Oct. 2012

THE JAPANESE JOURNAL OF ANTIBIOTICS

65—5335（ 45 ）Comparison of four reading methods of brothmicrodilution based on the Clinical andLaboratory Standards Institute M27-A3
method for Candida spp.
HIROMI MORISHIGE1*, YOKO MANO2, TOYOKO OGURI3
and NOBUHIKO FURUYA1,2
1

Graduate School of Health Care Science, Bunkyo Gakuin University,
Tokyo, Japan
2
Faculty of Health Science Technology, Bunkyo Gakuin University,
Tokyo, Japan
3
Clinical Laboratory, Kameda Medical Center, Chiba, Japan(Received for publication July 20, 2012)

This study aimed to compare the susceptibilities of 5 reference strains and 28
isolates of Candida spp., to micafungin, amphotericin B, Àucytosine, Àuconazole,
itraconazole, voriconazole, and miconazole, obtained by visually determined
minimum inhibitory concentration (MIC) using the agitation method (V-A), as
described in the Clinical and Laboratory Standards Institute M27-A3 document;
visual determinations without agitation (V-NA); and spectrophotometricdeterminations for the presence or absence of agitation (SP-A and SP-NA,
respectively). Our results indicate that when the V-NA, SP-A, and SP-NA—the 3alternative microdilution procedures for MIC endpoint determinations—werecompared with the V-A, excellent agreements were observed between the V-NA and
V-A rather than with the spectrophotometric methods (between the SP-A or SP-NA,
and V-A). Furthermore, many errors occurred while using the SP-A method in thepresence of agitation and some isolates showed major errors. Three of 5 isolates thatshowed very major errors between the spectrophotometric SP-A or SP-NA, and thereference V-A method were trailing isolates. Therefore, it was suggested that the
MICs of Candida spp. obtained by the V-NA method were more precise than thoseby the conventional SP-A method.

*Current af¿liation: Clinical Laboratory, Kameda Medical Center, Chiba, Japan336（ 46 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

65—5

Oct. 2012

Introduction
The increasing incidence of human immunode¿ciency virus-1 infection, the use of therapeutic modalities for advanced life support, and certain surgical procedures, such as organ transplantation and the implantation of prosthetic devices, have had considerable inÀuences on the expanding incidence of Candida infections1). Candida is the most frequently implicated agent ininvasive fungal infections, now ranking as the fourth most common cause of nosocomial bloodstream infections and accounting for 8% of all hospital-acquired bloodstream infections in the
United States of America. Candidemia is associated with an extremely high rate of motility2).
Therefore, antifungal drug susceptibility testing, which offers a guide to the selection and followup of treatment for accurate and reproducible results, has become more important due to the risein serious fungal infections and the concomitant emergence of resistance to antifungal agents3–5).
The Clinical and Laboratory Standards Institute (CLSI; formerly the NCCLS) has published an
approved reference method for antifungal susceptibilities of Candida species. In 2008, CLSI
developed an approved reference procedure (document M27-A3) through which a new level of
reproducibility was achieved6). Recent efforts have been directed to the problem of accurate determination of minimum inhibitory concentration (MIC) endpoints, particularly with the azole
antifungal agents tested against Candida spp7). According to the CLSI M27-A3, the microdilution
MIC endpoints are visually determined by the agitation of microdilution trays prior to the readingof the MICs. Recently, conducting the reading after agitation facilitates the use of a spectrophotometer for determining MIC endpoints. Since it easily lends itself to automation7), the spectrophotometric approach to MIC endpoint determination provides an objective, rapid MIC readingand eliminates the subjective judgments concerning minimal turbidity, which confounds antifungal susceptibility testing8). However, the description of MIC endpoint determination for theazoles in CLSI is overly complex and somewhat ambiguous for the microdilution procedure 3).
Therefore, the present study aimed to evaluate the performance of visual and spectrophotometric MIC readings for the presence or absence of agitation by determining the susceptibilitiesof 33 isolates of Candida spp., including 28 Candida albicans isolates.

