Medical Mycology Advance Access published May 3, 2016

Medical Mycology, 2016, 0, 1–17

doi: 10.1093/mmy/myw024
Advance Access Publication Date: 0 2016
Original Article

Original Article

Antifungal susceptibility of 175 Aspergillus

isolates from various clinical and environmental
sources

Downloaded from http://mmy.oxfordjournals.org/ at Universidad Nacional Autonoma de Mexico on May 27, 2016
Raquel Sabino1,∗ , Elisabete Carolino2 , Cristina Verı́ssimo1 ,
Marife Martinez3 , Karl V. Clemons3,4 and David A. Stevens3,4
1
National Institute of Health Dr. Ricardo Jorge – URSZ- Infectious Diseases Department, Lisbon, Portugal,
2
Scientific Area of Mathematics, Lisbon School of Health Technology – Polytechnic Institute of Lisbon,
Lisbon, Portugal, 3 California Institute for Medical Research, San Jose, CA, United States and 4 Stanford
University School of Medicine, Stanford, CA, United States
∗
To whom correspondence should be addressed. Raquel Sabino, National Institute of Health Dr- Ricardo Jorge, Av- Padre
Cruz, 1649-016 Lisboa, Portugal. Tel: +00351217519247; Fax: +00351217526400; E-mail: raquel.sabino@insa.min-saude.pt;
raquelsabino@hotmail.com
Received 16 October 2015; Revised 26 January 2016; Accepted 21 February 2016

Abstract
Some environmental Aspergillus spp. isolates have been described as resistant to anti-
fungals, potentially causing an emerging medical problem. In the present work, the an-
tifungal susceptibility profile of 41 clinical and 134 environmental isolates of Aspergillus
was determined using the CLSI microdilution method. The aim of this study was to com-
pare environmental and clinical isolates with respect to their susceptibility, and assess
the potential implications for therapy of isolates encountered in different environments.
To our knowledge, this is the first report comparing antifungal susceptibility profiles of
Aspergillus collected from different environmental sources (poultries, swineries, beach
sand, and hospital environment). Significant differences were found in the distribution of
the different species sections for the different sources. Significant differences were also
found in the susceptibility profile of the different Aspergillus sections recovered from the
various sources. Clear differences were found between the susceptibility of clinical and
environmental isolates for caspofungin, amphotericin B and posaconazole, with clinical
isolates showing overall greater susceptibility, except for caspofungin. In comparison to
clinical isolates, hospital environmental isolates showed significantly less susceptibility
to amphotericin B and posaconazole. These data indicate that species section identity
and the site from which the isolate was recovered influence the antifungal susceptibility
profile, which may affect initial antifungal choices.
Key words: Aspergillus, antifungal susceptibility, clinical, environment.

Introduction ity and mortality, especially of solid organ transplant pa-

tients or recipients of allogeneic hematopoietic stem cell
Aspergillus is one of the major fungal threats to im-
transplantation. To exacerbate this problem, intrinsic and
munocompromised patients,1 causing significant morbid-


