1 Department of Microbiology, 2 Section of Infectious Diseases, Department of Medicine, Hope Hospital, Eccles Old Road, Salford M6 8HD; 3 School of Medicine, University of Manchester, Manchester M13 9PL, UK
Received 12 April 2001; returned 20 June 2001; revised 4 February 2002; accepted 5 November 2002
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Abstract |
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Introduction |
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In this study, we compared three methods: the broth macrodilution method4 and two microtitre modifications of the NCCLS M27-A method5 using either RPMI supplemented with 1.8% glucose (RPMI-G) or YNBG as the test medium, to attempt to correlate results between methods. MICs were also compared according to different endpoint definitions; either no growth or optical density reduction by 50% or 80% compared with the drug-free control. Few inter-species comparisons have been carried out; therefore, we have compared results for six pathogenic Candida species.
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Materials and methods |
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The three methods were compared using 150 isolates of Candida belonging to six different species. These consisted of 144 clinical isolates from a variety of patient types and six American Type Culture Collection (ATCC) isolates: Candida albicans ATCC 24433, C. albicans ATCC 90028, Candida tropicalis ATCC 750, Candida krusei ATCC 6258, Candida glabrata ATCC 90030 and Candida parapsilosis ATCC 22019. The group comprised 65 C. albicans, 27 C. tropicalis, 21 C. krusei, 16 C. glabrata, 11 C. parapsilosis and 10 Candida lusitaniae. Stored isolates were subcultured on blood agar plates (Oxoid Ltd, Basingstoke, UK) at 37°C for 48 h to ensure purity before testing.
Flucytosine
Flucytosine was provided by Roche Products Limited, Hertfordshire, UK. The powder was dissolved in sterile distilled water to produce a stock solution of 1280 mg/L. This was then dispensed into aliquots and stored at 20°C in the dark until required.
Media
For the macrodilution susceptibility testing method and one of the microtitre methods, YNBG (Difco, Surrey, UK) was used. RPMI-1640 (Sigma, Dorset, UK) supplemented with 1.8% glucose, buffered with MOPS (Sigma) and adjusted to pH 7.0 (RPMI-G) was used for the other microtitre method.
Susceptibility testing
All three methods were carried out simultaneously.
Macrodilution method. The broth macrodilution method employed was that of the Report of a Working Group of the British Society for Mycopathology4 using 1 mL volumes of YNBG broth in 5 mL glass, loose-capped tubes. The final drug range was 0.0332 mg/L. Yeast suspensions were prepared by suspending single colonies in sterile distilled water. The final inoculum in the assay was 5 x 104 cfu/mL. A flucytosine-susceptible strain Candida kefyr San Antonio (SA) was included with each batch of test organisms as a control. In addition, a negative control was included to ensure the sterility of the medium and one tube for each isolate was left drug-free to act as a positive control. The tubes were incubated at 37°C for 48 h in air. The MIC was read visually, with the aid of an 80% inhibition standard tube,5 and was taken as the lowest drug concentration to inhibit 80% of the growth compared with the positive control.
Microtitre methods. The method used was a microtitre modification of the NCCLS M27-A method5 in flat-bottomed microtitre plates with either YNBG or RPMI-G broth. The final drug range was 0.0332 mg/L. The yeast suspensions used for the macrodilution method were then adjusted further, using the appropriate medium, to give a final inoculum of 0.52.5 x 103 cfu/mL. The C. kefyr SA strain was used as a control. Positive and negative controls were also included. The microtitre plates were incubated in a moist chamber at 37°C for 48 h. After incubation, the microtitre plates were shaken for 5 min to obtain a uniform suspension before reading. The growth in each well was measured by determining the optical density at 490 nm by spectrophotometry. Three different endpoints were then recorded; no growth by eye, and the drug concentrations that reduced the OD490 by either 50% or 80% when compared with the drug-free control.
Reproducibility
Twenty per cent of the isolates (30/150) were randomly selected and re-tested to establish the reproducibility of each method and each endpoint.
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Results |
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With NCCLS breakpoints,5 seven isolates were resistant, one intermediate and the remainder susceptible. With BSMM breakpoints,4 seven isolates were resistant, five intermediate and the remainder susceptible. The seven isolates were classed as resistant regardless of breakpoint used.
Comparisons within microtitre methods. When comparing the different endpoints, only two (3.1%) and five (7.7%) isolates showed differences of more than one doubling dilution with RPMI-G and YNBG microtitre methods, respectively.