Materials and Methods
Isolates
Thirty-three isolates of Candida spp. were tested. Five of these were American Type Culture
Collection strains (C. parapsilosis ATCC 90018, C. krusei ATCC 6258, C. albicans ATCC 90028,
C. albicans ATCC 24433, and C. tropicalis ATCC 750). Ten C. albicans isolates from the respiratory and urogenital systems were derived from a hospital in Tokyo, Japan. Seven C. albicans isolates (TIMM 3163-3166, 3209, 3309, and 3960) were kindly provided by the Teikyo University

Oct. 2012

THE JAPANESE JOURNAL OF ANTIBIOTICS

65—5

337（ 47 ）Institute of Medical Mycology and 9 isolates (IFM 46910, IFM 54354, IFM 57376, IFM 57378,
IFM 57388, IFM 57389, and IFM 57408-57410) by the Chiba University Research Center for
Pathogenic Fungi and Microbial Toxicoses. Sixteen isolates of C. albicans putatively indicatedresistance to all azoles. Two putative azole-trailing strains, C. tropicalis KYTE1 and KYTE2(kindly provided by Kyokuto Chemical, Tokyo, Japan), were also tested. Con¿rmation of species
identi¿cation was performed with API products (bioMérieux, Tokyo, Japan) as recommended by
the manufacturer. Each isolate was maintained in sterile water and subcultured in antimicrobialfree medium to ensure viability and purity prior to testing.
Antifungal agents and microdilution panels
The antifungal susceptibilities of Candida spp. isolates were determined by using microdilution panels (Yeast-like fungi DP, Eiken kit; Eiken Chemical Company, Ltd., Tokyo, Japan) containing two-fold serial dilutions of micafungin, amphotericin B, 5-Àuorocytosine (Àucytosine),Àuconazole, itraconazole, voriconazole, and miconazole. The tested drugs concentrations ranged
from 0.015 to 16 ȝg/mL for micafungin, 0.03 to 16 ȝg/mL for amphotericin B and miconazole,
0.12 to 64 ȝg/mL for Àucytosine and Àuconazole, and 0.015 to 8 ȝg/mL for itraconazole and voriconazole. The microdilution panels were shipped frozen and stored at −70°C until testing.
Antifungal susceptibility test methods
Inoculum suspensions of Candida spp. isolates were obtained from 24 h cultures on Sabouraud dextrose agar at 35°C. The turbidity of each yeast suspension was adjusted by the spectrophotometric method to match the turbidity of a 0.5 McFarland standard and diluted to a concentration of 0.5×103 to 2.5×103 CFU/mL with the ¿nal testing inoculum in RPMI 1640 medium(Sigma-Aldrich, Tokyo, Japan), buffered to pH 7.0 with 0.165 M morpholinopropanesulfonic acid
(MOPS; Kanto Chemical, Tokyo, Japan), and an aliquot of 100 ȝL was added to each well of the
microdilution panels. The panels were incubated in ambient air at 35°C and MICs determined at
24 h and 48 h for all species.
Determination of MICs
MIC results for micafungin were read following a 24 h of incubation, whereas MIC results
for amphotericin B, Àucytosine, Àuconazole, itraconazole, voriconazole, and miconazole wereread after 48 h of incubation. Optical densities of each microplate well before and after agitation
were measured either visually using inverted reading mirror or spectrophotometrically with theMultiskan FC Microplate Photometer (Thermo Scienti¿c, Yokohama, Japan) set at 640 nm. The
visual and spectrophotometric MIC values for the azoles, micafungin, and Àucytosine were de¿ned as the lowest drug concentration that resulted in a reduction of approximately 侒50% in turbidity in comparison with the drug-free growth control well. For amphotericin B, MIC values338（ 48 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

