1 Section of Microbiology and Infectious Diseases, Kaohsiung Veterans General Hospital, 386 Ta-Chung 1st Rd, Kaohsiung; 2 Department of Medical Technology, Foo-Yin Institute of Technology, Kaohsiung; 3 Graduate Institute of Environmental Education, National Kaohsiung Normal University; 4 Department of Medicine, National Yang Ming University, Taipei, Taiwan, ROC
Received 10 September 2003; returned 29 October 2003; revised 15 December 2003; accepted 18 December 2003
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Abstract |
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Methods: We evaluated 46 clinical strains of M. tuberculosis isolated from patients admitted to Kaohsiung Veterans General Hospital. Testing of MICs of ciprofloxacin and ethionamide was carried out by MGIT 960 and compared with the agar dilution method.
Results: Good agreement was found between MGIT 960 and agar dilution. The greatest concordance between the agar dilution and MGIT assay at ±1 and ±2 dilution was 80.4% and 97.8% for ciprofloxacin, and 82.6% and 93.5% for ethionamide, respectively.
Conclusion: MGIT 960 was found to be comparable to the current NCCLS standard method, agar dilution, and has the advantage of being rapid (obtaining results within 517 days, average 8.9 days) and easy to achieve standardization.
Keywords: minimum inhibitory concentration, M. tuberculosis, susceptibility testing
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Introduction |
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Tentative standard methods for the testing of MICs for M. tuberculosis include agar dilution and BACTEC methods (Becton Dickinson, Microbiology Systems, Sparks, MD, USA). Protocols have been described for carrying out antimicrobial susceptibility testing by the conventional proportion method on solid media and by BACTEC.36 Both methods require technical expertise for the interpretation of results and the agar dilution method has the additional disadvantage of requiring 3 weeks of incubation.7,8 BACTEC, although rapid, involves the use of radioisotopes. Alternative methods have been reported to provide rapid indirect drug susceptibility test results, such as the microplate Alamar Blue colourimetric (MAB) method.911 The MAB method is cheap and rapid, however, it requires a greater level of technical expertise to provide reliable results. Furthermore, the microplates do not have a tight seal which may represent a biohazard unless handled carefully in the laboratory.
In contrast, the MGIT 960 system (Becton Dickinson) uses a fluorescence quenching based oxygen sensor that can be read automatically. The system is robust, safe, simple and has been validated for first-line drug susceptibility tests. Reliable results showing an excellent correlation with reference methods on M. tuberculosis complex have been reported.6,1217 The MGIT technique has been thoroughly evaluated with qualitative measurement against the first-line drugs isoniazid, rifampicin, ethambutol, streptomycin, pyrazinamide,18 and quantitative measurement against the second-line drug, kanamycin.19 The purpose of this study is to investigate the reliability of the MGIT 960 system for the determination of MICs of ciprofloxacin and ethionamide for M. tuberculosis complex isolates.
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Materials and methods |
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Forty-six isolates of M. tuberculosis complex, collected in the period 19942000 were randomly selected to include 23 (50%) strains susceptible to the four first-line drugs, 17 multiple drug-resistant TB (MDRTB), and six strains with any drug resistance. All isolates were identified by BD ProbeTec ET ctb identification test using standard procedures as recommended by the manufacturer (Becton Dickinson). Testing of MICs of antituberculous drugs was carried out by both the agar dilution and MGIT 960 method in parallel.
Antimicrobial agents
The stock solutions of each drug were prepared in accordance with manufacturers instructions and kept in aliquots at 20°C. From these stock solutions, working solutions were made in distilled water to be incorporated into the 7H11 media. Ethionamide was purchased from Sigma and ciprofloxacin was obtained from Bayer (Leverkusen, Germany).
MIC determination by the agar dilution method
The agar dilution method was carried out according to recommended standard procedures.4,20 Briefly, 7H11 agar medium (Becton Dickinson) was prepared from a dehydrated base as recommended by the manufacturer. Some multiple drug-resistant M. tuberculosis strains may not grow or grow poorly on 7H10, while growing sufficiently on 7H11 agar. After the agar was autoclaved, oleic acidalbumindextrosecatalase (OADC) supplement (Becton Dickinson) and drugs were added at 5056°C by doubling dilutions except for isoniazid which was five-fold diluted. Four millilitres of each concentration of antimycobacterial-containing medium was dispensed into plastic quadrant Petri dishes. As a growth control, one quadrant in each plate was filled with 7H11 agar medium with no drug. An inoculum of each isolate was prepared in Middlebrook 7H9 broth, and the absorbance was adjusted until it was equivalent to that of a McFarland No. 1 Standard. Final suspensions were carried out by adding Middlebrook 7H9 broth to prepare 102 and 104 dilutions of the standardized suspensions. Upon solidification of the medium, the plates received 0.1 mL on each quadrant of the agar plates. The inoculated plates were then incubated at 37°C for 3 weeks. The MICs of each isolatedrug pair were the lowest concentration of the antimycobacterial agents that inhibited >99% of the colonies growing on the drug-free control. M. tuberculosis H37Rv strain and an MDR strain (clinical isolate) were used as control strains.
MIC determination by the MGIT 960 method
Appropriate solutions of drugs were added in a volume of 0.1 mL to an MGIT (Mycobacteria Growth Indicator Tube) tube to achieve the desired final concentrations. The range of concentrations used for each drug is the same as in the agar dilution method. An inoculum was made from an initial MGIT broth culture after 12 days of showing positive by the instrument (Day 1 to Day 2) or diluted 1:5 for Day 3 to 5 positive tube. Half a millilitre of the inoculum was inoculated into each drug-containing tube that had been supplemented with 0.8 mL of OADC and antimicrobial. The drug-free control vial was inoculated with a 1:100 dilution of the inoculum to represent 1% of the bacterial population. The vials were loaded and the MIC was determined to be the lowest dilution that was negative by automated reading in drug-containing tubes when the control vial turned positive. The MIC was compared to the agar dilution method.
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Results and discussion |
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MGIT 960 was found to be equivalent to the current NCCLS standard method, agar dilution and had the advantage of being reasonably fast (giving results in 517 days). The test was technically easy to perform, and the results were easy to interpret. Inoculum preparation and susceptibility turnaround times were satisfactory. Determination of MIC based on growth unit (GU) values obtained using the MGIT 960 is more objective, precise and less labour intensive than the agar dilution method. The latter method is prone to inter-individual variation and is more technique-dependent. Although further studies with larger isolate collections are required to confirm the optimal breakpoint concentration and the interpretation criteria, the MGIT 960 system appears to be an appropriate technique for carrying out MIC tests in areas where MDRTB is endemic, and where such tests are increasingly required.
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Acknowledgements |
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Footnotes |
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References |
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