C The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. 1
All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
2 Medical Mycology, 2016, Vol. 00, No. 00
acquired resistance have been documented and the number
of clinical strains resistant to the most common antifun-
gals is increasing rapidly in some geographic areas.2 Recent
observations suggest that resistance to triazoles among As-
pergillus spp. may be more common than previously ac-
knowledged;3 most resistant isolates harbor azole cross-
resistance.4
Variable antifungal susceptibility profiles among species
have been reported,5 and antifungal resistance in As-
pergillus strains tends to emerge with varying frequencies
in various centers and countries, especially in Europe.6 Re-
duced susceptibility of strains to antifungals has been shown
to be related to higher mortality rates in murine treatment
models and, presumably, in clinical treatment.7,8
Exposure of Aspergillus spp. to antifungal agents via
medical or agricultural use of these compounds appears to
have a major impact on acquisition of resistance to triazoles.
Azole fungicides are used commonly for plant protection by
the European Union member states. Apart from being se-
lected due to local use of azole fungicides, azole-resistant
Aspergillus spp. might be introduced in the environment
via the use of imported azole-treated commercial compost,
an ecological niche that has been suggested as a key com-
ponent in resistance development.9 Moreover, Aspergillus
isolates are frequently recovered from poultry farms; in
these settings, fungi can be exposed to antifungal drugs
regularly used for the prevention of avian mycosis. The
consequence of development of resistance is that patients
at risk can be exposed to and infected by azole-resistant
strains from the environment, which is one of the biggest
recent concerns of the mycological community. Regard-
ing other classes of antifungals, only sporadic resistance
has been detected, although innate amphotericin B resis-
tance of Aspergillus terreus10,11 and Aspergillus nidulans12
is common.
The majority of studies on Aspergillus spp. azole-
resistance have been focused on isolates from the As-
pergillus fumigatus section, Fumigati, whereas few data are
available on antifungal susceptibility patterns of the other
sections.13–15 Furthermore, the detection of azole-resistance
in environmental isolates has been performed but primarily
focused on the Fumigati section. Native resistance to certain
antifungals reinforces the importance of knowing the distri-
bution of antifungal susceptibilities of different species from
different environments.16 This gives insight into whether an
isolate of Aspergillus that one may encounter in various en-
vironments is more or less likely to be drug-susceptible or
resistant, and may also suggest where environmental ma-
nipulations (e.g., use of chemicals) have led to alterations in
the susceptibility of the local flora. Moreover, a profile of
resistance in various environments may be useful in guiding
immunocompromised patients with respect to places they
might particularly avoid.
The goal of the current study was to determine the an-
tifungal susceptibility profile of 175 isolates of Aspergillus
identified as belonging to different sections and collected
from different clinical and environmental sources. Thus,
this study aims to contribute to the knowledge of the epi-
demiology of antifungal resistance patterns of isolates of
Aspergillus collected from different sources and how the
environmental isolates could influence the overall situation
of antifungal resistance by Aspergillus spp.
Materials and methods
Aspergillus isolates
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A total of 175 isolates of various species of Aspergillus were
tested for their susceptibility to five antifungal drugs in this
study. Of these, 41 were from clinical sources, and 134 were
collected from environmental sources in Portugal. Clinical
isolates (one isolate per patient) included 20 from bronchial
secretions or sputum, seven from bronchoalveolar lavage
(BAL), seven from auricular exudates, two from biopsies
(skull and palate), one from ascites fluid, one from stool, one
from nasal sinus aspirate, and two from unknown sources.
Environmental isolates included 36 from sand, 39 from
poultries (air, surfaces and litter) and 29 from swineries (air,
surfaces, and litter), 23 from hospital environments (air and
surfaces), and seven from other sources (food, stones).
Identification of the species sections was based on mor-
phological observation, achieved through macro- and mi-
croscopic characteristics. Identification to species section
level of the 175 isolates was confirmed by sequencing the
ITS1-5.8S-ITS2 region of the ribosomal DNA after PCR
amplification using the universal fungal primers (ITS1 and
ITS4).18 For the Fumigati section (48 isolates), identifica-
tion to species was done by sequencing the calmodulin gene,
as described previously.17 In both cases, the obtained se-
quences were accepted only in case of ≥98% homology
with sequences deposited in the databases (National Cen-
ter for Biotechnology Information and CBS-KNAW Fungal
Biodiversity Centre) that were BLAST searched.
Antifungal agents
The antifungal agents studied were caspofungin (CAS;
Merck & Co. Inc, Whitehouse Station, NJ, USA),
amphotericin B (AMB; Bristol-Myers Squibb Co., Prince-
ton, NJ, USA), itraconazole (ITC; Janssen Pharmaceutica
N.V., Beerse, Belgium), voriconazole (VRC; Roerig Divi-
sion of Pfizer Inc., NY, USA), and posaconazole (PCZ;
Schering Corporation, Kenilworth, NJ, USA). They were
obtained as powders, and stock solutions were prepared in
100% dimethyl sulfoxide for azoles and AMB or in steril-
ized water for CAS. Stock solutions of each drug were kept
at −70◦ C.
Sabino et al.
Antifungal susceptibility testing
Isolates were subcultured on potato dextrose agar slants
(Becton, Dickinson and Company, Sparks MD, USA) for 5
to 7 days at room temperature. The antifungal susceptibil-
ity profiles were determined using a broth dilution reference
method for susceptibility testing of moulds. The M38-A218
protocol from the Clinical and Laboratory Standards In-
stitute was applied for determining the Minimal Inhibitory
Concentrations (MIC) for AMB, VRC, ITC, PCZ, and the
Minimal Effective Concentration (MEC) for CAS. The fi-
nal concentrations of drugs in the wells ranged from 0.5 to
8 μg/ml (ITC), 0.25 to 8 μg/ml (PCZ and VRC), 0.125 to
8 μg/ml (AMB), and 0.39 to 50 μg/ml (CAS). An internal
control strain (Candida kefyr strain SA) with known suscep-
tibility and MEC/MIC ranges was included in each run as a
positive control of the antifungals’ potency. Sabouraud dex-
trose agar plates (Becton, Dickinson and Company, Sparks
MD, USA) were inoculated with the final inoculum to check
the number of colony-forming units in the inoculum.18
Plates were incubated at 35◦ C and examined after 48 h
incubation. Absence of visual growth (for AMB, VRC, ITC,
and PCZ) or aberrant growth (for CAS) defined the MIC or
MEC, respectively. In case of upper end of range of values
of MEC or MIC, the tests were performed a second time to
confirm the previous results. A selected sample of isolates
was tested to assess reproducibility, and all the results varied
by one dilution or less.
Statistical analysis
Comparisons of MIC were done among the various clini-
cal and environmental groups of isolates, as well as species
section, using nonparametric statistical methods. A Mann–
Whitney U test was used to compare the antifungal drug
MICs,19 regardless of Aspergillus species, between two
groups: clinical and environmental isolates (all environ-
mental sources were considered as only one group). A
Kruskal–Wallis test19 was used to compare the MICs for
each antifungal among different sources or among different
species-sections. If significant differences were found with
the initial Kruskal–Wallis comparison, all pairs were then
compared using a Kruskal–Wallis multiple comparison
test.19 A goodness of fit χ 2 for homogeneity was used to de-
termine whether the distribution of the different species sec-
tions (e.g., Fumigati, Flavi, Circumdati, Versicolores, and
Nigri) in each of the sources was skewed. When the assump-
tions of χ 2 test were violated, the Contingency Chi-square
test by Monte Carlo simulation was used.19 A .05 signifi-
cance level was set to attain significance. The software SPSS
(Statistic Package for Social Sciences) V21.0 (IBM Corp.,
Armonk, NY, USA) was used to perform the statistical anal-
3
ysis. Geometric means (based on log2 transformations), me-
dian, and ranges were determined using Excel for Windows
XP.
Results
The distribution of clinical and environmental isolates is
detailed in Table 1.
Comparison of antifungal susceptibility of clinical
and environmental isolates
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The probability of encountering resistance as a dependent
variable of the source of the isolate was studied. The me-
dian and geometric mean (GM) MIC or MEC values for
each antifungal agent against the clinical or environmental
isolates, without regard to their species of Aspergillus, are
shown in Table 2.
Analysis of the susceptibility phenotypes by Mann–
Whitney U test demonstrated clear differences between clin-
ical and environmental isolates. For CAS, a significant dif-
ference (P = .047) was found between the MECs of clini-
cal and environmental isolates, with clinical isolates having
higher values. Although the MEC ranges were identical, the
geometric mean of clinical isolates was three times higher
than that of the environmental isolates (6.3 μg/ml and
2.6 μg/ml, respectively). Twenty-seven (66%) clinical iso-
lates had MEC values for CAS > 6.3 μg/ml, whereas 54
(40%) environmental isolates had MEC values above this
value. When different environmental sources were com-
pared, the MEC ranges were the same for CAS.
Significant differences were found between MICs of
AMB for clinical and environmental sources (P < .001);
environmental strains were less susceptible. Their MICs
ranged up to MIC > 8 μg/ml and the geometric mean was
higher that of the clinical strains (2.2 μg/ml vs. 1.5 μg/ml).
No clinical isolates had MICs > 2 μg/ml to AMB, as
opposed to 31 (23%) environmental isolates with MICs
> 2 μg/ml.
No significant differences (P > .05) were found when
comparing the clinical and environmental groups for their
susceptibility to ITC or VRC. Despite the slight differences
in the MIC ranges, the geometric mean values were similar.
For ITC, 21 (51%) clinical isolates and 72 (54%) of the
environmental isolates had an MIC > 2 μg/ml. For VRC, no
clinical isolate showed a MIC above 1 μg/ml, and only three
environmental isolates showed a MIC value of 4 μg/ml.
For PCZ, a significant difference (P < .001) was found
between the MICs from clinical or environmental sources.
The median and the geometric mean of environmental
strains had higher values. No isolates, other than four en-
vironmental ones, had MICs > 2 μg/ml to PCZ. Those
4 Medical Mycology, 2016, Vol. 00, No. 00

Table 1. Molecular identification and antifungal susceptibility of the Aspergillus isolates collected from different clinical and
environmental sources.

Source Sections MEC CAS MIC AMB MIC ICZ MIC VCZ MIC PCZ
(ITS sequencing) (μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml)

Sputum Fumigati 25 2 4 ≤0.25 ≤0.25

Sputum Fumigati 25 2 2 ≤0.25 0.5
Bronchial secretions Fumigati 25 1 2 ≤0.25 ≤0.25
Bronchial secretions Fumigati 25 1 2 ≤0.25 ≤0.25
Sputum Fumigati ≤0.39 1 4 ≤0.25 ≤0.25
Bronchoalveolar lavage Fumigati 25 1 4 ≤0.25 ≤0.25
Bronchoalveolar lavage Flavi ≥50 2 2 0.5 0.5
Bronchoalveolar lavage Flavi ≤0.39 1 2 ≤0.25 1
Sputum Flavi ≥50 2 2 ≤0.25 0.5

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Sputum Nigri ≤0.39 1 4 ≤0.25 1
Not referred Flavi 25 2 2 ≤0.25 0.5
Bronchial secretions Flavi ≥50 2 2 0.5 0.5
Stool Nigri ≤0.39 1 4 0.5 1
Bronchoalveolar lavage Fumigati 25 1 4 0.5 0.5
Auricular exudate Fumigati 25 1 4 ≤0.25 ≤0.25
Sputum Nigri ≤0.39 1 4 ≤0.25 1
Auricular pus Fumigati 1.6 2 4 ≤0.25 ≤0.25
Bronchial secretions Fumigati 25 2 2 ≤0.25 ≤0.25
Sputum Fumigati 12.5 2 4 ≤0.25 ≤0.25
Sputum Fumigati 12.5 2 4 0.5 0.5
Biopsy palate Fumigati 12.5 2 4 ≤0.25 ≤0.25
Bronchoalveolar lavage Versicolores 25 1 2 ≤0.25 0.5
Sputum Fumigati ≤0.39 2 4 ≤0.25 0.5
Bronchial secretions Fumigati 25 2 4 ≤0.25 ≤0.25
Sputum Fumigati 25 2 1 ≤0.25 ≤0.25
Bronchoalveolar lavage Fumigati 12.5 1 4 0.5 0.5
Sputum Fumigati 25 2 2 0.5 1
Ascitic fluid Fumigati 25 2 4 ≤0.25 ≤0.25
Ear exudate Flavi 25 2 2 ≤0.25 0.5
Bone biopsy Flavi ≥50 2 2 0.5 0.5
Bronchial secretions Flavi ≥50 2 2 0.5 0.5
Not referred Flavi ≤0.39 2 2 0.5 0.5
Ear exudate Flavi ≤0.39 1 1 ≤0.25 ≤0.25
Bronchial secretions Nigri ≤0.39 1 8 1 2
Nasal sinus aspirate Flavi ≤0.39 2 2 ≤0.25 0.5
Bronchoalveolar lavage Fumigati 12.5 2 4 1 0.5
Ear exudate Nigri ≤0.39 1 4 ≤0.25 1
Ear exudate Nigri ≤0.39 1 4 ≤0.25 1
Ear exudate Nigri ≤0.39 2 8 1 2
Bronchial secretions Fumigati ≥50 2 2 0.5 ≤0.25
Sputum Fumigati 12.5 2 1 ≤0.25 ≤0.25
Beach sand Fumigati 25 2 2 1 1
Beach sand Nigri ≤0.39 1 >8 0.5 2
Beach sand Nigri ≤0.39 1 4 ≤0.25 1
Beach sand Versicolores 3.1 4 4 1 1
Beach sand Circumdati ≤0.39 8 2 ≤0.25 2
Beach sand Versicolores ≤0.39 2 4 ≤0.25 1
Beach sand Circumdati 25 2 4 0.5 1
Beach sand Circumdati ≤0.39 >8 2 ≤0.25 ≤0.25
Beach sand Circumdati 0.78 >8 4 ≤0.25 1
Beach sand Nigri ≤0.39 1 4 0.5 1
Beach sand Nigri ≤0.39 1 4 ≤0.25 1
Beach sand Versicolores 25 2 2 ≤0.25 0.5
Sabino et al. 5