65—5

Oct. 2012were de¿ned as the lowest drug concentration for which the well was optically clear. The visualand spectrophotometric MICs for the presence or absence of agitation were represented as V-A,
V-NA, SP-A, and SP-NA, respectively.
Analysis of results
Reproducibility was ensured by testing each of the ¿ve reference strains a total of two times
by each of microdilution methods. CLSI-determined MICs (the V-A determined MICs) for thesequality control strains were within the recommended ranges. The CLSI interpretative breakpoints
for micafungin (susceptible [S], 侑2 ȝg/mL; nonsusceptible [NS], >2 ȝg/mL), amphotericin B (S,侑1 ȝg/mL), Àucytosine (S, 侑4 ȝg/mL; intermediate [I], 侒8–16 ȝg/mL; resistant [R], 侒32 ȝg/mL),Àuconazole (S, 侑8 ȝg/mL; susceptible-dose dependent [S-DD], 侒16–32 ȝg/mL; R, 侒64 ȝg/mL),
itraconazole (S, 侑0.12 ȝg/mL; S-DD, 侒0.25–0.5 ȝg/mL; R, 侒1 ȝg/mL), and voriconazole (S, 侑
1 ȝg/mL; S-DD, 2 ȝg/mL; R, 侒4 ȝg/mL) (CLSI subcommittee, 2008 meeting) were used to obtaincategorical agreement percentages among V-A, V-NA, SP-A, and SP-NA. The V-A was de¿ned
as the reference broth microdilution method in the CLSI M27-A3 document. The MIC range,
MIC50, and MIC90 (the lowest drug concentrations that inhibited 50% and 90% of isolates,
respectively) were determined for each drug tested. An agreement was reported when the MIC
results among the 4 methods were in exact agreement or within 1 two-fold dilution. Very major
errors were identi¿ed when R was indicated by the reference (V-A) MIC and S by one of theothers (V-NA, SP-A, and SP-NA). Major errors were identi¿ed when the isolate was classi¿ed as
S by the reference method and R by the other methods. Minor errors were identi¿ed when the
result of the reference readings was either S or R, and that of the others was S-DD or I.

Results
1. Comparison between the V-A, V-NA, SP-A, and SP-NA for the seven antifungal
agents against Candida spp. isolates
Table 1 summarized MICs of 33 Candida spp. isolates as determined V-A as described in
CLSI M27-A3. For FLCZ, three isolates were classi¿ed as S-DD and 7 isolates were classi¿ed as
R. For ITCZ, ¿ve isolates were classi¿ed as S-DD and 7 isolates were classi¿ed as R. For VRCZ,
eight isolates were classi¿ed as R and S-DD isolates were no observed.
Table 2 summarizes MICs of the 5 CLSI reference isolates to micafungin, amphotericin B,Àucytosine, Àuconazole, itraconazole, voriconazole, and miconazole as determined by the 4 reading methods following incubation for 48 h (only 24 h for micafungin). MICs that fell outside of
the CLSI recommended range included the Àuconazole MICs for C. albicans ATCC 90028 and
ATCC 24433, generated by SP-A and SP-NA; Àuconazole MIC for C. tropicalis ATCC 750, generated by SP-NA; and Àucytosine MIC for C. krusei ATCC 6285, generated by SP-A. AlthoughMicafungin MICs were determined following a 24 h incubation.

THE JAPANESE JOURNAL OF ANTIBIOTICS

1Table 1. Susceptibilities of Candida spp. to seven antifungal agents as determined with the reference CLSI M27-A3 method
following a 48 h incubation (n=33).

Oct. 2012
65—5339（ 49 ）340（ 50 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

65—5

Oct. 2012

Table 2. Comparison of broth microdilution MICs read by four methods for sevenantifungal agents against ¿ve reference strains following a 48 h incubation.

V-A: visual-agitation, V-NA: visual-no agitation, SP-A: spectrophotometric-agitation, SP-NA: spectrophotometric-noagitation1
Micafungin MICs were determined following a 24 h incubation. 2 NA, not available.

Oct. 2012

THE JAPANESE JOURNAL OF ANTIBIOTICS

65—5

341（ 51 ）both Àucytosine MIC for C. albicans ATCC 24433 by V-NA and itraconazole MIC for C. krusei
ATCC 6258 by SP-A were outside of the recommended range, each of those MICs was similar to
the reference (V-A) MIC (within 1 two-fold dilution).
2. Categorical agreement between the V-A, V-NA, SP-A, and SP-NA
Table 3 summarizes the categorical agreement among the V-A, V-NA, SP-A, and SP-NA for
33 isolates of Candida spp. against the 6 antifungals tested, except for miconazole which did nothave a de¿ned breakpoint by CLSI M27-A3. All the Candida spp. isolates were classi¿ed as S for

Table 3. Distribution of 33 organisms tested according to methods and susceptibility
pro¿le following a 48 h incubation.

V-A: visual-agitation, V-NA: visual-no agitation, SP-A: spectrophotometric-agitation, SP-NA: spectrophotometric-noagitation
1
intermediate interpretative category for flucytosine only.
2
Micafungin is classified as nonsusceptible and MICs were determined following a 24 h incubation.
3
There is no official breakpoint for amphotericin B MIC.
NA, not available.

