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J Clin Microbiol. Dec 2011; 49(12): 4322–4325.
PMCID: PMC3232968
Validation of 24-Hour Flucytosine MIC Determination by Comparison with 48-Hour Determination by the Clinical and Laboratory Standards Institute M27-A3 Broth Microdilution Reference Method [down-pointing small open triangle]
Shawn R. Lockhart,* Carol B. Bolden, Naureen Iqbal, and Randall J. Kuykendall
Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia
*Corresponding author. Mailing address: Mycotic Diseases Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd., Mailstop G-11, Atlanta, GA 30333. Phone: (404) 639-2569. Fax: (404) 639-3546. E-mail: gyi2/at/cdc.gov.
Received August 15, 2011; Revisions requested October 12, 2011; Accepted October 12, 2011.
Abstract
Flucytosine and itraconazole are the only antifungal agents for which the Clinical Laboratory and Standards Institute recommendations include MIC breakpoint readings at 48 h only. Here we show good essential and categorical agreement between the flucytosine MIC readings at 48 and 24 h for Candida species.
Candida infections are a major cause of morbidity and mortality, and, given that the incidence of candidemia as a cause of sepsis has increased substantially over the last 20 years, it is a major cause of concern in health care settings (14, 30). Candidemia infections increase the risk of patient mortality as well as both the length of stay and costs associated with hospitalization (12, 16). Early and appropriate antifungal therapy has been shown to have a positive impact on patient outcome and hospitalization costs (1, 10, 15, 20, 31). The combination of susceptibility testing and development of antibiograms represents one method to ensure that therapy is adequate and appropriate (2, 5, 11, 13). To this end, the Clinical and Laboratory Standards Institute (CLSI) has developed a set of standards for antifungal susceptibility testing by broth microdilution (3, 4). The standards include interpretive susceptibility breakpoints for fluconazole, itraconazole, voriconazole, caspofungin, micafungin, anidulafungin, and flucytosine (5FC). The original breakpoints were developed for MIC determinations performed after 48 h of incubation, but data have shown that MIC determinations at 24 h provide good essential and categorical agreement with 48-h values for many of the antifungal agents tested (6, 7, 8, 17, 24, 25). The CLSI has recently advocated determination of voriconazole MIC values after 24 h of incubation rather than 48 h (22), which brings voriconazole in line with the existing echinocandin, fluconazole, and amphotericin B recommendations for 24-h breakpoint determinations (3, 18, 23). The only antifungal agents with CLSI-recommended 48-h breakpoints for which 24-h breakpoints have not been established are itraconazole and 5FC.
Flucytosine is a fluorinated pyrimidine analog with excellent antifungal activity against Candida and Cryptococcus. Its mechanism of action is intracellular conversion to 5-fluorouridine triphosphate, which alters aminoacylation of tRNA and inhibits protein synthesis (29). Because of high rates of acquired resistance when used as monotherapy (28), 5FC is generally recommended for use in combination with other antibiotics, especially amphotericin B (19, 21).
We used previously gathered Candida surveillance data to evaluate the essential and categorical agreement between 5FC MIC values determined at 48 h and at 24 h. A total of 2,682 Candida bloodstream isolates collected as part of a population-based surveillance were included in the evaluation. All isolates were incident isolates; no two isolates of the same species were collected from the same patient. Species included Candida albicans (888), C. glabrata (821), C. parapsilosis (484), C. tropicalis (303), C. lusitaniae (42), C. dubliniensis (41), C. krusei (41), C. orthopsilosis (15), C. guilliermondii (11), C. metapsilosis (10), C. fermentati (6), C. nivariensis (4), C. bracarensis (3), C. catenulata (2), C. pararugosa (2), C. kefyr (1), C. famata (1), C. norvegensis (1), C. pelliculosa (1), and C. rugosa (1). MIC values were generated for the Candida species according to the M27-A3 methods recommended in the most recent guidelines of the Clinical and Laboratory Standards Institute (3) using frozen RPMI microbroth trays custom manufactured by TREK Diagnostics (Cleveland, OH). Results were read visually after 24 and 48 h of incubation and represented the lowest concentrations of drug that caused a significant decrease in growth compared to control well growth. Quality-control isolates C. parapsilosis ATCC 22019 and C. krusei ATCC 6058 were included on each day of testing. Table 1 summarizes the results (stratified by species) of 24-h and 48-h MIC testing performed with 5FC. With the exception of five isolates, the drug MIC values for each isolate were either equal at 24 h and 48 h or higher at 48 h (Fig. 1). The overall MIC50 values for all Candida isolates at 24 h and 48 h were 1 log2 dilution apart, with a 24-h MIC50 of 0.125 μg/ml and 48-h MIC50 of 0.25 μg/ml. The 24-h and 48-h MIC50 values were both 0.125 μg/ml for C. glabrata and were within 1 dilution for C. albicans, C. tropicalis, and C. parapsilosis. The overall MIC90 values for all Candida isolates at 24 h and 48 h (1.0 μg/ml at 24 h and 2.0 μg/ml at 48 h) were also 1 log2 dilution apart. The 24-h and 48-h MIC90 values were within 1 dilution for C. glabrata and C. parapsilosis and within 2 dilutions for C. albicans and C. tropicalis (Table 1). The 24-h MIC90 values determined in this study were very similar to the 48-h MIC90 values determined by Pfaller and colleagues for 8,803 Candida isolates from a worldwide collection (26).
