound for determining the susceptibility of MDR M. tuberculosis strains to several second-line anti-TB drugs in a microplate indicator-based method.12
We have been involved in the development of a rapid microplate method, the Resazurin Microtitre Assay (REMA) plate for the detection of MDR strains of M. tuberculosis.13 The method was successfully tested on clinical isolates against isoniazid and rifampicin with results obtained after 7 days. More recently, Martin et al. have applied this same method for DST of clinical isolates of M. tuberculosis against second-line anti-TB drugs with very good results.14
In this study, we have performed for the first time a comparative evaluation of the nitrate reductase assay, the MTT reduction test and the REMA plate for DST of 100 clinical isolates of M. tuberculosis to first-line drugs. The results obtained were compared with those of the PM on LJ medium.
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Materials and methods |
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One hundred clinical isolates of M. tuberculosis from the collection of the reference laboratory of the Instituto de Medicina Tropical Pedro Kouri, Havana, Cuba, were evaluated. Reference strains H37Rv (ATCC 27294), and rifampicin-resistant (ATCC 35838), isoniazid-resistant (ATCC 35822), ethambutol-resistant (ATCC 35837) and streptomycin-resistant (ATCC 35820) strains from the American Type Culture Collection, were used as susceptible and resistant controls. All strains were freshly sub-cultured on LJ medium before being used.
Anti-tuberculous drugs
Rifampicin, isoniazid, ethambutol and streptomycin were obtained as powder from SigmaAldrich (Bornem, Belgium). The stock solutions were prepared in advance at a concentration of 10 g/L in methanol for rifampicin, and 1 g/L in distilled water for the other drugs. Stock solutions were filter sterilized and kept at 20 °C for not more than 1 month. Working solutions of each drug were prepared at four-fold the highest concentration tested on the plates.
Drug susceptibility testing
The PM was carried out on LJ medium according to the standard procedures with the recommended critical concentrations of 40 mg/L for rifampicin, 0.2 mg/L for isoniazid, 2 mg/L for ethambutol and 4 mg/L for streptomycin.15
Nitrate reductase assay (NRA)
This method is based on the ability of M. tuberculosis to reduce nitrate to nitrite, which is routinely used for biochemical identification of mycobacterial species. The presence of nitrite can easily be detected with specific reagents, which produce a change in colour. The nitrate reductase assay uses the detection of nitrite as an indication of growth when used as a drug susceptibility test. The antibiotic was included in the LJ medium at a concentration of 40 mg/L for rifampicin, 0.2 mg/L for isoniazid, 2 mg/L for ethambutol and 4 mg/L for streptomycin; 1000 mg/L of potassium nitrate (KNO3) was also added. The inoculum was adjusted to a turbidity equivalent to that of a no. 1 McFarland Standard, and diluted 1:10 in phosphate buffered saline (PBS). The reagent mix consisted of 1 part 50% concentrated hydrochloric acid (HCl), 2 parts 0.2% sulfanilamide, and 2 parts 0.1% N-1-naphthylethylenediamine dihydrochloride. The method was carried out as described by Ängeby et al.9 For each strain, 200 µL of the undiluted inoculum was added to the antibiotic tube, and 200 µL of the 1:10 dilution to the drug-free tube as a growth control. The tubes were incubated at 37 °C. After 7 days, 500 µL of the reagent mixture was added to one drug-free tube. If any colour change appeared, all the tubes were developed with the reagent mixture; otherwise, the tubes were re-incubated and the procedure repeated at day 10 and day 14. An isolate was considered resistant if colour developed in the test tube (pink to red or purple) and this colour was greater than that appearing in the 1:10-diluted growth control.
Reagents for colorimetric methods
A stock solution of resazurin sodium salt powder (Acros Organic N.V., Geel, Belgium) was prepared at 0.01% in distilled water, filter sterilized and kept at 4 °C.
A stock solution of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (SigmaAldrich, Belgium) at a concentration of 5 g/L was prepared in PBS, pH 6.8, and kept at 4 °C in the dark. Formazan solubilization buffer was prepared by mixing 1:1 (v/v) 20% sodium dodecyl sulphate (SDS) and a solution of 50% N,N-dimethylformamide (DMF).