Table 1 – continued

Source Sections MEC CAS MIC AMB MIC ICZ MIC VCZ MIC PCZ
(ITS sequencing) (μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml)

Beach sand Circumdati ≤0.39 >8 4 0.5 1

Beach sand Fumigati 25 2 2 0.5 0.5
Beach sand Fumigati 25 2 4 0.5 1
Beach sand Nigri ≤0.39 1 4 ≤0.25 1
Beach sand Fumigati 12.5 2 2 0.5 0.5
Beach sand Nigri ≤0.39 1 4 ≤0.25 1
Beach sand Versicolores ≤0.39 2 4 ≤0.25 1
Beach sand Not identified ≤0.39 2 >8 1 8
Beach sand Circumdati 0.78 >8 4 ≤0.25 1
Beach sand Fumigati 12.5 2 2 ≤0.25 ≤0.25

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Beach sand Fumigati 25 4 1 0.5 0.5
Beach sand Fumigati 12.5 2 1 ≤0.25 0.5
Beach sand Circumdati 1.6 >8 4 ≤0.25 1
Beach sand Fumigati 25 2 2 0.5 0.5
Beach sand Nigri ≤0.39 1 4 ≤0.25 1
Beach sand Circumdati 1.6 >8 4 ≤0.25 1
Beach sand Flavi ≥50 2 2 ≤0.25 0.5
Beach sand Not identified 3.1 4 4 0.5 0.5
Beach sand Fumigati ≤0.39 4 4 ≤0.25 0.5
Beach sand Fumigati 25 2 2 0.5 0.5
Beach sand Nigri ≤0.39 1 4 0.5 1
Beach sand Aspergilli ≤0.39 1 1 ≤0.25 ≤0.25
Beach sand Circumdati 1.6 >8 4 ≤0.25 1
Beach sand Nigri 12.5 1 4 ≤0.25 1
Other environmental Fumigati 25 2 2 ≤0.25 ≤0.25
Other environmental Nigri ≤0.39 1 8 1 1
Other environmental Nigri ≤0.39 1 8 1 0.5
Other environmental Nigri ≤0.39 1 4 0.5 1
Other environmental Fumigati 25 2 2 ≤0.25 ≤0.25
Other environmental Fumigati 12.5 2 2 0.5 ≤0.25
Other environmental Fumigati 12.5 2 2 ≤0.25 ≤0.25
Poultry-Air Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Air Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Air Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Surface Flavi ≤0.39 2 2 ≤0.25 0.5
Poultry-Surface Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Air Flavi ≥50 2 2 0.5 1
Poultry-Surface Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Surface Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Air Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Air Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Litter Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Litter Fumigati 12.5 2 2 ≤0.25 ≤0.25
Poultry-Surface Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Litter Clavati ≤0.39 1 4 0.5 1
Poultry-Surface Versicolores 12.5 4 >8 4 2
Poultry-Surface Versicolores ≤0.39 4 4 0.5 1
Poultry-Litter Fumigati 12.5 2 2 ≤0.25 0.5
Poultry-Air Aspergilli ≤0.39 1 4 1 1
Poultry-Air Candidi ≤0.39 4 2 ≤0.25 1
Poultry-Air Versicolores ≤0.39 4 4 1 2
Poultry-Air Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Air Versicolores ≤0.39 4 >8 0.5 1
6 Medical Mycology, 2016, Vol. 00, No. 00

Table 1 – continued

Source Sections MEC CAS MIC AMB MIC ICZ MIC VCZ MIC PCZ
(ITS sequencing) (μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml)

Poultry-Air Fumigati 12.5 2 2 ≤0.25 0.5

Poultry-Litter Fumigati ≤0.39 2 2 ≤0.25 0.5
Poultry-Surface Fumigati 25 2 2 ≤0.25 0.5
Poultry-Surface Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Air Aspergilli 3.1 2 >8 2 >8
Poultry-Air Versicolores ≤0.39 1 4 ≤0.25 1
Poultry-Air Usti ≤0.39 8 8 1 2
Poultry-Surface Versicolores ≤0.39 1 4 ≤0.25 1
Poultry-Litter Fumigati ≤0.39 2 2 ≤0.25 1
Poultry-Air Versicolores 0.78 2 4 ≤0.25 1

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Poultry-Litter Candidi ≤0.39 1 0.5 ≤0.25 ≤0.25
Poultry-Litter Fumigati ≤0.39 2 2 ≤0.25 ≤0.25
Poultry-Surface Nigri ≤0.39 1 8 0.5 1
Poultry-Surface Fumigati ≤0.39 2 2 ≤0.25 1
Poultry-Litter Versicolores 25 2 4 ≤0.25 1
Poultry-Litter Candidi ≤0.39 1 0.5 ≤0.25 ≤0.25
Poultry-Surface Flavi ≥50 2 2 ≤0.25 0.5
Swinery-Air Flavi ≥50 2 2 ≤0.25 0.5
Swinery-Surface Nigri ≤0.39 1 4 ≤0.25 1
Swinery-Air Fumigati 12.5 2 2 ≤0.25 0.5
Swinery-Surface Candidi ≤0.39 2 2 ≤0.25 1
Swinery-Surface Circumdati ≤0.39 >8 4 ≤0.25 1
Swinery-Surface Circumdati ≤0.39 8 4 ≤0.25 1
Swinery-Air Candidi ≤0.39 2 2 ≤0.25 1
Swinery-Air Fumigati 12.5 2 4 0.5 1
Swinery-Surface Versicolores ≤0.39 2 4 ≤0.25 1
Swinery-Air Fumigati 25 2 2 ≤0.25 0.5
Swinery-Air Fumigati 25 2 2 ≤0.25 1
Swinery-Surface Nigri ≤0.39 1 8 0.5 1
Swinery-Surface Flavi ≥50 2 2 ≤0.25 0.5
Swinery-Air Versicolores ≤0.39 2 4 ≤0.25 1
Swinery-Surface Versicolores 1.6 4 4 ≤0.25 1
Swinery-Air Flavi ≥50 2 2 ≤0.25 0.5
Swinery-Air Flavi 25 2 2 ≤0.25 0.5
Swinery-Air Usti 0.78 4 >8 2 >8
Swinery-Air Versicolores ≤0.39 4 4 ≤0.25 1
Swinery-Air Clavati 12.5 ≤0.5 4 1 2
Swinery-Air Flavi ≥50 2 2 ≤0.25 0.5
Swinery-Air Candidi 1.6 1 1 ≤0.25 ≤0.25
Swinery-Surface Versicolores ≤0.39 2 4 ≤0.25 1
Swinery-Surface Flavi 25 2 2 ≤0.25 0.5
Swinery-Air Versicolores ≤0.39 2 4 ≤0.25 1
Swinery-Air Nigri ≤0.39 1 8 ≤0.25 1
Swinery-Surface Usti ≤0.39 2 8 4 >8
Swinery-Surface Flavi ≥50 2 2 ≤0.25 0.5
Swinery-Surface Versicolores 12.5 4 4 ≤0.25 1
Hospital environment-Air Nigri ≤0.39 1 4 ≤0.25 1
Hospital environment-Surface Nigri ≤0.39 1 4 ≤0.25 1
Hospital environment-Air Nigri ≤0.39 1 4 ≤0.25 1
Hospital environment-Air Nigri ≤0.39 1 4 0.5 1
Hospital environment-Air Flavi ≤0.39 2 2 0.5 0.5
Sabino et al. 7