342（ 52 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

65—5

Oct. 2012

micafungin by the 4 methods. For amphotericin B, 87.9% of Candida spp. isolates were classi¿ed
as S, as determined by the SP-A and 90.9% by each of other methods. As for the result withÀucytosine, the categorical agreement between the SP-NA-determined MICs and the other 3
methods MICs was low (3.0%) due to a minor error resulting from an isolate shifting from S to
S-DD. For the azoles, 21.2–24.2% of Candida spp. isolates were classi¿ed as R by the V-A.
However, these isolates determined by SP-A and SP-NA were classi¿ed as S and showed a very
major error (from 12.1% for itraconazole and voriconazole to 15.2% for Àuconazole, and 3.0%
for itraconazole to 9.1% for voriconazole, respectively). For voriconazole, categorical agreementof SP-NA was the highest (81.8%). Although only 1 isolate determined by V-NA for voriconazole
exhibited a very major error (3.0%), the highest categorical agreements were observed with V-NA
for Àuconazole and itraconazole rather than through the determination by the other spectrophotometric method.
3. The percentages of agreement between the V-A, V-NA, SP-A, and SP-NA for sevenantifungal agents
Table 4 summarizes the percentages of agreement between the V-A, V-NA, SP-A, and
SP-NA for all organism-drug combinations following a 48 h incubation (only 24 h for micafungin). Complete agreement (100%) noted within 1 two-fold dilution between the 4 methods was
obtained for the micafungin and amphotericin B susceptibilities of 33 Candida spp. isolates. The
agreements among the 4 methods for Àucytosine were very strong and varied from 93.9% (between the V-A and SP-NA) to 100% (between the V-A and V-NA). When the azoles were tested,
the agreements within 1 two-fold dilution among the 4 methods were as follows: 69.7–72.7%,
72.7–81.8%, 69.7–72.7%, and 66.7–69.7% for Àuconazole, itraconazole, voriconazole, and miconazole, respectively. The poorest agreement was observed between the V-A and SP-A for all
the azoles.

Discussion
The recently published CLSI standard method for susceptibility testing of yeasts6) speci¿esvisual endpoints after agitation of microdilution plates, as this method has good-to-excellent
reproducibility in a multilaboratory comparison9) and resulted in clear-cut endpoints that werereproducible7,10). However, in the present study, we showed that it was dif¿cult to determine the
MICs of the azole-high resistant isolates by the CLSI method due to signi¿cant turbidity in all thewells. Moreover, some laboratories often determine MICs by spectrophotometry after constantly
agitating microplates in a shaker, due to the simplicity of this procedure11–16). In the present study,
we compared antifungal susceptibilities of 33 Candida spp. to the 7 antifungal agents by thebroth microdilution method (the V-A) based on the CLSI M27-A3 to the MICs obtained by theOct. 2012

THE JAPANESE JOURNAL OF ANTIBIOTICS

65—5

343（ 53 ）Table 4. Percents agreement between the V-A, V-NA, SP-A and SP-NA for sevenantifungal agents following a 48 h incubation (n=33).

V-A: visual-agitation, V-NA: visual-no agitation, SP-A: spectrophotometric-agitation, SP-NA: spectrophotometric-noagitation1
Micafungin MICs were determined following a 24 h incubation.
0: Modal MICs (V-NA, SP-A, SP-NA) were equal to V-A MICs.
1: Modal MICs (V-NA, SP-A, SP-NA) were 1 dilution, 2-fold, higher than V-A MICs.
−1: Modal MICs (V-NA, SP-A, SP-NA) were 1 dilution, 2-fold, lower than V-A MICs.
2: Modal MICs (V-NA, SP-A, SP-NA) were 2 dilutions, 4-fold, higher than V-A MICs.
−2: Modal MICs (V-NA, SP-A, SP-NA) were 2 dilutions, 4-fold, lower than V-A MICs.

other 3 methods: V-NA, SP-A, and SP-NA.
In the present study, the highest agreement for almost all antifungal agents was observed
between V-NA and the CLSI reference method (the V-A) rather than among the SP-A, SP-NA,
and V-A, as PFALLER et al. and LOZANO-CHIU et al. have reported7,17). However, the spectrophotometric approach was noted to provide precise, objective MIC endpoints, particularly for the azole
antifungal agents18,19), and determination of MICs spectrophotometrically has shown good agree-344（ 54 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