REMA
The REMA plate method was carried out as described by Palomino et al.13 Briefly, the inoculum was prepared from a fresh LJ medium in 7H9-S broth, adjusted to a McFarland tube No.1 and diluted 1:20; 100 µL was used as the inoculum. One hundred microlitre volumes of 7H9-S broth were dispensed in each well of a sterile 96-well flat bottom plate (Becton Dickinson) and serial two-fold dilutions of each drug were prepared directly on the plate by adding 100 µL of the working solution of each drug to achieve the final concentrations. The range of concentrations tested were 0.0622.0 mg/L for rifampicin, 0.0311.0 mg/L for isoniazid, 132 mg/L for ethambutol, and 0.258 mg/L for streptomycin. Then, 100 µL of the inoculum was added to each well. A growth control containing no antibiotic and a sterile control without inoculum were also included for each isolate. Two hundred microlitres of sterile water was added to all perimeter wells to avoid evaporation during the incubation. The plates were covered and replaced in their original plastic bags and incubated at 37 °C in a normal atmosphere. After 7 days of incubation, 30 µL of resazurin solution was added to each well and the plate was re-incubated overnight. A change in colour from blue (oxidized state) to pink (reduced) indicated the growth of bacteria and the minimal inhibitory concentration (MIC) was defined as the lowest concentration of each drug that prevented this change in colour.
MTT assay
This method was carried out as described by Abate et al.10 The inoculum was prepared as described above for the REMA plate method and the drug concentration ranges used were the same. Preparation of the 96-well plates was identical as described for the REMA plate. After 7 days of incubation at 37 °C, 10 µL of the MTT solution (5 g/L) was added to each well and the plate was re-incubated overnight. If a violet precipitate (formazan) appeared, 50 µL of the SDS/DMF solution was added to the wells and the plate re-incubated for 3 h. A change in colour from yellow to violet indicated growth of bacteria and the MIC was interpreted as in the REMA plate.
Data analysis
Analysis of data was carried out with MedCalc software (MedCalc, Belgium). Receiver operating characteristic (ROC) curve analysis was also carried out with MedCalc.
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Results |
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Discussion |
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For rifampicin and isoniazid, both methods showed 100% sensitivity and more than 96% specificity. These results are very important since rifampicin and isoniazid are the two most important drugs used in the treatment of TB. Results for ethambutol and streptomycin, two drugs known to be difficult to test, showed a sensitivity higher than 92% but the specificity was low (5889%). On the other hand, using the NRA, very good sensitivity and specificity (94100%) were found for all drugs. Thus, solid medium seems to give more reliable results for ethambutol and streptomycin. This could be one possible explanation for the high number of discordant results obtained with ethambutol and streptomycin where the antibiotics might have deteriorated faster in the liquid medium, especially in the case of ethambutol. More studies are needed, however, to assess the discordant results obtained with streptomycin.
In summary, this study shows that using these three low cost methods for rapid detection of rifampicin and isoniazid resistance, a high level of agreement with the PM was obtained. These new alternative methods seem to have the potential to provide rapid detection of resistance to isoniazid and rifampicin, do not need any sophisticated equipment, are simple to perform, reduce the time to report first results compared to classical conventional methods and could be implemented in laboratories with limited resources. NRA seems to be an inexpensive alternative method for rapid and accurate detection of resistance to all four first-line drugs and has the advantage of being performed on the classical LJ media. MTT and REMA need more standardization for ethambutol and streptomycin and due to their performance in liquid media, MTT and resazurin assays have the disadvantage from the point of view of biosafety that manipulation of plates could generate aerosols. A clinical trial of these new rapid methods is warranted to analyse the possibility of implementing them in low-resource countries.
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Acknowledgements |
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References |
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2 . World Health Organization. (2004). Anti-tuberculosis Drug Resistance in the World: Report No. 3. Prevalence and Trends. The WHO/IUATLD Global Project on Anti-tuberculosis Drug Resistance Surveillance. WHO/CDS/TB/2004. WHO, Geneva, Switzerland, in press.
3 . Kent, P. T. & Kubica, G. P. (1985). Public health mycobacteriology. A guide for the level III laboratory. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Atlanta, GA, USA.