Table 1 – continued

Source Sections MEC CAS MIC AMB MIC ICZ MIC VCZ MIC PCZ
(ITS sequencing) (μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml)

Hospital environment-Surface Flavi ≥50 2 2 0.5 1

Hospital environment-Surface Flavi ≥50 2 2 0.5 1
Hospital environment-Surface Flavi ≤0.39 2 2 0.5 1
Hospital environment-Air Flavi ≥50 2 2 ≤0.25 0.5
Hospital environment-Air Nidulantes ≤0.39 2 2 ≤0.25 0.5
Hospital environment-Air Not identified ≤0.39 4 4 0.5 0.5
Hospital environment-Air Circumdati ≤0.39 2 4 0.5 1
Hospital environment-Air Circumdati ≤0.39 2 4 ≤0.25 1
Hospital environment-Air Versicolores ≤0.39 2 4 1 2
Hospital environment-Air Aspergilli 1.6 4 8 0.5 1

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Hospital environment-Air Circumdati 6.3 >8 4 ≤0.25 1
Hospital environment-Air Circumdati ≤0.39 >8 4 ≤0.25 1
Hospital environment-Air Circumdati 0.78 >8 4 ≤0.25 1
Hospital environment-Surface Versicolores 25 4 >8 4 2
Hospital environment-Air Versicolores ≤0.39 4 4 0.5 2
Hospital environment-Surface Versicolores 6.3 2 4 0.5 1
Hospital environment-Surface Versicolores ≤0.39 2 4 ≤0.25 0.5
Hospital environment-Air Versicolores ≤0.39 2 4 0.5 1

four isolates showed MICs ≥ 8 μg/ml. In addition, these
same isolates had cross-resistance with ITC (MICs ≥
8 μg/ml); these four isolates were collected from beach sand
(one isolate), poultry (one isolate), or swineries (two iso-
lates). One isolate, collected from a swinery surface, also
showed a reduced susceptibility to VRC (MIC = 4 μg/ml)
(Table 1).
Comparison of antifungal susceptibility of
isolates collected from each source analyzed
Because we had large numbers of isolates from distinct
sources, we compared the susceptibility of the isolates from
the various sources with each other. When comparing the
MECs of CAS for isolates collected amongst all the sources,
significant differences were found (χ K2 W(4) = 9.510,
P = .050), specifically between hospital environmental (P
= .008) or beach sand (P = .048) compared to clinical
isolates. In both instances, clinical isolates showed higher
MEC values (Table S1). Among environmental isolates, a
significant difference was also found between hospital en-
vironmental and isolates from poultries (P = .023), with
the latter isolates having four-times higher MECs to CAS.
Twenty (51%) isolates collected from poultries had a MEC
above 6.3 μg/ml (Table S1).
Significant differences were also found when compar-
ing the MICs for AMB of isolates collected among all the
sources (χ K2 W(4)= 13.097, P = .011), especially between
clinical isolates and isolates from the hospital environment
(P = .029) or isolates from beach sand (P = .049). Interest-
ingly, 12 (33%) environmental isolates with MIC > 2 μg/ml
for AMB were collected from beach sand (Table S1,
Table 2). For collections from both these environments,
the clinical isolates had lower MICs for AMB.
A significant difference was found for PCZ (χ K2 W(4)=
30.857, P ≤ .001) when comparing all the sources. Namely,
between clinical isolates and those from hospital environ-
ment (P < .001), swineries (P < .001), or beach sand (P =
.001). In these comparisons, the environmental isolates had
higher MICs (Table S1).
No significant differences in MIC values were found
among the isolates from different specific sources for the
antifungals ITC and VRC (data not shown).
Comparison of antifungal susceptibility from
strains belonging to different species-sections,
regardless of the source
Determination of species section for each of the isolates in
these studies was done by DNA sequencing. These results
showed that nine species sections were represented among
the 175 isolates. The MIC or MEC values for the most rep-
resentative species sections (i.e., Fumigati, Flavi, Nigri, Ver-
sicolores, Circumdati) were determined. The group “other
species sections” includes the species sections Usti, Clavati,
Aspergilli and Candidi (Table 3). Multiple comparison tests
showed significant differences among the MIC or MEC of
those sections (Table S2).
Regardless of the source, the sections Fumigati and
Flavi presented higher MEC values for CAS than did the
other species sections (geometric mean of 10.4 μg/ml and
8 Medical Mycology, 2016, Vol. 00, No. 00

Table 2. Minimal inhibitory concentrations (MIC) or minimal effective concentrations (MEC) of various antifungal drugs against
clinical and environmental isolates of Aspergillus.

Clinical Hospital Poultries Swineries Beach sand Other environments

(N = 41) (N = 23) (N = 39) (N = 29) (N = 35) (N = 8)

MEC or MIC parameters CAS MEC (μg/ml)

Median 25 0.39 12.50 1.60 1.19 18.75
Geometric mean 6.2 1.2 4.4 2.9 2.2 17.7
Range ≤0.39–>50 ≤0.39–>50 ≤0.39–>50 ≤0.39–>50 ≤0.39–>50 12.5–25
No.isolates(%) with MIC > 6.3 μg/ml 27 (65.8) 4 (17.4) 20 (51.3) 13 (44.8) 13 (36.1) 4(100.0)
P-value clinical vs. environmental isolates 0.047∗
AMB MIC (μg/ml)
Median 2 2 2 2 2 2

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Geometric mean 1.5 2.4 2.0 2.1 2.4 2.0
Range 1–2 1–>8 1–8 ≤0.5–>8 1–>8 2
No.isolates(%) with MIC > 2 μg/ml 0 (0.0) 7 (30.4) 6 (15.4) 6 (20.4) 12 (33.3) 0 (0.0)
P-value clinical vs. environmental isolates <0.001∗
ITC MIC (μg/ml)
Median 4 4 2 4 4 2
Geometric mean 2.8 3.5 2.6 3.1 3.0 2.0
Range 1–8 2–>8 ≤0.5–>8 1–>8 1–>8 2
No.isolates(%) with MIC > 2 μg/ml 21 (51.2) 17 (73.9) 13 (33.3) 16 (55.2) 23 (63.9) 0 (0.0)
P-value clinical vs. environmental isolates .4
VRC MIC (μg/ml)
Median 0.3 0.5 0.25 0.25 0.25 0.25
Geometric mean 0.3 0.4 0.3 0.3 0.3 0.3
Range ≤0.25–1 ≤0.25–4 ≤0.25–4 ≤0.25–4 ≤0.25–1 ≤0.25–0.5
No.isolates(%) with MIC > 2 μg/ml 0 (0.0) 1 (4.3) 1 (2.6) 1 (3.4) 0 (0.0) 0 (0.0)
P-value clinical vs. environmental isolates .8
PCZ MIC (μg/ml)
Median 0.5 1 0.5 1 1 0.25
Geometric mean 0.5 0.9 0.7 0.9 0.8 0.2
Range ≤0.25–2 0.5–2 ≤0.25–>8 ≤0.25–>8 ≤0.25–8 0.25
No.isolates(%) with MIC > 2 μg/ml 0 (0.0) 0 (0.0) 1 (2.6) 2 (6.9) 1 (2.8) 0 (0.0)
P-value clinical vs. environmental isolates <0.001∗
∗
Statistically significant differences at 5% significance level using the Mann–Whitney U test.

