1 Instituto de Medicina Tropical Pedro Kouri, La Habana, Cuba; 2 Mycobacteriology Unit, Institute of Tropical Medicine, Nationalestraat, 155, Antwerp, B-2000 Belgium
Received 17 February 2004; returned 27 April 2004; revised 4 May 2004; accepted 10 May 2004
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Abstract |
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Methods: A panel of 20 coded Mycobacterium tuberculosis strains was tested blindly by the low cost methods: nitrate reductase, MTT and resazurin assays, and compared with the results obtained with the gold standard methods: the proportion method on LöwensteinJensen medium and the BACTEC TB 460 system. We have also tested two commercial tests: MGIT and INNO LiPA Rif.TB kit.
Results: Complete agreement was observed among all methods.
Conclusion: These three simple methods might become inexpensive alternative procedures for rapid detection of rifampicin resistance in low-resource countries.
Keywords: M. tuberculosis , MTT , resazurin , nitrate reductase assay , rifampicin
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Introduction |
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The purpose of our study was to evaluate the performance of three rapid low cost methods for the detection of rifampicin resistance: resazurin, MTT and nitrate reductase assays. We compared the results with those obtained by the gold standard methods: the PM on LJ medium and the BACTEC 460 system, with a panel of 20 coded M. tuberculosis strains tested in a blind manner to avoid influencing the results of the different methods. In parallel, we have tested this same panel by the MGIT system and determined the rpoB mutations with the INNO-LiPA Rif.TB kit.
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Materials and methods |
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A panel of 20 clinical isolates of M. tuberculosis originating from Peru with known drug susceptibility patterns was studied. Ten were MDR and 10 were susceptible strains. Reference strains H37Rv (ATCC number 27294) and a rifampicin-resistant strain (ATCC number 35838), from the American Type Culture Collection, were used as the reference susceptible and resistant controls, respectively. All strains were freshly subcultured on LJ medium before use and tested in a blind manner for all different methods.
Antituberculous drug
Rifampicin was obtained as powder from SigmaAldrich (Bornem, Belgium). The stock solution was prepared in advance at a concentration of 10 g/L in methanol, filter sterilized and kept at 20°C for not more than 1 month.
Drug susceptibility testing
The PM was carried out on LJ medium according to the standard procedure with the recommended critical concentration of rifampicin 40 mg/L.4,5
BACTEC 460 system
The standard protocol was followed according to the manufacturer's instructions with a BACTEC 460 instrument (Becton Dickinson, Sparks, MD, USA) using a final rifampicin concentration of 2 mg/L. A loopful of M. tuberculosis from fresh LJ medium was transferred to a tube containing several glass beads. The turbidity of the bacterial suspension was adjusted to McFarland tube no. 0.5 in distilled water. To determine the 1% proportion of resistance, the inoculum used in the control vial was 100 less than that used for the drug containing vials. All vials were tested daily after inoculation and incubation at 37°C. The rate of increase in the growth index (GI) or the amount of change over that of the previous day, called GI, was compared for the control vial and the vial containing the rifampicin. If the daily
GI in the drug vial was greater than that in the control vial, the strain was considered to be resistant to rifampicin, and if the
GI was inferior, the strain was considered to be susceptible.
MGIT
The MGIT test was carried out as described by Palomino et al.9 Briefly, MGIT tubes were supplemented with 0.5 mL of oleic acid/albumin/dextrose/catalase (OADC), and rifampicin was added to obtain a final concentration of 1 mg/L. A growth control tube was prepared without antibiotic. The inoculum was adjusted to McFarland 0.5 and further diluted 1:5 with sterile saline. The tubes were inoculated with 0.5 mL of the inoculum diluted 1:5. A positive control tube was prepared by adding 5 mL of a 0.4% sodium sulphite solution to an empty MGIT tube and an uninoculated MGIT tube was used as a negative control. After 3 days of incubation at 37°C, the fluorescence of the MGIT tubes was read on a 365 nm UV transilluminator. The growth control tube was compared to the positive and negative controls. Positivity was indicated by bright orange fluorescence at the bottom of the tube and an orange reflection at the meniscus. Negative tubes showed very little or no fluorescence. When fluorescence appeared in the growth control tube, it was considered as day 0 for the interpretation of the drug-containing tube. A strain was considered as susceptible if the drug tube did not fluoresce within 2 days of the positivity of the growth control and as resistant if the drug tube was positive within 2 days of the positivity of the growth control.
