Medical Mycology 2000, 38, 23–26 Accepted 9 July 1999

Comparison of the minimum fungicidal concentration

of amphotericin B determined in filamentous fungi by
macrodilution and microdilution methods
I. PUJOL,* C. AGUILAR,† J. FERNÁNDEZ-BALLART‡ & J. GUARRO†
*Laboratori de Microbiologia, Hospital Universitari de Sant Joan de Reus, 43201 Reus, Tarragona, Spain; †Unitat de
Microbiologia, Facultat de Medicina, Universitat Rovira i Virgili, 43201 Reus, Tarragona, Spain; ‡Unitat de Medicina
Preventiva, Facultat de Medicina, Universitat Rovira i Virgili, 43201 Reus, Tarragona, Spain

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Minimum fungicidal concentration (MFC) determination could be useful in severe
fungal infections in immunocompromised patients. No reference tests to determine
the MFC are available, and both macro- and microdilution methods are commonly
used. In this study, discrepancies between minimum inhibitory concentrations
(MICs) and MFCs of amphotericin B against 58 isolates of filamentous fungi other
than Aspergillus (46 Fusarium spp., six Paecilomyces spp. and six Scopulariopsis
spp.), obtained with macro- and microdilution methods, were evaluated. Addition-
ally, the agreement between MFCs obtained by both methods were analysed. In
general MFCs were higher than the corresponding MICs overall using the macrodi-
lution method. MFCs were more than one dilution higher than MICs in 52·3% of the
cases in the macrodilution test and in 20·5% of the cases in microdilution test. The
degree of agreement between MFCs obtained with the two methods was of 70·4%
(Kappa coefficient of 0·5). In general, the macrodilution method showed higher
MFC values than the microdilution method. Differences of up to six drug dilutions
were observed between MFCs obtained by both methods.
Keywords amphotericin B, broth dilution method, filamentous fungi, minimum
fungicidal concentration

Introduction out to develop techniques for filamentous fungi [2–5].

However, in spite of the fact that it has not been demon-
The incidence of opportunistic fungal infections has risen
strated that the minimum inhibitory concentration (MIC)
dramatically over the last few years. The treatment of
would be more predictive for clinical outcome than mini-
most of these infections is not yet fully developed. It is
mum fungicidal concentration (MFC), all these ap-
important to devise reference methods for antifungal
proaches are mostly based on obtaining reproducible
susceptibility testing in 6itro which can serve as guidance
MIC values with no estimation of MFC.
for clinical treatment. The National Committee for Clini-
Tests for bactericidal activity of antimicrobials have
cal Laboratory Standards (NCCLS) has devoted a great
been recommended in several clinical situations, espe-
deal of effort to the development of a reference method
cially in infections in immunocompromised patients [6].
for yeasts [1] and some studies are currently being carried
MFC determination could be useful in severe fungal
infections where the defence mechanisms of the host are
Correspondence: Prof. J. Guarro, Unitat de Microbiologia, Facul-
tat de Medicina, Universitat Rovira i Virgili, Carrer Sant Llorenç,
usually compromised. No reference tests to determine the
21, 43201 Reus, Spain. Tel.: +34 77 759359; fax: + 34 77 759322; MFC of antifungal drugs are available, and both macro-
e-mail: umb@astor.urv.es and microdilution methods are frequently used. At

