Evaluation of MIDITECH automated colorimetric MIC reading for antimicrobial susceptibility testing

Rainer Gattringera, Milan Niksb, Richard Ostertágc, Konstantin Schwarza, Hrvoje Medvedovica, Wolfgang Graningera and Apostolos Georgopoulosa,*

a Department of Internal Medicine I, Division of Infectious Diseases and Chemotherapy, University of Vienna, Austria; b Institute of Microbiology, School of Medicine, Comenius University, Bratislava; c Department of Computer Science, Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovak Republic


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The MIDITECH colorimetric susceptibility test with automated reading is a modification of the standard broth microdilution method that uses a 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye for detecting viable bacteria. The method can be applied to non-fastidious aerobic Gram-negative bacteria, staphylococci and Enterococcus faecalis. To assess the reliability of this method, we compared susceptibility data obtained by this test with standard NCCLS microdilution assay results. For this purpose, 15 antibiotics and a well characterized set of 527 Gram-negative and Gram-positive bacterial isolates collected and stored at the Division of Infectious Diseases and Chemotherapy (Vienna General Hospital, Austria), yielding 5751 organism–antibiotic combinations, were analysed in duplicate. The overall essential agreement (±1 log2 dilution) between the MIDITECH and NCCLS methods was 96.18 ± 0.67%. The colorimetric assay compared with the reference method produced MICs 2 log2 dilutions and 2 log2 dilutions in 2.34% and 1.48% comparisons, respectively. For 326 Gram-negative bacteria, the absolute interpretative agreement of both methods ranged from 87.12% for ampicillin–sulbactam to 99.85% for meropenem (mean 94.86%); 417 (4.92%) minor, three (0.05%) major and 15 (0.63%) very major errors were found. For 127 staphylococci and 74 E. faecalis isolates, the absolute interpretative agreement ranged from 90.80% for ciprofloxacin to 100% for vancomycin and linezolid (mean 96.96%); 81 (2.77%) minor, three (0.15%) major and eight (0.83%) very major errors were found. For most of the clinically important aerobically growing pathogens, the MIDITECH colorimetric test provided reliable quantitative susceptibility data. The main advantage of this method is simple performance, automated reading and data processing without expensive investments.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Quantitative antibiotic susceptibility testing would be very useful to clinical microbiology laboratories because pharmacokinetic/pharmacodynamic surrogate parameters can be correctly calculated using MIC values.1 An accepted and frequently used method for MIC testing is the broth microdilution method described by the National Committee for Clinical Laboratory Standards (NCCLS).2 This method is convenient to use, can be standardized and can be prepared ‘in house’ or purchased from commercial manufacturers. However, automation of this method requires sensitive optical devices. Since a low optical signal is generated by growth of some bacteria, nephelometers, ELISA readers and/or sophisticated image-processing software are needed for correct automated reading of broth microdilution trays.3 Incorporation of a fluorogenic oxidation– reduction substrate to the culture medium has recently been used successfully to increase the growth signal in the NCCLS susceptibility microdilution method.4,5 However, automated reading of this test requires the use of a fluorometer, as for other tests that use fluorescent substrates.6 In addition to fluorescent dyes, other oxidation–reduction compounds yielding coloured end-products have been proposed for susceptibility testing.7,8 Of these, 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) produces the highest optical signal in the visible spectrum. This tetrazolium derivative was introduced in 1983 by Mosmann9 for determination of cell growth and viability. Recently, this principle has also been used successfully for susceptibility testing of yeasts and filamentous fungi.10,11

In this study, we assessed the reliability of results obtained by a commercial modification of the NCCLS broth microdilution method with automated reading based on MTT tetrazolium colour growth indicator (Bel-MIDITECH, Bratislava, Slovak Republic), compared with the standard NCCLS microdilution assay results. For this purpose, a well characterized set of 527 Gram-negative and Gram-positive bacterial strains (Vienna testing set) collected and stored at the Division of Infectious Diseases and Chemotherapy (Vienna General Hospital, Austria) was used.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates and control strains

