In Vitro Fluconazole and Voriconazole Susceptibilities of Candida Bloodstream Isolates in Korea: Use of the CLSI and EUCAST Epidemiological Cutoff Values
2013; 33(3): 167-173
Ann Lab Med 2021; 41(6): 559-567
Published online November 1, 2021 https://doi.org/10.3343/alm.2021.41.6.559
Copyright © Korean Society for Laboratory Medicine.
Suhak Jeon, M.D.1 , Jong Hee Shin, M.D., Ph.D.1 , Ha Jin Lim, M.D.1 , Min Ji Choi, Ph.D.1 , Seung A Byun, M.S.1 , Dain Lee, M.S.1 , Seung Yeob Lee, M.D.2 , Eun Jeong Won, M.D.3 , Soo Hyun Kim, M.D.1,4 , and Myung Geun Shin, M.D.1
1Department of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hospital, Gwangju, Korea; 2Department of Laboratory Medicine, Jeonbuk National University Medical School and Jeonbuk National University Hospital, Jeonju, Korea; Departments of 3Parasitology and Tropical Medicine and 4Microbiology and Laboratory Medicine, Chonnam National University Medical School, Hwasun, Korea
Correspondence to: Jong Hee Shin, M.D., Ph.D.
Department of Laboratory Medicine, Chonnam National University Medical School, 42 Jebong-ro, Dong-gu, Gwangju 61469, Korea
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Given the increased fluconazole resistance (FR) among Candida isolates, we assessed the suitability of disk diffusion susceptibility testing (DDT) for the early detection of FR using well-characterized Candida isolates.
Methods: In total, 188 Candida isolates, including 66 C. albicans (seven Erg11 mutants), 69 C. glabrata (33 Pdr1 mutants), 29 C. parapsilosis (15 Erg11 mutants), and 24 C. tropicalis (eight Erg11 mutants) isolates, were tested in this study. FR was assessed using DDT according to the standard CLSI M44-ED3 method, except that two cell suspensions, McFarland 0.5 (standard inoculum) and 2.5 (large inoculum), were used, and the inhibition zones were read at 2-hour intervals from 10 hours to 24 hours.
Results: DDT results for the standard inoculum were readable after 14 hours (C. albicans, C. glabrata, and C. tropicalis) and 20 hours (C. parapsilosis) for >95% of the isolates, whereas the results for the large inoculum were readable after 12 hours (C. glabrata and C. tropicalis), 14 hours (C. albicans), and 16 hours (C. parapsilosis) for >95% of the isolates. Compared with the results produced using the CLSI M27-ED4 broth microdilution method, the first readable results from the DDT method for each isolate exhibited an agreement of 97.0%, 98.6%, 72.4%, and 91.7% for the standard inoculum and 100%, 98.6%, 96.6%, and 95.8% for the large inoculum for C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis, respectively.
Conclusions: DDT using large inoculum may detect FR rapidly and reliably in the four most common Candida species.
Keywords: Candida, Fluconazole, Resistance, Disk diffusion, CLSI, Antifungal susceptibility testing
Disk diffusion testing (DDT), one of the oldest approaches to susceptibility testing, is still widely used in clinical laboratories because of its low cost and technical simplicity . DDT has recently been used for the direct testing of bacterial or fungal susceptibility in positive blood cultures [9–11]. Methods for shortening the incubation times for DDT have recently been reported for several common bacterial pathogens, enabling faster reporting of antimicrobial susceptibility test results from positive blood cultures [9, 12]. However, methods for shortening the incubation times for DDT for the detection of FR
We evaluated the performance of the DDT for the early detection of FR in four common
In total, 188 clinical isolates (186 from blood, one from urine, and one from pus) of four common
The diameter of the growth inhibition zone was measured across the area in which growth was prominently reduced. The interpretive criteria for DDT are as follows: an inhibition zone diameter ≥17 mm indicates FS, a diameter of 14–16 mm indicates F-SDD, and a diameter ≤13 mm indicates FR for
Categorical agreement (CA) was determined as the percentage of isolates classified into the same FS or FR categories using both DDT and the CLSI M27 BMD method. Errors were classified as a very major error (VME) when an isolate classified as FR by the reference BMD method was categorized as FS by DDT, a major error (ME) when an isolate classified as FS by the BMD method was classified as FR by DDT, or a minor error for all other discordances. The chi-square or Fisher’s exact test was used to compare CA rates, and Student’s
Table 1 shows the CA rates between fluconazole susceptibility results obtained using the DDT and BMD methods for standard inoculum at 24 hours. The CA rates, determined using revised species-specific CBPs, between the two methods were 97.0%, 98.6%, 89.7%, and 95.8% for
Table 3 shows the proportion (%) of readable isolates (forming uniformly circular inhibition zones with sufficient growth) of the four
Excellent CA (>90%) between DDT and the BMD method for assessing fluconazole susceptibility was observed for each incubation time tested for nearly all
Table 4 shows the average first readable times for DDT for all 188
Table 5 shows the distribution of DDT inhibition zone diameters obtained at the first readable time for the 188
With an increase in FR among
The standardized DDT (CLSI M44-ED1 or ED2), which uses McFarland 0.5 inoculum and a 24-hour incubation period, has been extensively validated in comparison with the CLSI M27 BMD reference method for the assessment of fluconazole sensitivity in common
In a large study of FR in 1,586
To date, only three studies have directly evaluated DDT use for assessing fluconazole susceptibility in
DDT results were readable after 14 hours (
We found that DDT using both standard and large inocula produced readable results after 14 hours of incubation for >95% of
When inhibition zone diameters at the first reading for each isolate were recorded, DDT using the standard inoculum showed overlap between wild-type isolates and FR variant populations for both
Shin JH designed the study; Jeon S performed laboratory measurements; Lim HJ, Choi MJ, Byun SA, and Lee D collected and identified clinical isolates, and performed molecular studies; Shin JH, Won EJ; and Jeon S wrote the preliminary manuscript; Shin JH, Jeon S, and Lim HJ analyzed the data; Shin JH revised the manuscript; Lee SY, Won EJ, Kim SH, and Shin MG provided valuable comments and recommendations. All authors revised and accepted the final version of the manuscript.
No potential conflicts of interest relevant to this article were reported.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1A2C1004644).