MEC applies only to caspofungin.

19.5 μg/ml, respectively; Table 3). These values were 10 or
20 times higher than those obtained with isolates belonging
to other species-sections. Isolates from the Circumdati com-
plex showed, on average, three-times higher median MIC
values to AMB (geometric mean = 6.2 μg/ml) than the oth-
ers species-sections (Table 3). Thirteen isolates (81%) in
the Circumdati complex had a MIC to AMB ≥ 8 μg/ml
(Table 4). A MIC value of 8 μg/ml for AMB was detected
for only one other isolate, which belonged to the Usti sec-
tion. That isolate was collected from the air at one poultry
farm and showed a MIC value of 8 μg/ml also for ITC.
The susceptibility to ITC differed among species sections,
with higher values for Nigri complex (geometric mean =
4.9 μg/ml), followed by Versicolores (geometric mean =
4.1 μg/ml), Circumdati (geometric mean = 3.7 μg/ml) and
the other species sections (geometric mean = 3.0 μg/ml).
Interestingly, 27 (100%) isolates in the Nigri complex and
24 (92%) isolates in the Versicolores complex had MIC
values ≥4 μg/ml for ITC (Table 4). The isolates belonging
to Fumigati and Flavi presented the lowest MIC values to
ITC (geometric mean = 2.4 and 2.0 μg/ml, respectively).
For VRC, the Versicolores and Usti sections had isolates
with the highest MIC values to VRC (4 μg/ml). One of the
four isolates that presented a MIC value of ≥8 μg/ml for
both ITC and PCZ could not be identified to the species
level but the other three were identified as belonging to the
Usti and Aspergilli sections. Two other isolates, identified
as A. versicolor and A. sidowii, presented ITC MICs val-
ues ≥8 μg/ml, together with high MICs to VRC (4 μg/ml)
(Table 4).
Sabino et al. 9

Table 3. Distribution of minimal inhibitory concentrations (MIC) and minimal effective concentrations (MEC)(μg/ml) among the
different Aspergillus sections.

Fumigati Flavi Nigri Versicolores Circumdati Other sections

(N = 48) (N = 39) (N = 27) (N = 26) (N = 16) (N = 19)

CAS
Median 18.75 50.00 ≤0.39 ≤0.39 0.59 0.39
Geometric mean 10.36 19.49 0.44 1.26 0.89 0.70
Range ≤0.39–50 ≤0.39–50 ≤0.39–12.5 ≤0.39–25 ≤0.39–25 ≤0.39–12.5
AMB
Median 2.00 2.00 1.00 2.00 >8.00 2.00
Geometric mean 1.86 1.93 1.03 2.41 6.17 1.93
Range 1–4 1–2 1–2 1–4 2–>8 ≤0.5–8

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ITC
Median 2.00 2.00 4.00 4.00 4.00 4.00
Geometric mean 2.41 1.96 4.91 4.11 3.67 2.99
Range 1–4 1–2 4–>8 2–>8 1–2 ≤0.5–>8
VRC
Median ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.25 0.50
Geometric mean 0.33 0.30 0.38 0.41 0.28 0.55
Range ≤0.25–1 ≤0.25–0.5 ≤0.25–1 ≤0.25–4 ≤0.25–0.5 ≤0.25–4
PCZ
Median 0.50 0.50 1.00 1.00 1.00 1.00
Geometric mean 0.43 0.54 1.05 1.05 0.96 1.12
Range ≤0.25–1 ≤0.25–1 0.5–2 0.5–2 ≤0.25–2 ≤0.25–>8

MEC applies only to caspofungin.

Distribution of species sections in different
environments
The analysis of differences in encountering resistance in dif-
ferent environments just elucidated appear to be dependent
on differences in resistance among species-sections, and the
different distribution of species sections in various environ-
ments. The goodness of fit χ 2 for homogeneity test was
used to verify that the distribution of the different sections
(Fumigati, Flavi, Circumdati, Versicolores, and Nigri) for
the different sources was not homogeneous (χ42 = 19.833,
P = .001). Regarding these data, the Contingency Chi-
square test by Monte Carlo simulation was used to analyze
the existence of significant differences in species-sections’
distribution among different sources. It was concluded that
there were significant differences among the different envi-
ronments (P = .000105, C.I.95% = (0.00009; 0.00012)).
Section Fumigati was more common in clinical (53.7%)
and beach sand (20.8%) sources, the Flavi species section
was more frequently found among clinical (28.2%) and
poultry (38.5%) isolates, whereas Circumdati species sec-
tion was more frequent among the isolates recovered from
beach sand (56.3%) and the hospital environment (31.3%).
Isolates belonging to the Versicolores species section were
more frequently found in isolates recovered from poultry
houses (30.8%) or swineries (26.9%), and the isolates be-
longing to the species section Nigri were more frequent in
the beach sand isolates (33.3%) or clinical (25.9%) sources
(Table 4).
Differences in susceptibility of species sections
from different sources
To evaluate if there were differences in the susceptibil-
ity profile of the species-sections recovered from the var-
ious sources, we applied a Kruskal–Wallis test. Statisti-
cally significant differences were found for Fumigati and
Flavi species-sections). Regarding the Fumigati complex,
differences were found for AMB (χ K2 W (3) = 10.381, P =
0.016), with isolates from beach sand having the highest
MIC and the clinical ones the lowest (P = .015); for ITC
(χ K2 W (3) = 8.853, P = 0.031), with clinical isolates having
the highest MIC and those from the poultry and beach sand
(P = .015 and P = .02, respectively) the lowest; for VRC
(χ K2 W (3) = 10.329, P = 0.016), with isolates from beach
sand having the highest MIC and the poultry ones the
lowest (P = .011); and for PCZ (χ K2 W (3) = 14.581, P =
0.002) with the isolates collected from swineries and beach
sand having the highest MIC and the clinical isolates the
 10

Table 4. Distribution of minimal inhibitory concentrations (MIC) and minimal effective concentrations (MEC)(μg/ml) among the different Aspergillus sections and among
sources.
Fumigati (N = 48) Flavi (N = 39)
Sand Other Sand Other
Clinical Hospital Poultries Swineries beach environments Clinical Hospital Poultries Swineries beach environments
(N = 22) (N = 0) (N = 8) (N = 4) (N = 10) (N = 4) (N = 11) (N = 5) (N = 15) (N = 7) (N = 1) (N = 0)
MEC or MIC parameters CAS MEC (μg/ml) CAS MEC (μg/ml)
Median 25 – 6.4 18.7 25 18.7 25 50 50 50 50 –
Geometric mean 12.9 – 2.4 17.7 13.4 17.7 7.5 7.2 36.2 41.0 50 –
Range ≤0.39–50 – ≤0.39–25 12.5–25 ≤0.39–25 12.5–25 ≤0.39–50 ≤0.39–50 ≤0.39–50 25–50 50 –
No. isolates (%) with MIC>6.3 μg/ml 19 (86) – 4 (50) 4 (100) 9 (90) 4 (100) 7 (64) 3 (60) 1 (7) 7 (100) 1 (100) –

AMB MIC (μg/ml) AMB MIC (μg/ml)