65—5

Oct. 2012

ment with the standardized visual reading for Candida spp., as previously reported by other researchers7,12,20–22). Moreover, NISHIYAMA et al. demonstrated the Japanese Society for Medical
Mycology (JSMM) method, in which the endpoint was to read as 50% inhibitory concentration(IC50) and use a spectrophotometer, was superior in both stability and reproducibility, as compared to the CLSI method in which growth was assessed by visual observation23). Additional
studies will be required to compare between V-NA method and JSMM method.
Similar results were observed for almost all reference isolates; these MICs determined by the
V-NA method were clustered within 1 two-fold dilution, compared with MICs of the V-A method,
and were within the established reference MIC range, unlike MICs of the SP-A or SP-NA
method. However, MICs derived by the SP-NA varied widely among the MICs obtained by the 4
methods.
The V-NA-determined MICs for amphotericin B were 2 two-fold dilutions lower than the
V-A MICs for 2 C. albicans clinical isolates (data not shown). For amphotericin B the V-A MIC
was de¿ned as the lowest drug concentration required for an optically clear agitated well,whereas the V-NA MIC value indicated precipitation in the well without agitation. The discrepancy might be due to the turbidity not being observed optically when a small quantity of the precipitation was agitated in the well of the V-NA MIC. Despite the presence or absence of agitation,
spectrophotometric MICs of amphotericin B for these 2 isolates were identical to the V-A MICs.
These results indicate that MIC readings determined by the V-NA may be more precise than thosedetermined by the other MIC readings.
For micafungin, amphotericin B, and Àucytosine, the rates of errors were low because almost
all isolates were classi¿ed as S. For the azoles, the susceptibilities of Candida spp. isolates werevaried due to the variability of each method. The percentage of errors, particularly very major errors, obtained from the SP-A method was higher than that from the other methods. However, by
the V-NA method, only 1 isolate for voriconazole showed a very major error. Three of 5 isolates
that showed very major errors between the spectrophotometric SP-A or SP-NA, and the reference
V-A method were trailing isolates.
In one study24), the rates of apparent in vitro resistance to Àuconazole and itraconazole
among bloodstream isolates of C. albicans and C. tropicalis—derived from visual reading of
MIC end points after 48 h of incubation—were higher than those based on spectrophotometric
MIC endpoints. The results of the present study are similar to those reported by ARTHINGTONSKAGGS et al. 24) Therefore, it was suggested that optical density varied due to the large amount ofprecipitation in each well because almost all trailing isolates were determined after 48 h incubation. Therefore, our results showed that MIC determinations of the V-NA method at 48 h may be
more appropriate than those of the other methods against Candida spp., except for trailing isolates. However, for trailing isolates, agitation of the broth in the CLSI method has been shown to
reduce the trailing-endpoint effect, although the incubation time in the study by ANAISSIE et al.

Oct. 2012

THE JAPANESE JOURNAL OF ANTIBIOTICS

65—5

345（ 55 ）was 24 h instead of the standard 48 h25). In the present study, the reduction of the trailing MIC
endpoints by agitation was not observed for most of the isolates at 48 h. When 3 trailing isolates
that showed a very major error between the spectrophotometric SP-A and the reference V-A
method were determined by V-A and V-NA method after 24 h of incubation, the MICs by V-NA
were similar to V-A MICs within 1 two-fold dilution (data no shown). Therefore, our results also
showed that trailing MIC determinations of the V-NA method at 24 h may be more appropriate
than those of the other methods against Candida spp.
In conclusion, the microdilution MIC endpoints for Candida spp. based on CLSI M27-A3
are visually determined by the agitation of microdilution trays prior to the reading of the MICs.
Because it is dif¿cult to perform the present CLSI method in routine clinical testing, the spectrophotometric approach has recently been adopted by multilaboratories. In the present study, we
compared the susceptibilities of Candida spp. to 7 antifungal agents obtained by the V-A method
described in CLSI M27-A3 document and the V-NA, SP-A, and SP-NA methods. The results
showed a highly signi¿cant agreement between the V-A and V-NA method regardless of the presence or absence of agitation, whereas many errors occurred while using the SP-A method in thepresence of agitation. Moreover, some isolates determined by the SP-A method showed a very
major error, which was suspected to be due to the accuracy of MIC determination by spectrophotometry. Therefore, the V-NA method, for which both trailing and no-trailing endpoints areobserved for Candida spp., may be easier and more precise than the reference V-A method andthe conventional SP-A method.