© 2000 ISHAM
 24 Pujol et al.
present, no study has been conducted to estimate the
agreement between both methods to determine the MFC
of antifungal drugs. In a previous work, we compared the
influence of the broth macro- and microdilution methods
on the MICs of different antifungals against some
filamentous fungi, and a good agreement was reached [4].
In this study we compared the MFC values of ampho-
tericin B obtained with the macro- and microdilution
methods against 58 isolates of filamentous fungi other
than Aspergillus. Discrepancies between MICs and MFCs
obtained in each of the methods were also evaluated.
Materials and methods
Antifungal agent
Fungizone (E.R. Squibb & Sons, Barcelona, Spain), the
commercial preparation of amphotericin B, was used. An
initial stock solution containing 1000 mg ml − 1 of drug
was prepared with sterile distilled water and was stored at
−20 °C until used.
Test organisms
Fifty-eight isolates of filamentous fungi were used in this
study. The isolates included 46 strains of Fusarium spp.
(14 F. oxysporum, nine F. solani, seven F. 6erticillioides,
four F. dimerum, four F. equiseti, three F. proliferatum,
one F. subglutinans, one F. chlamydosporum, one F.
a6enaceum, one F. graminearum and one F. semitectum);
six Paecilomyces spp. (two P. fumoso-roseus, one P. 6ari-
otii, one P. marquandii, one P. carneus and one P.
ni6eus); six Scopulariopsis spp. (two S. koningii, one S.
acremonium, one S. carbonaria and two Scopulariopsis
spp.). P. 6ariotii ATCC 36257 (American Type Culture
Collection, Manassas, VA, USA) was included as the
control organism. The isolates were stored as a suspen-
sion in water at 4 °C and were subcultured onto potato
dextrose agar slants at 25 °C for 10 – 15 days when the
experiment was performed. The majority of isolates of
Fusarium were tested twice on different days and the
remaining strains were tested once.
MFC determination
MICs of amphotericin B were determined by both broth
macro- and microdilution tests as described previously [4]
using RPMI 1640 medium buffered with morpholine-
propanelsulfonic acid (pH 7·0). The inoculum was stan-
dardized spectrophotometrically (at 530 nm and 95% of
transmittance) and viable cell concentrations were deter-
mined by enumeration of the cfu per millilitre on
Sabouraud glucose agar plates (SGA). The amphotericin
B concentrations tests ranged 0·07–36·94 mg ml − 1. The
temperature of incubation in both methods was 25 °C.
MICs were read after 48 h of incubation for Fusarium
spp. and 72 h for Paecilomyces spp. and Scopulariopsis
spp. The MIC was defined as the lowest amphotericin B
concentration at which no growth could be seen. After
MIC readings, tubes and microplates were agitated using
a vortex and an automatic plate shaker, respectively.
Then, 100 ml and 10 ml aliquots were removed from each
growth-negative tube (macrodilution) and well (microdi-
lution), respectively, and were spread on SGA petri
dishes. The plates were incubated at 25 °C and the fungal
colonies grown were counted after approximately 3–4
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days of incubation. The MFC was defined as the lowest
drug concentration from which 5 10 colonies (macrodi-
lution) or 5 1 colony (microdilution) were visible on the
agar plate.
Analysis of results
The MFCs obtained with the two methods were com-
pared. Both on-scale and off-scale MFC values were
included in the analysis. The high off-scale MFCs
(\ 36·94 mg ml − 1) were converted to the next highest
concentration (73·88 mg ml − 1), and the low off-scale
MFCs (5 0·07 mg ml − 1) were left unchanged
(0·07 mg ml − 1). When skips (uneven patterns) were
present, the MIC or MFC endpoint was the higher drug
concentration. Differences between MFC values obtained
by both methods of no more than one dilution (one tube
or well) were used to calculate the percentage of agree-
ment. The Kappa test was used to calculate the degree of
agreement. Kappa coefficients greater than 0·75 represent
excellent agreement, coefficients below 0·40 represent
poor agreement and coefficients between 0·4 and 0·75
represent fair to good agreement.
Results and discussion
It is generally accepted that one of the most important
steps in the realization of bactericidal and fungicidal tests
is inoculum concentration [7]. In this study, using the
spectrophotometric method at 95% transmittance, a wide
variation in the quantification of cfu per millilitre of
inoculum was observed, differing among the genera stud-
ied. The range was from B1 × 103 to 1 – 5 ×105 cfu ml − 1
for Fusarium, from 5–9× 103 to 5 – 9 ×105 cfu ml − 1 for
Paecilomyces and from 5–9× 103 to 5 – 9 ×104 cfu ml − 1
for Scopulariopsis isolates. This was similar to results
obtained with other genera of fungi [4]. This variation
could be due to the fact that the fungal suspensions
obtained were really mixtures of conidia and hyphal
© 2000 ISHAM, Medical Mycology, 38, 23 – 26