Median 2 – 2 2 2 2 2 2 2 2 2 –
Geometric mean 1.6 – 2 2 2.3 2 1.8 2 2 2 2 –
Range 1–2 – 2 2 2–4 2 1–2 2 2 2 2 –
No. isolates (%) with MIC > 2 μg/ml 0 (0) – 0 (0) 0 (0) 2 (20) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) –
ITC MIC (μg/ml) ITC MIC (μg/ml)
Median 4 – 2 2 2 2 2 2 2 2 2 –
Geometric mean 2.9 – 2 2.4 2 2 1.9 2 2 2 2 –
Range 1-4 – 2 2–4 1–4 2 1–2 2 2 2 2 –
No. isolates (%) with MIC > 2 μg/ml 14 (64) – 0 (0) 1 (25) 2 (20) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) –
VRC MIC (μg/ml) VRC MIC (μg/ml)
Median 0.25 – 0.25 0.25 0.5 0.25 0.25 0.5 0.25 0.25 0.25 –
Geometric mean 0.31 – 0.25 0.30 0.43 0.30 0.34 0.43 0.26 0.25 0.25 –
Range ≤0.25–1 – ≤0.25 ≤0.25–0.5 ≤0.25–1 ≤0.25–0.5 ≤0.25–0.5 ≤0.25–0.5 ≤0.25–1 ≤0.25 ≤0.25 –
No. isolates (%) with MIC > 2 μg/ml 0 (0) – 0 (0) 0 (0) 2 (20) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) –
PCZ MIC (μg/ml) PCZ MIC (μg/ml)
Median 0.25 – 0.5 0.75 0.5 0.25 0.5 1 0.5 0.5 0.5 –
Geometric mean 0.32 – 0.5 0.70 0.54 0.25 0.5 0.75 0.52 0.5 0.5 –
Range ≤0.25-1 – ≤0.25-1 0.5-1 ≤0.25-1 ≤0.25 ≤0.25-1 0.5-1 0.5-1 0.5 0.5 –
No. isolates (%) with MIC > 2 μg/ml 0 (0) – 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) –

Nigri (N = 27) Versicolores (N = 26)

Clinical (N = 7) Hospital Poultries Swineries Sand beach Other environments Clinical Hospital Poultries Swineries Sand beach Other environments
(N = 4) (N = 1) (N = 3) (N = 9) (N = 3) (N = 1) (N = 6) (N = 8) (N = 7) (N = 4) (N = 0)

CAS MEC (μg/ml) CAS MEC (μg/ml)

Median 0.39 0.39 0.39 0.39 0.39 0.39 25 0.39 0.40 0.39 1.7 –
Geometric mean 0.39 0.39 0.39 0.39 0.57 0.39 25 1.2 1.1 0.78 1.8 –
Range ≤0.39 ≤0.39 ≤0.39 ≤0.39 ≤0.39–12.5 ≤0.39 25 ≤0.39–25 ≤0.39–25 ≤0.39–12.5 ≤0.39–25 –
No. isolates (%) with MIC > 6.3 μg/ml 0 (0) 0 (0) 0 (0) 0 (0) 1 (14) 0 (0) 1 (100) 1 (17) 2 (25) 1 (14) 1 (25) –
Medical Mycology, 2016, Vol. 00, No. 00

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Table 4 – continued
Sabino et al.

AMB MIC (μg/ml) AMB MIC (μg/ml)

Median 1 1 1 1 1 1 1 2 3 2 2 –
Geometric mean 1.1 1 1 1 1 1 1 2.5 2.4 2.7 2.4 –
Range 1–2 1 1 1 1 1 1 2–4 1–4 2–4 2–4 –
No. isolates (%) with MIC > 2 μg/ml 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (33) 4 (50) 3 (43) 1 (25) –
ITC MIC (μg/ml) ITC MIC (μg/ml)
Median 4 4 8 8 4 8 2 4 4 4 4 –
Geometric mean 4.9 4 8 6.3 4.32 6.3 2 4.5 4.8 4 3.4 –
Range 4–>8 4 8 4–8 4–>8 4–8 2 4–>8 4–>8 4 2–4 –
No. isolates (%) with MIC > 2 μg/ml 7 (100) 4 (100) 1 (100) 3 (100) 9 (100) 3 0 (0) 6 (100) 8 (100) 7 (100) 3 (75) –
VRC MIC (μg/ml) VRC MIC (μg/ml)
Median 0.25 0.25 0.5 0.25 0.25 1 0.25 0.5 0.4 0.25 0.25 –
Geometric mean 0.41 0.3 0.5 0.31 0.31 0.79 0.25 0.7 0.5 0.25 0.35 –
Range ≤0.25–1 ≤0.25–0.5 0.5 ≤0.25–0.5 ≤0.25–0.5 0.5–1 ≤0.25 ≤0.25–4 ≤0.25–4 ≤0.25 ≤0.25–1 –
No. isolates (%) with MIC > 2 μg/ml 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (17) 1 (13) 0 (0) 0 (0) –
PCZ MIC (μg/ml) PCZ MIC (μg/ml)
Median 1 1 1 1 1 1 0.5 1.5 1 1 1 –
Geometric mean 1.2 1 1 1 1.1 0.79 0.5 1.29 1.2 1 0.84 –
Range 1–2 1 1 1 1–2 0.5–1 0.5 0.5–2 1–2 1 0.5–1 –
No. isolates (%) with MIC > 2 μg/ml 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) –

Circumdati (N = 16) Other sectionsa (N = 19)

Clinical Hospital Poultries Swineries Sand beach Other environments Clinical Hospital Poultries Swineries Sand beach Other environments
(N = 0) (N = 5) (N = 0) (N = 2) (N = 9) (N = 0) (N = 0) (N = 3) (N = 7) (N = 6) (N = 2) (N = 1)

CAS MEC (μg/ml) CAS MEC (μg/ml)

Median – 1 – 0.39 0.78 – – 0.39 0.39 0.6 0.39 3.1
Geometric mean – 0.75 – 0.39 1.2 – – 0.62 0.52 0.99 0.39 3.1
Range – ≤0.39–6.3 – ≤0.39 ≤0.39–25 – – ≤0.39–1.6 ≤0.39–3.1 ≤0.39–12.5 ≤0.39 3.1
No. isolates (%) with MIC > 6.3 μg/ml – 0 (0) – 0 (0) 1 (11) – – 0 (0) 0 (0) 1 (17) 0 (0) 0 (0)
AMB MIC (μg/ml) AMB MIC (μg/ml)
Median – 8 – 8 8 – – 4 1 2 1.5 4
Geometric mean – 4.6 – 8 6.9 – – 3.17 1.8 1.6 1.4 4
Range – 2–>8 – 8–>8 2–>8 – – 2–4 1–8 0.5–4 1–2 4
No. isolates (%) with MIC > 2 μg/ml – 3 (12) – 2 (100) 8 (89) – – 2 (67) 2 (29) 1 (17) 0 (0) 1 (100)
ITC MIC (μg/ml) ITC MIC (μg/ml)
Median – 4 – 4 4 – – 4 4 3 4.5 4
Geometric mean – 4 – 4 3.4 – – 4 2.4 3.2 2.8 4
Range – 4 – 4 2–4 – – 4 ≤0.5–>8 1–>8 1–>8 4
No. isolates (%) with MIC > 2 μg/ml – 4 (80) – 2 (100) 7 (78) – – 3 (100) 4 (57) 3 (50) 1 (50) 1 (100)
11

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12 Medical Mycology, 2016, Vol. 00, No. 00

0 (0)

0 (0)
lowest MIC (P = .024 and P = .027, respectively) (data not

0.5
0.5
0.5

0.5
0.5
0.5
shown).
In the Flavi complex, significant differences were
found for VRC (χ K2 W (4) = 15.205, P = 0.004) and PCZ

≤0.25–1

≤0.25–8
1 (50)
0 (0)
0.6
0.5

1.4
4
(χ K2 W (3) = 10.161, P = 0.038). For VRC, clinical isolates
showed significantly higher MICs than isolates recovered
from poultries and swineries (P = .023 and P = .041, re-

≤0.25–>8
≤0.25–4 spectively) and isolates collected from hospital environment
1 (17)