A total of 527 bacterial clinical isolates, prospectively and consecutively collected during a period of 3 years (1998–2000), was tested. The following Gram-positive and Gram-negative bacterial isolates were included: 40 methicillin-susceptible Staphylococcus aureus, 54 methicillin-resistant S. aureus, 33 Staphylococcus epidermidis, 74 Enterococcus faecalis, 41 Acinetobacter strains (A. baumanii, A. lwoffii, A. calcoaceticus), 26 Enterobacter aerogenes, 30 Enterobacter cloacae, 29 Citrobacter freundii, 48 Escherichia coli, 48 Klebsiella pneumoniae, 24 Klebsiella oxytoca, 30 Pseudomonas aeruginosa, 25 Proteus vulgaris and 25 Stenotrophomonas maltophilia. The bacteria were identified using API 20 E, API 20 NE, API Staph and API 20 Strep identification strips (bioMérieux, Marcy l’Étoile, France) and stored in a liquid-nitrogen freezer. The control strains used in this study were E. coli ATCC 25922 and ATCC 35218, P. aeruginosa ATCC 27853, S. aureus ATCC 29213 and E. faecalis ATCC 29212.

Antimicrobial agents

Standard antimicrobial powders obtained from various manufacturers were used. MIDITECH panels (see the assay description) were supplied dehydrated, with separate frozen indicator solution. The antimicrobial agents were tested with the following serial two-fold dilutions: ampicillin (Gram-negative 64–0.5 mg/L, Gram-positive 32–0.25 mg/L), ampicillin–sulbactam (Gram-negative 64/32– 0.5/0.25 mg/L, Gram-positive 32/16–0.25/0.125 mg/L), co-amoxiclav (Gram-negative 64/32–0.5/0.25 mg/L, Gram-positive 32/16–0.25/0.125 mg/L), cefuroxime (Gramnegative 64–0.5 mg/L, Gram-positive 32–0.25 mg/L), cefotaxime (64–0.5 mg/L), ceftazidime (64–0.5 mg/L), cefepime (32–0.25 mg/L), ciprofloxacin (4–0.125 mg/L), gentamicin (32–0.25 mg/L; 1000–8 mg/L when testing E. faecalis), imipenem (32–0.25 mg/L), linezolid (8–0.25 mg/L), meropenem (32–0.25 mg/L ), netilmicin (32–0.25 mg/L), oxacillin (4–0.125 mg/L; final NaCl concentration, 2% v/w), piperacillin (128–1 mg/L), trimethoprim–sulfamethoxazole (4/76–0.03/0.59 mg/L) and vancomycin (32–0.25 mg/L).

Inoculum preparation

Bacterial isolates were removed from storage, streaked on to Columbia agar plates supplemented with 5% sheep blood (bioMérieux) and incubated for 18–24 h at 35°C in ambient air. A working bacterial suspension was prepared by suspending 3–5 isolated colonies in 3 mL of Mueller– Hinton broth. The turbidity of this suspension was carefully adjusted photometrically to equal that of a 0.5 McFarland standard. For the test, the final inoculum was further diluted in Mueller–Hinton broth to achieve a final concentration of c. 5 x 105 cfu/mL.

NCCLS broth microdilution

The MICs were determined by a standardized broth microdilution method using cation-supplemented Mueller– Hinton broth according to NCCLS guidelines.2 The inoculated microdilution plates were incubated for 16–20 h at 35°C (24 h for oxacillin and vancomycin with staphylococci and enterococci, respectively) in ambient air. For the reading of microtitration plates, a magnifying mirror was used. Standard quality control ATCC strains with known MICs were included in each run.

MIDITECH MTT assay

The MIDITECH assay is a modification of the NCCLS broth microdilution method intended for routine testing of aerobic Gram-negative non-fastidious bacteria, staphylococci and E. faecalis.12 Instead of a traditional microtitration tray as described in the NCCLS method, this technique uses a plastic panel with multiple non-transparent nylon microchambers. The microchambers contain antibiotics and the panels are provided dehydrated. To each test microchamber was added 22 µL of bacterial suspension, prepared in Mueller–Hinton broth (5 x 105 cfu/mL). The panel was sealed in a plastic bag and incubated in an inverted position for the same time as recommended for the NCCLS method. At the end of the incubation time, 4 µL of MTT indicator solution was added to each microchamber and the panel was incubated for another 30 min (60 min for E. faecalis). The reactions were read visually (yellow colour = no growth, purple colour = growth), but a table scanner (610S; UMAX, Hsinchu, Taiwan) is routinely used. The panel was digitally analysed by the MIDITECH system software. The growth intensity in each chamber was compared with negative and positive controls. The collected data can be further processed by MIDITECH's interpretative reading module and a pharmacokinetic/pharmacodynamic module. In this study, only raw data produced by the reading module (MIDITECH Analyser, version 2/96) were compared with those obtained by the visual reading of the NCCLS standard broth microdilution method.