Comparison between all methods. Correlation between all three methods was good with over 75% of isolates having MICs within two doubling dilutions (Table 3). A small percentage of major discrepancies were found when employing either NCCLS (4.6%) or BSMM (1.5%) breakpoints, and these showed varied differences (Table 4).
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With either NCCLS5 or BSMM4 breakpoints four isolates were resistant, two intermediate and the remainder susceptible.
Comparisons within microtitre methods. Comparison of the three endpoints showed that seven (25.9%) and eight (29.6%) isolates differed by more than one doubling dilution with RPMI-G and YNBG microtitre methods, respectively.
Comparison between all methods. Overall, the correlation between the different tests was good, with almost 60% of isolates having MICs within two doubling dilutions (Table 3). Again, a small number of major discrepancies were seen when using either NCCLS (11.1%) or BSMM (3.7%) breakpoints, and these showed varied differences (Table 4).
C. krusei (n = 21)
With NCCLS breakpoints,5 all isolates were intermediate. With BSMM breakpoints,4 nine isolates were resistant and 12 intermediate. No isolate was classed as susceptible regardless of breakpoint chosen.
Comparisons within microtitre methods. Comparison of the three endpoints found that 10 (47.6%) and 16 (76.2%) isolates showed differences greater than one doubling dilution for RPMI-G and YNBG microtitre methods, respectively.
Comparison between all methods. Correlation between the three methods was very poor, with <5% of isolates having MICs within two doubling dilutions. Over 70% of isolates showed differences of four or five dilutions (Table 3). RPMI-G results were significantly higher for most isolates compared with the other two methods. Perhaps surprisingly, there were no major discrepancies when NCCLS breakpoints were utilized. However, when using BSMM breakpoints, nine (42.8%) isolates showed discrepancies, all of which were resistant in RPMI-G, susceptible in YNBG microtitre and either susceptible or intermediate with the macrodilution broth method (Table 4).
C. glabrata (n = 16)
With NCCLS breakpoints,5 all isolates were susceptible. With BSMM breakpoints,4 three isolates were intermediate and the remainder susceptible. No isolate was classed as resistant regardless of breakpoint used.
Comparisons within microtitre methods. The different endpoints showed a difference of more than one doubling dilution for only one isolate (6.2%) with the YNBG microtitre method. The RPMI-G microtitre method showed no differences between endpoints.
Comparison between all methods. Correlation between methods was poor, with only 18.8% of isolates having MICs within two doubling dilutions (Table 3). The poor correlation resulted mainly from the broth macrodilution method since these results were usually higher than those of either of the microtitre methods. However, since most isolates were susceptible with either breakpoint or any methods, only one major discrepancy was found with BSMM breakpoints (Table 4). This isolate was susceptible or intermediate by either microtitre method, but resistant by the broth macrobroth dilution method. No major discrepancies were found when using NCCLS breakpoints.
C. parapsilosis (n = 11)
With either NCCLS5 or BSMM4 breakpoints all isolates were susceptible.
Comparisons within microtitre methods. Comparison between the different endpoints showed differences of greater than one doubling dilution for six (54.5%) and one (9.1%) isolate with RPMI-G and YNBG microtitre methods, respectively.
Comparison between all methods. The three methods exhibited a poor correlation, with only 18.2% of strains having MICs within two doubling dilutions. The majority of isolates (72.7%) had MICs that differed by three dilutions (Table 3). The YNBG microtitre results were notably lower than those of the other methods; however, no major discrepancies were seen with either breakpoint.
C. lusitaniae (n = 10)
With NCCLS breakpoints,5 six isolates were resistant and the remainder susceptible. With BSMM breakpoints,4 six isolates were resistant, one intermediate and the remainder susceptible.
Comparisons within microtitre methods. When analysing the different endpoints, two (20%) and four (40%) isolates were different by more than one doubling dilution with RPMI-G and YNBG microtitre methods, respectively.
Large endpoint variations were noted with this species, with some MICs increasing dramatically as the stringency of the endpoint increased.
Comparison between all methods. Correlation between the three methods showed that 50% of isolates had MICs within two doubling dilutions (Table 3). Three major discrepancies (30%) were seen with both NCCLS and BSMM breakpoints (Table 4). These three isolates were resistant by RPMI-G microtitre, susceptible by YNBG microtitre and either susceptible (NCCLS breakpoints) or intermediate (BSMM breakpoints) in the broth macrodilution test.