References
1) EDWARDS, J. E., Jr.: Candida species. Mandell, Douglas, and Bennettʼs principles and practice of
infectious diseases. In: MANDELL, G. L., J. E. BENNETT, R. DOLIN (ed.), 6th ed. Churchill Livingstone, Philadelphia, Pa., 2938〜2957, 2005
2) GONZÁLEZ, G. M.; M. ELIZONDO & J. AYALA: Trends in species distribution and susceptibility ofbloodstream isolates of Candida collected in Monterrey, Mexico, to seven antifungal agents: results of a 3-year (2004 to 2007) surveillance study. J. Clin. Microbiol. 46: 2902〜2905, 2008
3) ARTHINGTON-SKAGGS, B. A.; W. LEE-YANG, M. A. CIBLAK, et al.: Candidemia Active SurveillanceGroup. Comparison of visual and spectrophotometric methods of broth microdilution MIC end
point determination and evaluation of a sterol quantitation method for in vitro susceptibility testing of Àuconazole and itraconazole against trailing and nontrailing Candida isolates. Antimicrob.
Agents Chemother. 46: 2477〜2481, 2002
4) KOGA-ITO, C. Y.; J. P. LYON & M. A. RESENDE: Comparison between E-test and CLSI broth microdilution method for antifungal susceptibility testing of Candida albicans oral isolates. Rev. Inst.
Med. Trop. Sao Paulo 50: 7〜10, 2008
5) VANDENBOSSCHE, I.; M. VANEECHOUTTE, M. VANDEVENNE, et al.: Susceptibility testing of Àuconazoleby the NCCLS broth macrodilution method, E-test, and disk diffusion for application in the routine laboratory. J. Clin. Microbiol. 40: 918〜921, 2002
6) Clinical and Laboratory Standards Institute: Reference method for broth dilution antifungal sus-

346（ 56 ）

7)

8)

9)

10)

11)

12)

13)

14)

15)

16)

17)

18)

19)