Statistical analysis

All tests were carried out in duplicate. For comparison and statistical analysis, each of these tests was considered as an individual observation; therefore, two observations were obtained for each combination of isolate and antibiotic agent.

Comparison of interpretative results (susceptible, intermediate and resistant breakpoints according to NCCLS criteria) was done by calculating minor, major, very major and essential errors and their rates according to Murray et al.13 Percentages of individual errors were calculated as follows. Minor errors: percentage of isolates interpreted by the tested method as resistant or susceptible when the isolate was categorized as intermediate by standard test, together with isolates recognized as intermediate by the tested method and recorded as resistant or susceptible by the standard test, calculated with the sum of all tested isolates as the denominator. Major errors: percentage of susceptible isolates falsely determined by the tested method as resistant, calculated with number of susceptible isolates as the denominator. Very major errors: percentage of resistant isolates falsely determined by the tested method as susceptible, calculated with the number of resistant isolates as the denominator. Essential errors: percentage of isolates with major and very major errors, calculated with the sum of susceptible and resistant isolates as the denominator. Percentage of absolute agreement was calculated as the sum of isolates with complete interpretative agreement, using the sum of all tested isolates as the denominator. Percentage of essential agreement was calculated for all isolates except those with major and very major errors, using the sum of all tested isolates as the denominator.

The degree of agreement between the MIDITECH assay and the NCCLS microdilution test was defined as the proportion of MICs determined by the MIDITECH test that fell within NCCLS results ±1 log2 dilutions (accuracy limits of the test) for each reading. MICs possibly smaller than the lowest tested concentration were all considered equal to this concentration. Similarly, MICs greater than the highest tested concentration were all considered equal to the next highest concentration. Significance of possible differences between both methods has been evaluated by the Wilcoxon signed rank directional test on the log2 results obtained by compared tests.14 The sign of the W value reflects the orientation of shifts of the MTT assay towards lower (-) or higher (+) values.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to compare the new MIDITECH tetrazolium broth microdilution MIC method with automated colorimetric reading with the NCCLS standard broth microdilution test. A total of 15 antimicrobial agents for defined 326 Gram-negative and 201 Gram-positive bacterial isolates (yielding 5751 organism–antibiotic combinations) were analysed in duplicate. The overall essential agreement (± 1 log2 dilution) between the two methods was 96.18 ± 0.67 %. The MIDITECH assay compared with the reference NCCLS method produced MICs >=2 log2 dilutions and >=2 log2 dilutions in 2.34% and 1.48% comparisons, respectively.

Gram-negative bacteria

A total of 326 Gram-negative bacteria representing 10 clinically common species were tested for susceptibility to 13 antibiotics. The results are shown in Tables 1 and 2GoGo. Table 1Go shows the numbers of susceptible, intermediate and resistant isolates tested, together with the distribution of interpretative errors of the MIDITECH assay. The absolute interpretative agreement for both methods ranged from 87.12% for ampicillin–sulbactam to 99.85% for meropenem. A total of 417 (4.92%) minor, three (0.05%) major and 15 (0.63%) very major errors were observed. Minor errors were highest for ampicillin–sulbactam, co-amoxiclav, cefotaxime and cefuroxime. The highest number of essential errors (major and very major) was seen with trimethoprim–sulfamethoxazole (1.23%) and with ampicillin– sulbactam (0.86%). No essential errors were observed for ceftazidime, cefepime and gentamicin, and there were relatively few minor errors for ciprofloxacin. The best agreement was demonstrated for both carbapenems.