Table 1.
Table 1.
Susceptibility of Candida bloodstream isolates to flucytosine determined by CLSI broth microdilution readings at both 24 and 48 h
Fig. 1.
Fig. 1.
Correlation of 24-h and 48-h flucytosine MIC values determined using 2,682 isolates of Candida spp. Current 48-h MIC breakpoints are indicated by vertical and horizontal lines. Each value represents the number of isolates with the corresponding 24-h and (more ...)
The MIC values for each isolate were considered to be in essential agreement when the MIC value at 24 h and the MIC value at 48 h were within 2 log2 dilutions. Essential agreement ranged from 90.9% for C. metapsilosis to 99.8% for C. glabrata, with an overall essential agreement of 97.3% (Table 1). Resistance to 5FC in this set of isolates, as determined using the CLSI M27-A3 48-h criterion of ≥32 μg/ml, was low (4). The 24-h MIC values indicated that 0.9% of the isolates were resistant; the 48-h MIC values indicated that 1.6% of the isolates were resistant. The highest resistance value determined for the four most common species was 2.7%, for C. albicans; and the lowest resistance value was 0%, for C. parapsilosis. Categorical agreement was defined using the interpretive criteria of CLSI M27-A3 with the 48-h MIC values as the gold standard. A very major error was identified when the 48-h MIC value indicated that the isolate was resistant while the 24-h MIC that indicated the isolate was susceptible; a major error was identified when the 48-h MIC indicated that the isolate was susceptible while the 24-h MIC indicated that the isolate was resistant; and a minor error was identified when there was a single categorical shift. Categorical agreement ranged from a high of 99.8% for C. parapsilosis to 87.8% for C. krusei, with an overall categorical agreement of 98.5%. Very major errors occurred only among C. albicans isolates and comprised only 0.3% of the overall comparisons. Minor errors ranged from 12.2% to 3.7% and mostly resulted from determinations performed with isolates that exhibited susceptibility at 24 h but intermediate susceptibility (MIC = 8 to 16 μg/ml) at 48 h. For analysis of correlation (see Fig. 1) between the 24-h and 48-h MICs determined for each isolate (Table 2), we used linear regression calculations and Pearson's correlation coefficient. For statistical analysis, values that were off the scale at the high end were left at the highest value and those off the scale at the low end were converted to the next lowest log2 value. A suitable value was obtained for the comparison of all isolates (R = 0.814) with the best correlation for C. glabrata (R = 0.99) and the worst for C. parapsilosis (R = 0.74), although the value was still within the suitable range (R > 0.7). Although there have been no previously published direct comparisons of 24-h and 48-h MIC readings for 5FC, Pfaller et al. (27) and Espinel-Ingroff et al. (9) made such comparisons when validating the Senstitre YeastOne panel. Espinel-Ingroff found that the values at 24 h and 48 h were generally the same and that the 24 h CLSI readings correlated well with the 24-h YeastOne readings. Pfaller did not directly compare 24-h and 48-h readings in his manuscript, but he found that the 24-h YeastOne readings correlated best with the 48-h CLSI readings and indicated that accurate 24-h readings of 5FC MIC values may be achievable.
Table 2.
Table 2.
Categorical agreement between 24-h and 48-h flucytosine MICs using the CLSI 48-h breakpointsa
In summary, all antifungal agents with CLSI interpretive breakpoints currently have approved 24-h breakpoints except for 5FC and itraconazole. We set out to determine whether 24-h MIC readings for 5FC would be usable. The flucytosine MIC values for all species of Candida can be determined by CLSI broth microdilution at 24 h with the same accuracy as the values determined by 48-h readings. Using the 48-h MIC values as the gold standard, the 24-h MIC values accurately predicted resistance 99.7% of the time; therefore, we recommend the adoption of 24-h MIC determinations as part of the standard protocol.
Acknowledgments
The findings and conclusions of this article are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.
We acknowledge the CDC Antimicrobial Resistance Working Group for funding this study.
We acknowledge the contributions of Shirley McClinton, Joyce Peterson, Eszter Deak, Lalitha Gade, Angela Ahlquist, and Tom Chiller at the CDC and the Emerging Infections Programs of Georgia (Monica Farley, coordinator) and Maryland (Lee Harrison, coordinator).
Footnotes
[down-pointing small open triangle]Published ahead of print on 19 October 2011.
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