THE JAPANESE JOURNAL OF ANTIBIOTICS

65—5

Oct. 2012

ceptibility testing of yeasts; Approved standard-third edition M27-A3. CLSI, Wayne, PA, USA,2008
PFALLER, M. A.; S. A. MESSER & S. COFFMANN: Comparison of visual and spectrophotometricmethods of MIC endpoint determinations by using broth microdilution methods to test ¿ve antifungal agents, including the new triazole D0870. J. Clin. Microbiol. 33: 1094〜1097, 1995
CUENCA-ESTRELLA, M. et al.: Comparative evaluation of two standardized methodologies (NCCLS
and EUCAST) for antifungal susceptibility testing [abstract J-570]. In: Program and abstracts of
the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL. Washington, DC: Am. Soc. Microbiol. p. 373, 2001
ANAISSIE, E. J.; V. L. PAETZNICK, L. G. ENSIGN, et al.: Microdilution antifungal susceptibility testing of Candida albicans and Cryptococcus neoformans with and without agitation: an eightcenter collaborative study. Antimicrob. Agents Chemother. 40: 2387〜2391, 1996
ANAISSIE, E.; V. PAETZNICK & G. P. BODEY: Fluconazole susceptibility testing of Candida albicans:microtiter method that is independent of inoculum size, temperature, and time of reading. Antimicrob. Agents Chemother. 35: 1641〜1646, 1991
YAMAZAKI, T.; Y. INAGAKI, T. FUJII, et al.: In vitro activity of isavuconazole against 140 reference
fungal strains and 165 clinically isolated yeasts from Japan. Int. J. Antimicrob. Agents 36: 324〜
331, 2010RODRÍGUEZ-TUDELA, J. L.; J. BERENGUER, J. V. MARTÍNEZ-SUÁREZ, et al.: Comparison of a spectrophotometric microdilution method with RPMI-2% glucose with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro susceptibility testingof amphotericin B, Àucytosine, and Àuconazole against Candida albicans. Antimicrob. Agents
Chemother. 40: 1998〜2003, 1996
CUENCA-ESTRELLA, M.; D. RODRIGUEZ, B. ALMIRANTE, et al.: Barcelona Candidemia Project Study
Group. In vitro susceptibilities of bloodstream isolates of Candida species to six antifungal
agents: results from a population-based active surveillance programme, Barcelona, Spain, 2002–
2003. J. Antimicrob. Chemother. 55: 194〜199, 2005
ISHIDA, K.; J. C. FERNANDES RODRIGUES, S. CAMMERER, et al.: Synthetic arylquinuclidine derivatives
exhibit antifungal activity against Candida albicans, Candida tropicalis and Candida parapsilopsis. Ann. Clin. Microbiol. Antimicrob. 10: 3, 2011
ARENDRUP, M.; B. LUNDGREN, I. M. JENSEN, et al.: Comparison of Etest and a tablet diffusion testwith the NCCLS broth microdilution method for Àuconazole and amphotericin B susceptibility
testing of Candida isolates. J. Antimicrob. Chemother. 47: 521〜526, 2001
CUENCA-ESTRELLA, M.; C. B. MOORE, F. BARCHIESI, et al.: AFST Subcommittee of the European
Committee on Antimicrobial Susceptibility Testing: Multicenter evaluation of the reproducibilityof the proposed antifungal susceptibility testing method for fermentative yeasts of the Antifungal
Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility
Testing (AFST-EUCAST). Clin. Microbiol. Infect. 9: 467〜474, 2003
LOZANO-CHIU, M.; S. ARIKAN, V. L. PAETZNICK, et al.: Optimizing voriconazole susceptibility testing of Candida: effects of incubation time, endpoint rule, species of Candida, and level of Àuconazole susceptibility. J. Clin. Microbiol. 37: 2755〜2759, 1999
BARCHIESI, F.; M. DEL POETA, V. MORBIDUCCI, et al.: Turbidimetric and visual criteria for determining the in vitro activity of six antifungal agents against Candida spp. and Cryptococcus neoformans. Mycopathologia 124: 19〜25, 1993
PRICE, M. F.; M. T. LAROCCO & L. O. GENTRY: Fluconazole susceptibilities of Candida species andOct. 2012

20)

21)

22)

23)

24)

25)

THE JAPANESE JOURNAL OF ANTIBIOTICS

65—5

347（ 57 ）distribution of species recovered from blood cultures over a 5-year period. Antimicrob. Agents
Chemother. 38: 1422〜1424, 1994
NGUYEN, M. H. & C. Y. YU: InÀuence of incubation time, inoculum size, and glucose concentrations on spectrophotometric endpoint determinations for amphotericin B, Àuconazole, and itraconazole. J. Clin. Microbiol. 37: 141〜145, 1999
ESPINEL-INGROFF, A.; J. L. RODRIGUEZ-TUDELA & J. V. MARTINEZ-SUAREZ: Comparison of two alternative microdilution procedures with the National Committee for Clinical Laboratory Standards
reference macrodilution method M27-P for in vitro testing of Àuconazole-resistant and -susceptible isolates of Candida albicans. J. Clin. Microbiol. 33: 3154〜3158, 1995
ODDS, F. C.; L. VRANCKX & F. WOESTENBORGHS: Antifungal susceptibility testing of yeasts: evaluation of technical variables for test automation. Antimicrob. Agents Chemother. 39: 2051〜2060,
1995
NISHIYAMA, Y.; M. ABE, R. IKEDA, et al.: A study for testing the antifungal susceptibility of yeast by
the Japanese Society for Medical Mycology (JSMM) method. The proposal of the modi¿ed
JSMM method 2009. Jpn. J. Med. Mycol. 51: 153〜163, 2010ARTHINGTON-SKAGGS, B. A.; W. LEE-YANG, M. A. CIBLAK, et al.: Candidemia Active SurveillanceGroup. Comparison of visual and spectrophotometric methods of broth microdilution MIC end
point determination and evaluation of a sterol quantitation method for in vitro susceptibility testing of Àuconazole and itraconazole against trailing and nontrailing Candida isolates. Antimicrob.
Agents Chemother. 46: 2477〜2481, 2002
REVANKAR, S. G.; W. R. KIRKPATRICK, R. K. MCATEE, et al.: Interpretation of trailing endpoints in
antifungal susceptibility testing by the National Committee for Clinical Laboratory Standards
method. J. Clin. Microbiol. 36: 153〜156, 1998