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Table 1. Results for 326 strains of Gram-negative bacteria by interpretative categories for broth microdilution method and errors and agreement (double estimation) between MTT and standard NCCLS microdilution
 

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Table 2. Distribution of differences in the log2 MICs for 326 Gram-negative bacteria (double estimation) determined by MTT versus NCCLS broth microdilution
 
In Table 2Go the distribution of differences in log2 MICs, the percentage of essential agreement and W and P values from the Wilcoxon signed rank directional test for Gram-negative bacteria are shown. Overall agreement at ±1 log2 dilution for Gram-negative bacteria was 96.3% ± 0.65% and ranged from 94.17 ± 0.85% for ampicillin–sulbactam to 99.08 ± 0.50% for ampicillin. In a detailed statistical analysis, ±1 log2 dilution differences were also considered. For ampicillin, cefuroxime, imipenem, meropenem and gentamicin, the MTT assay results showed significant shifts towards lower values; for co-amoxiclav, cefepime and trimethoprim–sulfamethoxazole, MICs were shifted towards higher values when compared with the NCCLS standard test.

Gram-positive strains

A total of 127 staphylococci and 74 E. faecalis isolates were tested in duplicate with nine antibiotics. NCCLS criteria have been applied to E. faecalis isolates only for ampicillin, gentamicin (high concentrations), ciprofloxacin, vancomycin and linezolid.2 The absolute interpretative agreement ranged from 90.80% for ciprofloxacin to 100% for vancomycin and linezolid (Table 3Go). A total of 81 (2.77%) minor, three (0.15%) major and eight (0.83%) very major errors were found. Two very major errors were found when testing S. aureus (one methicillin-resistant isolate) against oxacillin and two in testing for high-concentration gentamicin resistance in E. faecalis. The minor errors were highest for ciprofloxacin and for both aminoglycosides when staphylococci were tested at low antibiotic concentrations.


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Table 3. Results for 201 strains of Gram-positive bacteria by interpretative categories for broth microdilution method and errors and agreement (double estimation) between MTT and standard NCCLS microdilution
 
The overall agreement at ±1 log2 dilutions for Gram-positive bacteria was 95.87% ± 0.73 % and ranged from 92.04% ± 0.93% for ampicillin to 99.50% ± 0.68% for linezolid (Table 4Go). For ampicillin, cefuroxime, linezolid and vancomycin, a significant shift in the MIDITECH results towards lower MICs could be observed. For oxacillin and trimethoprim–sulfamethoxazole, the MTT data were shifted towards higher values.


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Table 4. Distribution of differences in the log2 MICs for 201 Gram-positive bacteria (double estimation) determined by MTT versus broth microdilution
 
ATCC reference strains were included daily in the test procedure. Their respective MICs were consistently within recommended NCCLS limits for the two methods.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The MIDITECH colorimetric MIC method has recently been developed for automated routine quantitative susceptibility testing of aerobic non-fastidious Gram-negative bacteria, staphylococci and E. faecalis. Reading of this test is facilitated by the oxidase-reductase colour tetrazolium MTT metabolic growth indicator. Addition of indicator at the end of the incubation period ensures low non-specific reactivity and accurate endpoint reading of the test. A robust colour signal can be read using reflected light and simple scanning technology, even if the culture vessel is not transparent. The use of MTT tetrazolium indicator limits the assay applicability to the above-mentioned bacteria.12 Anaerobes, fastidious bacteria, streptococci and enterococci other than E. faecalis did not consistently produce enough coloured formazan from the MTT.

To assess the reliability of the MIDITECH method, we compared this technique with the standard NCCLS broth microdilution assay by using a well characterized set of Gram-negative and Gram-positive bacteria (Vienna testing set). Overall resistance rates for this set of bacteria (4.28% intermediate and 28.38% resistant) were found to be very similar to those published for the challenge susceptibility testing set of difficult-to-test bacteria from the Centers for Disease Control and Prevention.4,5 Only E. faecalis isolates could be included in this study, because of MIDITECH assay limitations for enterococci. All these isolates were susceptible to ampicillin.

Gram-negative non-fermenting bacteria (Pseudomonas species and S. maltophilia) are, in methodological studies, commonly considered as ‘difficult to test’ and were evaluated separately from other Gram-negative bacilli.3,4,15 In the present study, the overall agreement between methods for these isolates (absolute agreement 70.98%, essential agreement 96.43% ± 0.64%) was very close to that for all 326 Gram-negative strains. Therefore, the data of all Gram-negative bacteria are presented as a single group.