Fig. 1 Percentage of agreement between MICs and
MFCs obtained by broth macro- and microdilution
tests
fragments and the different proportions of them can
produce different counts. Despite the fact that in this
study the influence of the inoculum size was avoided
because the same inoculum was used in both methods,
the MFC values represented different killing rates de-
pending on the inoculum size assayed. Considering that
the final inoculum size of approximately 80% of the
strains tested in both methods showed an interval of
1– 5 × 103 to 5 – 9 × 104 cfu ml − 1, the MFC represented a
killing of ]90%.
Some differences between MICs and MFCs were ob-
served (Fig. 1), the latter being generally higher than the
former for each genus assayed and overall using the
macrodilution method. MFCs were more than one dilu-
tion higher than MICs in 52·3% of the cases in the
macrodilution test and 20·5% in the microdilution test.
Discrepancies of this nature between MICs and MFCs of
amphotericin B have also been reported by other investi-
gators [8,9]. Higher MFCs may be accounted for because
amphotericin B becomes less effective after prolonged
incubation in culture media [10].
The range of MFCs and the minimum concentration
of amphotericin B required to kill 50 and 90% of viable
organisms (MFC50 and MFC90, respectively) obtained by
the macro- and microdilution methods for each genus are
Table 1 Minimum fungicidal concentrations obtained by macro- and microdilution methods
Macrodilution
Fungi Tests n Range
Fusarium spp. 76 0·29–\36·94
Scopulariopsis spp. 6 36·94–\36·94
Paecilomyces spp. 6 2·31–\36·94
Data are presented as mg ml−1.
MFC50, minimum concentration of drug required to kill 50% of viable organisms;
MFC90, minimum concentration of drug required to kill 90% of viable organisms.
© 2000 ISHAM, Medical Mycology, 38, 23 – 26
Macro- and microbroth antifungal testing 25
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shown in Table 1. In general, the MFCs obtained with
the macrodilution method were higher than those ob-
tained with the microdilution one. In the three genera of
fungi studied, the MFCs showed a relatively broad range
using both methods, except in the case of Scopulariopsis
spp. in the macrodilution method where high resistance
was always observed. The MFC90 values were off-scale in
both methods for all groups tested (except in the case of
Fusarium).
Table 2 shows the differences between MFCs obtained
with the two methods, which agreed in 70·4% of the cases
(62/88 comparisons), giving a moderate agreement
(Kappa coefficient of 0·52). However, as illustrated, dif-
ferences of up to six drug dilutions were also observed,
which is in contrast to a previous work where good
agreement was found between MICs of amphotericin B
using both methods [4]. Similar results were obtained
when all off-scale MFC results were excluded from the
analysis of agreement (data not shown). The same vari-
ables that influence the MICs can also affect the MFCs,
although other factors such as the type of test tube or the
microtiter plates used, the correct mixing of tubes or
wells prior to subculturing and the time of incubation of
recovery media can also exert a remarkable influence on
results [7].
Microdilution
MFC50 MFC90 Range MFC50 MFC90
2·31 36·94 0·58–\36·94 1·16 9·24
\36·94 \36·94 1·16–\36·94 18·47 \36·94
9·24 \36·94 0·58–\36·94 2·31 \36·94
 26 Pujol et al.
Table 2 Differences between MFCs obtained by macro- and
microdilution methods for 58 isolates of filamentous fungi
No. dilutions of difference No. of comparisons (%)
0 39 (44·3)
1 23 (26·1)
2 14 (15·9)
3 6 (6·8)
4 3 (3·4)
5 2 (2·2)
6 1 (1·1)
In conclusion, after comparing the results obtained
with the two methods of antifungal susceptibility testing
applied, where possible, according the guidelines of the
NCCLS, we have demonstrated that the influence of the
method on MFC is more important than in the case of
MIC. Studies on which of the two methods correlate
better with clinical response are required, especially in
certain types of infections where the MFCs may be more
predictive than MICs.
Acknowledgements
This work was supported by a grant from the Fundació
Ciència i Salut of Spain. We thank Dr F.C. Odds
(Janssen Research Foundation, Beerse, Belgium) for his
critical comments.
References
1 National Committee for Clinical Laboratory Standards. Refer-
ence Method for Broth Dilution Antifungal Susceptibility Test-
ing for Yeasts: Approved Standard. Document M27-A.
Villanova, PA: National Committee for Clinical Laboratory
Standards, 1997.
2 Espinel-Ingroff A, Dawson K, Pfaller M, et al. Comparative
and collaborative evaluation of standardization of antifungal
susceptibility testing for filamentous fungi. Antimicrob Agents
Chemother 1995; 39: 314 – 319.
3 Espinel-Ingroff A, Bartlett M, Bowden R, et al. Multicenter
evaluation of proposed standardized procedure for antifungal
susceptibility testing of filamentous fungi. J Clin Microbiol
1997; 35: 139 – 143.
4 Pujol I, Guarro J, Llop C, Soler L, Fernández-Ballart J.
Comparison study of broth macrodilution and microdilution
antifungal susceptibility tests for the filamentous fungi. Antimi-
crob Agents Chemother 1996; 40: 2106 – 2110.
Downloaded from https://academic.oup.com/mmy/article/38/1/23/948767 by guest on 27 February 2021
5 Pujol I, Guarro J, Sala J, Riba MD. Effects of incubation
temperature, inoculum size, and time of reading on broth
microdilution susceptibility test results for amphotericin B
against Fusarium. Antimicrob Agents Chemother 1997; 41: 808–
811.
6 Swenson JM, Hindler JA, Peterson LR. Special tests for detect-
ing antibacterial resistance. In: Murray PR, Baron EJ, Pfaller
MA, Tenover FC, Yolken RH, eds. Manual of Clinical Micro-
biology. Washington DC: American Society for Microbiology,
1995: 1356 – 1367.
7 Taylor PC, Schoenknecht FD, Sherris JC, Linner EC. Determi-
nation of minimum bactericidal concentrations of oxacillin for
Staphylococcus aureus: influence and significance of technical
factors. Antimicrob Agents Chemother 1983; 23: 142 – 150.
8 Colombo AL, McGough DA, Rinaldi MG. Discrepancies be-
tween MIC and MLC values of amphotericin B against isolates
of Aspergillus species. Mycopathologia 1994; 128: 129 – 133.
9 Denning DW, Hanson LH, Perlman AM, Stevens DA. In vitro
susceptibility and synergy studies of Aspergillus species to con-
ventional and new agents. Diagn Micr Infec Dis 1992; 15:
21 – 34.
10 Cheung SC, Medoff G, Schlessinger D, Kobayashi GS. Stabil-
ity of amphotericin B in fungal culture media. Antimicrob
Agents Chemother 1975; 8: 426 – 428.
© 2000 ISHAM, Medical Mycology, 38, 23 – 26