4 . Roberts, G. D., Goodman, N. L., Heifets, L. et al. (1983). Evaluation of the BACTEC radiometric method for recovery of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis from acid-fast smear-positive specimens. Journal of Clinical Microbiology 18, 68996.[ISI][Medline]
5 . McNerney, R., Kiepiela, P., Bishop, K. S. et al. (2000). Rapid screening of Mycobacterium tuberculosis for susceptibility to rifampicin and streptomycin. International Journal of Tuberculosis and Lung Disease 4, 6975.[ISI][Medline]
6 . Rossau, R., Traore, H., De Beenhouwer, H. et al. (1997). Evaluation of the INNO-LiPA Rif. TB assay, a reverse hybridization assay for the simultaneous detection of Mycobacterium tuberculosis complex and its resistance to rifampin. Antimicrobial Agents and Chemotherapy 41, 20938.[Abstract]
7 . Palomino, J. C., Traore, H., Fissette, K. et al. (1999). Evaluation of Mycobacteria Growth Indicator Tube (MGIT) for drug susceptibility testing of Mycobacterium tuberculosis. International Journal of Tuberculosis and Lung Disease 3, 3448.[ISI][Medline]
8 . Foongladda, S., Roengsanthia, D., Arjrattanakool, W. et al. (2002). Rapid and simple MTT method for rifampicin and isoniazid susceptibility testing of Mycobacterium tuberculosis. International Journal of Tuberculosis and Lung Disease 6, 111822.[ISI][Medline]
9
.
Ängeby, K. A., Klintz, L. & Hoffner, S. E. (2002). Rapid and inexpensive drug susceptibility testing of Mycobacterium tuberculosis with a nitrate reductase assay. Journal of Clinical Microbiology 40, 5535.
10 . Abate, G., Mshana, R. N. & Miorner, H. (1998). Evaluation of a colorimetric assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) for rapid detection of rifampicin resistance in Mycobacterium tuberculosis. International Journal of Tuberculosis and Lung Disease 2, 10116.[ISI][Medline]
11
.
Mshana, R. N., Tadesse, G., Abate, G. et al. (1998). Use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide for rapid detection of rifampin-resistant Mycobacterium tuberculosis. Journal of Clinical Microbiology 36, 12149.
12 . Morcillo, N., Di Giulio, B., Testani, B. et al. (2004). A microplate indicator-based method for determining drug-susceptibility of multidrug-resistant Mycobacterium tuberculosis to antimicrobial agents. International Journal of Tuberculosis and Lung Disease 8, 2539.[ISI][Medline]
13
.
Palomino, J. C., Martin, A., Camacho, M. et al. (2002). Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 46, 27202.
14
.
Martin, A., Camacho, M., Portaels, F. et al. (2003). Resazurin microtiter assay plate testing of Mycobacterium tuberculosis susceptibilities to second-line drugs: rapid, simple, and inexpensive method. Antimicrobial Agents and Chemotherapy 47, 36169.
15 . Canetti, G., Froman, F., Grosset, J. et al. (1963). Mycobacteria: laboratory methods for testing drug sensitivity and resistance. Bulletin of the World Health Organization 29, 56578.[ISI][Medline]
16 . Palomino, J. C. (2000). Novel rapid antimicrobial susceptibility tests for Mycobacterium tuberculosis. In Multidrug-resistant Tuberculosis (Bastian, I. & Portaels, F. Eds), pp. 10012. Kluwer Academic Publishers, Dordrecht.
17 . Traore, H., Fissette, K., Bastian, I. et al. (2000). Detection of rifampicin resistance in Mycobacterium tuberculosis isolates from diverse countries by a commercial line probe assay as an initial indicator of multidrug resistance. International Journal of Tuberculosis and Lung Disease 4, 4814.[ISI][Medline]
18
.
Caviedes, L., Delgado, J. & Gilman, R. H. (2002). Tetrazolium microplate assay as a rapid and inexpensive colorimetric method for determination of antibiotic susceptibility of Mycobacterium tuberculosis. Journal of Clinical Microbiology 40, 18734.
19
.
Banfi, E., Scialino, G. & Monti-Bragadin, C. (2003). Development of a microdilution method to evaluate Mycobacterium tuberculosis drug susceptibility. Journal of Antimicrobial Chemotherapy 52, 796800.
20
.
Lemus, D., Martin, A., Montoro, E. et al. (2004). Rapid alternative methods for detection of rifampicin resistance in Mycobacterium tuberculosis. Journal of Antimicrobial Chemotherapy 54, 1303.