The overall agreement of the MIDITECH colorimetric assay with the NCCLS broth microdilution test based on MICs for a total of 5751 organism–antibiotic combinations tested in duplicate showed a 96.18% ± 0.67% essential agreement (±1 log2 dilution). The MIDITECH assay produced MICs differing from the NCCLS method by more than ±2 log2 dilutions in only 0.37% comparisons. The detailed statistical analysis of differences showed a significant shift in MIDITECH MICs towards lower values for nine, towards higher values for five and no significant shift for nine of 23 antibiotic–bacteria combinations tested. These shifts were generally within ±1 log2 dilution accuracy limit and showed no obvious relation to an individual antibiotic or resistance mechanism. Therefore, technical variables during antibiotic dilution, distribution and test procedure, rather then systemic errors, were probably responsible for the differences observed.

The absolute overall interpretative agreement observed for Gram-negative and Gram-positive bacteria was 95.42%. The essential interpretative agreement was 99.75%. The highest essential interpretative error rate for Gramnegative bacteria and trimethoprim–sulfamethoxazole is obviously related to the lack of an intermediate interpretative concentration (all errors have to be referred as essential) for this antibiotic combination. An increased number of interpretative errors was documented also for ampicillin–sulbactam. Despite good agreement between compared methods at ±1 log2 dilutions, we encountered 12.11% minor and 0.86% very major interpretative errors for this antibiotic combination; 11.99% of minor errors were due to lower MICs determined by the MIDITECH method. A similar situation was found for co-amoxiclav, but with fewer errors. Minor errors for other ß-lactam antibiotics in this study were almost equally distributed between higher and lower concentrations (data not shown).

To increase the sensitivity of the viability measurements, the MIDITECH test uses oxidase–reductase chemistry similar to that of the Alamar method.4,5 Baker et al.,4 in comparing this test with the NCCLS technique, encountered 16.4% minor and 6.2% essential interpretative errors for ampicillin–sulbactam. In their study, the Alamar method produced higher MICs of ß-lactam antibiotics if the resistant organism produced inducible or extended-spectrum ß-lactamase. The MIDITECH MICs for Gram-negative bacteria were rather underestimated, even for resistant isolates. We can only speculate that the addition of a sensitive growth indicator at the end of incubation, compared with its incorporation with the initial inoculum, reflects more precisely endpoint viability in the bacterial culture exposed overnight to an antibiotic. In the present study, we did not analyse exactly the underlying resistance mechanisms. Further studies will be necessary to elucidate the possible relationship between resistance mechanisms, induction and metabolic events in the microdilution MIC test.

The overall results for Gram-positive bacteria obtained by the MIDITECH assay were similar to those demonstrated for Gram-negative isolates. The highest minor error rate for ciprofloxacin (9.20%) was not unexpected, because almost all E. faecalis MICs were close or equal to the intermediate interpretative breakpoint for this antibiotic. Equally, MICs for many staphylococci corresponded to the netilmicin intermediate breakpoint and may explain many minor interpretative errors, despite a good essential agreement for this antibiotic. Of 74 E. faecalis isolates tested, 33 showed high-concentration gentamicin resistance by the NCCLS reference method (breakpoint 500 mg/L). Of these, the MIDITECH assay assessed one isolate incorrectly with an MIC of 250 mg/L, resulting in a 3.03% (very major) interpretative error. All five E. faecalis isolates with vancomycin MICs >= 32 mg/L were identified correctly by the MIDITECH test.

Oxacillin testing of staphylococci raised a special concern. The last revision of interpretative breakpoints facilitated, in particular, testing of coagulase-negtive staphylococci.16,17 Despite this, even new modifications of some commercial systems do not reach 100% agreement with the NCCLS method when testing oxacillin.3,5 In the present study, the MIDITECH assay failed to identify oxacillin resistance in one S. aureus isolate (1.25% very major error). All other S. aureus and S. epidermidis isolates were classified correctly.

The MIDITECH test was found to be ‘user friendly’, especially for large series of samples with multiple antibiotics. The principal disadvantage of this colorimetric method is the inability to test all medically relevant bacteria. However, for most clinically important aerobic pathogens, including those considered ‘difficult to test’, the MIDITECH modification of the NCCLS microdilution test provided reliable quantitative susceptibility data. The most attractive factors for the routine application of this method are its low cost and its capability for automating results. For many clinical microbiology laboratories, the need for automated reading and processing of extensive MIC data might favour the use of this method for routine work.