2 (33)
VRC MIC (μg/ml)
0.6
0.7

1.5
1.8
PCZ MIC (μg/ml)
presented significantly higher MICs than poultry isolates
(P = .001). For PCZ, hospital environmental isolates had
the highest MIC and were significantly different from clin-
ical (P = .037) or poultry isolates (P = .012) (data not
≤0.25–>8
≤0.25–1

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shown). Regarding the species-sections Circumdati, Ver-
1 (14)
0 (0)
0.55
0.5

1
1

sicolores and Nigri, no statistically significant differences

were found when analyzing the distribution of their suscep-
tibility profile among different sources (P > .05) (data not
shown).
≤0.25–0.5

0.5–1
0 (0)

0 (0)
0.40

0.63

These differences in susceptibility of the species sections

0.5

0.5

from different sources may be related to differences in vari-

ous species comprising the same complex and with different
susceptibility profiles, and how these species may be dis-
–
–
–

–
–

–
–

tributed differently among sections in different locales. To

assess this possibility, molecular determination of species
–
–
–
–

–
–
–
–

was performed for the isolates that belong to the species-

complex most frequently found in this study, Fumigati. We
≤0.25–0.5

≤0.25–1

found only three out of 48 (6%) isolates were not A. fumi-

0 (0)

0 (0)
0.25
0.29

0.92
1

gatus sensu stricto. Their susceptibility profile did not show

higher MICs in comparison to the remaining isolates of the
same complex (Table 5).
VRC MIC (μg/ml)

PCZ MIC (μg/ml)

Overall, these data are indicative that species complex

≤0.25
0 (0)

0 (0)
0.25
0.25

1
1
1

identity and site from where the isolate was recovered in-
fluence the antifungal susceptibility profile.
-
–
–
–
–

–
–
–

Discussion
≤0.25–0.5

Little is known about the epidemiology of antifungal sus-

0 (0)

0 (0)
0.25
0.28

ceptibility patterns of Portuguese isolates of Aspergillus spp.

1
1
1

and few studies involving Portuguese isolates belonging to

Other sections include Usti, Clavati, Aspergilli and Candidi.

different Aspergillus sections and collected from different

environments have been performed. In work by Lockhart
–
–
–
–

–
–
–
–

et al.20 , one of the azole-resistant isolates of A. fumigatus,

with an unknown mechanism of resistance, included in the
No. isolates (%) with MIC > 2 μg/ml

No. isolates (%) with MIC > 2 μg/ml

ARTEMIS Global Surveillance Study was collected in Por-

tugal.
MEC applies only to caspofungin.

We addressed the possible influence of environmental

isolates on the overall situation of Aspergillus antifungal
Table 4 – continued

resistance and the comparison among clinical and environ-

mental groups in this study. The environmental sources se-
Geometric mean

Geometric mean

lected were based on the criteria of probable higher As-

pergillus contamination or level of exposure (poultries,
Median

Median
Range

Range

swineries, beach sand) but also based on the high rele-

vance to human health (hospital environment). AMB, ITC,


a
Sabino et al. 13

Table 5. Minimal inhibitory concentrations (MIC) and minimal effective concentrations (MEC) (μg/ml) of the Fumigati isolates
identified to species by sequencing.

Source Sections Species identification CAS MEC AMB MIC ICZ MIC VCZ MIC PCZ MIC
(ITS sequencing) (Calmodulin sequencing) (μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml)

Clinical Fumigati A. fumigatus sensu stricto 25 2 4 ≤0.25 ≤0.25

Clinical Fumigati A. fumigatus sensu stricto 25 2 2 ≤0.25 0.5
Clinical Fumigati A. fumigatus sensu stricto 25 1 2 ≤0.25 ≤0.25
Clinical Fumigati A. fumigatus sensu stricto 25 1 2 ≤0.25 ≤0.25
Clinical Fumigati A. fumigatus sensu stricto ≤0.39 1 4 ≤0.25 ≤0.25
Clinical Fumigati A. fumigatus sensu stricto 25 1 4 ≤0.25 ≤0.25
Clinical Fumigati A. fumigatus sensu stricto 25 1 4 0.5 0.5
Clinical Fumigati A. fumigatus sensu stricto 25 1 4 ≤0.25 ≤0.25
Clinical Fumigati A. fumigatus sensu stricto 1.6 2 4 ≤0.25 ≤0.25