Reproducibility
Of the 20% of isolates (30/150) re-tested with all seven endpoints (210 results), 15 (7.1%) results in nine isolates differed by more than one dilution and one (0.5%) by more than two dilutions. This was an isolate of C. albicans tested using the YNBG microtitre method, with a 50% endpoint that rose from 4 to >32 mg/L.
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Discussion |
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In 1982, the first subcommittee of the NCCLS on antifungal susceptibility testing was formed. This subcommittee was charged with developing a reference method that might improve inter-laboratory reproducibility. A comparison of macrodilution and microdilution tests with flucytosine with RPMI-1640 showed equivalence or lower MICs with microdilution, especially with C. tropicalis.10,11 Other collaborative work indicated better inter-laboratory agreement (6590%) with flucytosine using a smaller inoculum, a 2 day incubation period and a 1+ turbidity growth endpoint.12 Agreement with C. lusitaniae was worse than other species and C. krusei was not included. Selection of quality control isolates involved repetitive testing of 10 candidate strains and three Candida isolates were selected with a range of flucytosine MICs.8,13,14 Agreement for C. albicans with the supplementation of the RPMI-1640 with 1.8% glucose was 98.1% in a microtitre format.15 Alteration of pH barely affected the results.16
In this study, we identified a number of resistant isolates of C. albicans (10.8%), C. tropicalis (14.8%) and C. lusitaniae (60%). In addition, we found all C. krusei isolates to be intermediate to flucytosine. This figure is three times higher than that previously published.6 These rates of reduced susceptibility were in contrast to that found by others,6 where much lower levels of resistance were observed. However, the previous study was carried out in Spain where flucytosine is rarely used; therefore our results may reflect greater use of this drug in the UK. Interestingly though, C. glabrata was always found to be susceptible in our study, compared with over 25% of isolates showing intermediate resistance in the Spanish study.6 C. parapsilosis was also fully susceptible in the present study, thus agreeing with the findings of others.6
Most published studies of flucytosine have compared the NCCLS method5 with commercial tests, such as Etest,17 Sensititre YeastOne Colorimetric Antifungal Panel18 and Fungitest,19 with varying degrees of success. None of these tests utilizes YNB broth as the test medium; however, YNB broth has previously been used to carry out flucytosine susceptibility testing against Candida species.20,21 Galgiani et al.21 showed that variations in the formulation of YNB did not affect the susceptibility testing of flucytosine to any significant extent. In addition, YNB broth has previously been used to test flucytosine against Cryptococcus neoformans,22 where it was found to give the widest range of MICs compared with other media, which included RPMI-1640 with and without additional glucose. It is also noted in the NCCLS document5 that YNB broth may improve clinical relevance when testing C. neoformans. Nevertheless, no comparisons between the methods of the NCCLS5 and BSMM4 with any Candida species have been published to date.
A few studies have shown a correlation with flucytosine susceptibility testing and outcome in vivo. Stiller et al.23 found a correlation between a broth macrodilution method and in vivo response in C. albicans. Isolates with an MIC of <12.5 mg/L responded much better to treatment than those isolates with an MIC of 12.5 mg/L or greater. In 1986, Radetsky et al.24 showed agreement with animal and clinical outcome with a broth microdilution method similar to the NCCLS microtitre method. They tested an isolate of C. albicans that was considered susceptible to flucytosine by both laboratory (MIC 2 mg/L) and clinical criteria, and an isolate of C. tropicalis that was considered resistant to flucytosine by the same criteria (MIC > 16 mg/L). Mice were infected and treated with 150 mg/kg per day flucytosine. All of the mice infected with the C. albicans isolate were still alive after 30 days, whereas all the mice infected with the C. tropicalis isolate had died within 3 days. Similar and more comprehensive results were obtained by Anaissie et al.7 with multiple isolates of C. albicans, C. krusei and C. lusitaniae.
For C. albicans and, to a slightly lesser extent, C. tropicalis, many different testing formats appeared to give comparable results. Variation was limited in C. glabrata and C. parapsilosis, but few isolates had elevated MICs. Considerable variation in MICs was seen with C. krusei and C. lusitaniae.
In summary, substantial in vitro/in vivo correlation work needs to be undertaken to establish optimum susceptibility testing procedures with flucytosine for non-albicans Candida, particularly C. krusei.
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Acknowledgements |
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Footnotes |
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References |
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23 . Stiller, R. L., Bennett, J. E., Scholer, H. J., Wall, M., Polak, A. & Stevens, D. A. (1983). Correlation of in vitro susceptibility test results with in vivo response: flucytosine therapy in a systemic candidiasis model. Journal of Infectious Diseases 147, 10707.[ISI][Medline]
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