    Notes
 
* Correspondence address. Department of Internal Medicine I, Division of Infectious Diseases and Chemotherapy, 3P, Vienna General Hospital, Waehringer-Guertel 18-20, A-1090 Vienna, Austria. Tel: +431-40-400-5139; Fax: +431-40-400-5167; E-mail: apostolos.georgopoulos{at}akh-wien.ac.at Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Craig, W. A. (1993). Qualitative susceptibility testing versus quantitative MIC tests. Diagnostic Microbiology and Infectious Diseases 16, 231–6.[ISI][Medline]

2 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA.

3 . Canton, R., Perez-Vazques, M., Oliver, A., Sanchez Del Saz, B., Gutierrez, M. O., Martinez-Ferrer, M. et al. (2000). Evaluation of the Wider System, a new computer-assisted image-processing device for bacterial identification and susceptibility testing. Journal of Clinical Microbiology 38, 1339–46.[Abstract/Free Full Text]

4 . Baker, C. N., Banerjee, S. N. & Tenover, F. C. (1994). Evaluation of Alamar colorimetric MIC method for antimicrobial susceptibility testing of Gram-negative bacteria. Journal of Clinical Microbiology 32, 1261–7.[Abstract]

5 . Baker, C. N. & Ternover, F. C. (1996). Evaluation of Alamar colorimetric broth microdilution susceptibility testing method for staphylococci and enterococci. Journal of Clinical Microbiology 34, 2654–9.[Abstract]

6 . Ferraro, M. J. & Jorgensen, J. H. (1999). Susceptibility testing instrumentation and computerised expert systems for data analysis and interpretation. In Manual of Clinical Microbiology, 7th edn, (Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. & Yolken, R. C., Eds), pp. 1593–1600. American Society for Microbiology, Washington, DC.

7 . Bartlett, R. C. & Mazens, M. F. (1979). Rapid antimicrobial susceptibility testing using tetrazolium reduction. Antimicrobial Agents and Chemotherapy 15, 769–74.[ISI][Medline]

8 . Johnson, T. L., Forbes, B. A., O'Connor-Scarlet, M., Machinski, A. & McClatchey, K. D. (1985). Rapid method of MIC determination utilizing tetrazolium reduction. American Journal of Clinical Pathology 83, 374–8.[ISI][Medline]

9 . Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. Journal of Immunological Methods 65, 55–63.[ISI][Medline]

10 . Clancy, C. J. & Nguyen, M. H. (1997). Comparison of a photometric method with standardised methods of antifungal susceptibility testing of yeasts. Journal of Clinical Microbiology 35, 2878–82.[Abstract]

11 . Meletiadis, J., Meis, J. F. G. M., Mouton, J. W., Donnelly, J. P. & Verweij, P. E. (2000). Comparison of NCCLS and 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) methods of in vitro susceptibility testing of filamentous fungi and development of a new simplified method. Journal of Clinical Microbiology 38, 2949–54.[Abstract/Free Full Text]

12 . Bel-MIDITECH. (1997). MIDITECH antimiocrobial susceptibility testing system. Procedure Manual. Bel-MIDITECH, Bratislava, Slovak Republic.

13 . Murray, P. R., Niles., A. C. & Heeren, R. L. (1987). Comparison of a highly automated 5-h susceptibility testing system, the Cobas-Bact, with two reference methods: Kirby–Bauer disk diffusion and broth microdilution. Journal of Clinical Microbiology25, 2372–7.[ISI][Medline]

14 . Lehmann, E. L. (1975). Nonparametrics: Statistical Methods Based on Ranks. Holden Day, San Francisco, CA.

15 . Carroll, K. C., Cohen, S., Nelson, R., Campbell, D. M., Claridge, J. D., Garrison, M. W. et al. (1998). Comparison of various in vitro susceptibility methods for testing Stenotrophomonas maltophilia. Diagnostic Microbiology and Infectious Diseases 32, 229–35.[ISI][Medline]

16 . McDonald, C. L., Maher, W. E. & Fass, R. J. (1995). Revised interpretations of oxacillin MICs for Staphylococcus epidermidis based on mecA detection. Antimicrobial Agents and Chemotherapy 39, 982–4.[Abstract]

17 . National Committee for Clinical Laboratory Standards. (1999). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.

Received 4 June 2001; returned 24 September 2001; revised 2 November 2001; accepted 12 November 2001





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