INNO LiPA Rif.TB assay
The commercial INNO LiPA Rif.TB kit (Innogenetics, Ghent, Belgium) was used according to the instructions of the manufacturer. A full loop of bacteria was resuspended in 400 µL of TE buffer, heated at 95°C for 5 min, followed by a short centrifugation at 3000g. Then, 2 µL of the supernatant was transferred to the PCR amplification mixture. M. tuberculosis strain H37Rv was used as a control. The rpoB gene was amplified with specific biotin-labelled primers by using 1 U of Taq DNA polymerase per reaction mixture in a thermocycler. The amplification was checked by 2% agarose gel electrophoresis. The amplicon appeared as a single band with a length of 260 bp. The PCR product was then denatured and hybridized to a strip with 10 specific oligonucleotide probes. The reactivities of an amplified fragment with the S-type probes for the wild-type (probes S1 through S5) were used to detect the mutations that lead to rifampicin resistance in M. tuberculosis. Four probes (R-type probes) are specifically designed to hybridize to the sequences of the four most frequently observed mutations: R2 (Asp-516Val), R4a (His-526
Tyr), R4b (His-526
Asp) and R5 (Ser-531
Leu). When all the wild-type S probes gave a positive signal and all the R probes reacted negatively, the M. tuberculosis isolate was considered susceptible to rifampicin. When at least one negative signal was obtained with the wild-type S probes, the isolate was considered rifampicin-resistant (Inno-LiPA Rif.TB S patterns).
Colorimetric method reagents
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 was kept at 4°C in the dark. Formazan solubilization buffer was prepared by mixing 1:1 (v/v) 20% SDS and a solution of 50% N,N-dimethylformamide (DMF).
Resazurin microtitre assay
The resazurin microtitre assay (REMA) plate method was carried out as described by Palomino et al.15 Briefly, the inoculum was prepared from fresh LJ medium in 7H9-S medium, adjusted to a McFarland tube no. 1, then diluted 1:20 and 100 µL was used as an inoculum. One hundred microlitres of 7H9-S broth was dispensed in each well of the sterile 96-well plate (flat bottom; Becton Dickinson) and serial two-fold dilutions of rifampicin were prepared directly on the plate by adding 100 µL of the working solution of drug to achieve the final concentration. The rifampicin concentration range used was 0.0622.0 mg/L. Then, 100 µL of inoculum was added to each well. A growth control well containing no drug and a sterile control 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 plate was covered, sealed in a plastic bag 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 reincubated overnight. A change in colour from blue (oxidized state) to pink (reduced) indicated the growth of bacteria and the MIC was defined as the lowest concentration of drug that prevented this change in colour.
MTT assay
This method was carried out as described by Abate et al.12 The inoculum was prepared as described above for the REMA plate method and the rifampicin concentration range used was also the same (0.0622.0 mg/L). The procedure to prepare the 96-well plate was identical to the REMA plate assay. 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 reincubated overnight. If a violet precipitate (formazan) appeared in the MTT well, 50 µL of the SDS/DMF solution was added to these wells and the plate reincubated for 3 h. A change in colour from yellow to violet indicated the growth of bacteria and the MIC was interpreted as in the REMA plate assay.
Nitrate reduction 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 colour change. The nitrate reduction assay uses the detection of nitrite as an indication of growth when used as a drug susceptibility test. The rifampicin was included in the LJ medium at a concentration of 40 mg/L with 1000 mg/L potassium nitrate (KNO3). The inoculum turbidity was adjusted to a McFarland tube no. 1 and diluted 1:10 in PBS. The reagent mixture 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.20 For each strain, 200 µL of the undiluted suspension was inoculated into the antibiotic tube, and 200 µL of the 1:10 dilution into the drug-free tube. The tubes were incubated at 37°C. After 7 days, 500 µL of reagent mixture was added to one drug-free tube. If any colour occurred, all the tubes were developed by the reagent mixture, otherwise the tubes were reincubated and the procedure was repeated at day 10 and day 14. A strain was considered resistant if there was a colour change in the antibiotic tube greater than the 1:10-diluted growth control.
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Results |
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Discussion |
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Acknowledgements |
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Footnotes |
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References |
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