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Clinical Fumigati A. fumigatus sensu stricto 25 2 2 ≤0.25 ≤0.25
Clinical Fumigati A. fumigatus sensu stricto 12.5 2 4 ≤0.25 ≤0.25
Clinical Fumigati A. fumigatus sensu stricto 12.5 2 4 0.5 0.5
Clinical Fumigati A. fumigatus sensu stricto 12.5 2 4 ≤0.25 ≤0.25
Clinical Fumigati A. fumigatus sensu stricto ≤0.39 2 4 ≤0.25 0.5
Clinical Fumigati A. fumigatus sensu stricto 25 2 4 ≤0.25 ≤0.25
Clinical Fumigati A. fumigatus sensu stricto 25 2 1 ≤0.25 ≤0.25
Clinical Fumigati A. fumigatus sensu stricto 12.5 1 4 0.5 0.5
Clinical Fumigati A. fumigatus sensu stricto 25 2 2 0.5 1
Clinical Fumigati A. fumigatus sensu stricto 25 2 4 ≤0.25 ≤0.25
Clinical Fumigati A. fumigatus sensu stricto 12.5 2 4 1 0.5
Clinical Fumigati A. fumigatus sensu stricto >50 2 2 0.5 ≤0.25
Clinical Fumigati A. fumigatus sensu stricto 12.5 2 1 ≤0.25 ≤0.25
Beach sand Fumigati A. lentulus 25 2 2 1 1
Beach sand Fumigati Neosartorya fischeri 25 2 2 0.5 0.5
Beach sand Fumigati A. fumigatus sensu stricto 25 2 4 0.5 1
Beach sand Fumigati A. fumigatus sensu stricto 12.5 2 2 0.5 0.5
Beach sand Fumigati A. fumigatus sensu stricto 12.5 2 2 ≤0.25 ≤0.25
Beach sand Fumigati Neosartorya fischeri 25 4 1 0.5 0.5
Beach sand Fumigati A. fumigatus sensu stricto 12.5 2 1 ≤0.25 0.5
Beach sand Fumigati A. fumigatus sensu stricto 25 2 2 0.5 0.5
Beach sand Fumigati A. fumigatus sensu stricto ≤0.39 4 4 ≤0.25 0.5
Beach sand Fumigati A. fumigatus sensu stricto 25 2 2 0.5 0.5
Other environment Fumigati A. fumigatus sensu stricto 25 2 2 ≤0.25 ≤0.25
Other environment Fumigati A. fumigatus sensu stricto 25 2 2 ≤0.25 ≤0.25
Other environment Fumigati A. fumigatus sensu stricto 12.5 2 2 0.5 ≤0.25
Other environment Fumigati A. fumigatus sensu stricto 12.5 2 2 ≤0.25 ≤0.25
Poultry Fumigati A. fumigatus sensu stricto 12.5 2 2 ≤0.25 ≤0.25
Poultry Fumigati A. fumigatus sensu stricto 12.5 2 2 ≤0.25 0.5
Poultry Fumigati A. fumigatus sensu stricto 12.5 2 2 ≤0.25 0.5
Poultry Fumigati A. fumigatus sensu stricto ≤0.39 2 2 ≤0.25 0.5
Poultry Fumigati A. fumigatus sensu stricto 25 2 2 ≤0.25 0.5
Poultry Fumigati A. fumigatus sensu stricto ≤0.39 2 2 ≤0.25 1
Poultry Fumigati A. fumigatus sensu stricto ≤0.39 2 2 ≤0.25 ≤0.25
Poultry Fumigati A. fumigatus sensu stricto ≤0.39 2 2 ≤0.25 1
Swine Fumigati A. fumigatus sensu stricto 12.5 2 2 ≤0.25 0.5
Swine Fumigati A. fumigatus sensu stricto 12.5 2 4 0.5 1
Swine Fumigati A. fumigatus sensu stricto 25 2 2 ≤0.25 0.5
Swine Fumigati A. fumigatus sensu stricto 25 2 2 ≤0.25 1
14
VRC, PCZ, and CAS have been approved for the treatment
of aspergillosis, but AMB MICs > 2 μg/ml and ITC MICs
> 8 μg/ml are associated with treatment failure and clinical
resistance to these agents.21 In our study, 18% of the strains
analyzed (i.e., 31/134 environmental and 0/41 clinical) had
AMB MICs values > 2 μg/ml, whereas 10% (15/134 en-
vironmental and 2/41 clinical isolates) had ITC MICs ≥
8 μg/ml. Notwithstanding, no isolate belonging to Fumi-
gati had ITC MICs ≥ 8 μg/ml, which was in contrast to
what was reported by other authors.22–26 However, a MIC
value of >2 μg/ml to AMB, ITC, or VRC, or >0.25 μg/ml
for PCZ is used to classify a strain as resistant by Alastruey-
Isquerdo et al.14 Thus, if we consider ITC MICs > 2 μg/ml
as a cutoff for resistance, 53% (72/134 environmental and
21/41 clinical) now would be considered resistant, with 17
(35%) Fumigati isolates included in this category. None
of the clinical isolates showed MICs ≥ 8 μg/ml to VRC
or PCZ and 4/134 environmental isolates showed MICs ≥
8 μg/ml to PCZ. VRC seems to be the most effective an-
tifungal in vitro, with only three strains having MICs ≥
4 μg/ml, which is in accordance to what has been reported
by other authors.27–29
These data raise our awareness of the problem of resis-
tance and to the eventual emergence of resistant strains, es-
pecially those from environmental sources. Our frequency
of azole-resistant isolates is higher than that obtained in
some other countries,21,30,31 as corroborated in another
recent Portuguese study.32 These differences can probably
be explained by the large number of isolates belonging to
non-Fumigati sections, as well as by the large number of
environmental isolates tested in our study. Centers in the
United Kingdom and the Netherlands have described partic-
ularly high frequencies of resistant isolates (15% and 10%,
respectively), and a significant increase in azole resistance,
but specifically for A. fumigatus.34,35
Overall, when clinical and environmental strains were
compared, significant differences were found among sus-
ceptibilities to CAS, AMB and PCZ. Teixeira et al.36 also
compared the MIC values of environmental Aspergillus spp.
strains with clinical isolates and in contrast to our findings,
found that there were no statistically significant differences
except for ITC MIC values of environmental strains, which
were statistically higher than clinical isolates, in opposition
to our results.
Similar to our findings, Araújo et al.16 detected signifi-
cant differences between clinical and environmental strains,
but only in non-fumigatus strains, with clinical isolates of
A. flavus showing significantly higher MIC values to AMB
and ITC. In contrast, our study revealed differences in the
geometric mean values (μg/ml) obtained when testing clin-
ical and environmental isolates of Fumigati section to CAS
(12.9 vs. 7.5), AMB (1.6 vs. 2.1), ITC (2.9 vs. 2.0), and PCZ
Medical Mycology, 2016, Vol. 00, No. 00
(0.32 vs. 0.45), respectively. Regarding non-fumigatus iso-
lates, those clinical isolates belonging to the Flavi complex
presented a significantly lower geometric mean MEC value
to CAS when compared to environmental isolates (7.5 vs.
37.6 μg/ml, respectively). These differing results between
publications may reflect the source of the tested environ-
mental strains. In the study performed by Araújo et al.16 , al-
most all environmental strains were collected from the hos-
pital environment, whereas in our study the source of envi-
ronmental strains was diverse, with different selective pres-
sures. In specific environments (e.g., poultries), Aspergillus
spp. can be regularly exposed to several other, industrially
Downloaded from http://mmy.oxfordjournals.org/ at Universidad Nacional Autonoma de Mexico on May 27, 2016
used, antifungal drugs [Wang D, Gricourt M, Arné P, et
al. May avian farms constitute a source of azole resistant
Aspergillus fumigatus? 5th Advances Against Aspergillosis,
Istanbul, Turkey, 2012, Abstr. No. 8], resulting in higher
MICs when exposed to similar molecules, and thus often in-
ducing resistance to clinical antifungals. To our knowledge,
this is the first report comparing antifungals susceptibilities
from Aspergillus spp. collected in different environmental
sources.
Isolates from the hospital environment frequently
showed high MIC values to ITC (74% with MICs
> 2 μg/ml). In comparison to clinical isolates, hospital en-
vironmental isolates showed significantly reduced suscepti-
bility to AMB and PCZ. Thus, in contrast to Araújo et al.16 ,
resistant strains of Aspergillus spp. appear to be frequent
in this environment, which implies a potentially dangerous
environment, especially to immunocompromised patients.
Several cases of cross-resistance were identified during
our study. All of them were detected in environmental
strains. The mechanisms of resistance to the triazoles have
been studied best in A. fumigatus and involve mutations
in the cyp51A gene encoding the target enzyme, as well as
efflux pumps and the overexpression of cyp51A3 . The po-
sition of the mutation may confer resistance to one, two or
more azoles.35,37 Cross-resistance patterns in isolates with
M220 alterations appear to be related to a reduced suscep-
tibility to PCZ and/or VRC as well as ITC.22,38 In our study
one isolate presented an interesting resistance pattern, with
a MIC ≥8 μg/ml to both ITC and AMB. This isolate belongs
to the Usti complex. Future species identification based on
calmodulin and β-tubulin sequencing is required in order
to detect cryptic species from the Aspergillus ustus section.
The more recently described Aspergillus calidoustus39 is
characterized by poor susceptibilities to several antifungal
classes,39,40 which may explain our result.
Epidemiological cutoff values (ECVs) have been recently
suggested as a means of distinguishing wild-type (WT) iso-
lates of Aspergillus spp. from those that may exhibit ac-
quired resistance mechanisms. ECVs were proposed for
ITC, PCZ, VRC, and AMB against A. fumigatus, A. flavus,
Sabino et al.
A. nidulans, A. niger, A. terreus, and A. versicolor.5,6 Ac-
cording to these ECVs, the percentages of non-WT (not
susceptible) isolates for AMB, ITC, VRC, and PCZ, respec-
tively, in our study were as follows: A. fumigatus (5%,
92%, 0% and 15%), A. flavus (0%, 97%, 0%, and 97%),
A. niger (0%, 100%, 0%, and 96%), and A. versicolor
(39%, 92%, 8%, and 19%). The strains analyzed in our
study include not only the sensu stricto species but probably
also cryptic species within the studied sections, which may
heighten the MIC values obtained for a section and may also
explain differences in the percentages of non-WT isolates
in our work, when compared to published data from other
authors.16,21 Furthermore, the great majority of our strains
came from different environmental sources, subjected to
different and unknown pressures, which could lead to local
shifts in the epidemiology of antifungal resistance patterns.
According to our obtained values, VRC seems to be the
most active agent against all the species-sections studied,
with only two strains (Versicolores complex) considered as
non-WT. Low VRC MICs were also described in other re-
ports from Spain41 and Greece42 and contrast with studies
published in the United States where 4.1% of the tested
Aspergillus isolates had MICs of >4 μg/ml to VRC,43 or
with a recent Dutch survey, where 82 clinical azole-resistant
isolates were all resistant to ITC (MIC > 2 μg/ml), 79%
were resistant to VRC (MIC > 2 μg/ml), and 65% to PCZ
(MIC > 0.25 μg/ml) (EUCAST breakpoints).44 These data,
taken together, suggest geographic heterogeneity in As-
pergillus resistance trends.42
Our data showed that isolates belonging to Fumigati
complex appear more susceptible to AMB than are isolates
in the other sections (except for Nigri) and to ITC (except
for Flavi), in opposition to what was found by Arabatzis
et al.42 Furthermore, regarding the susceptibilities to CAS,
and also in contrast to the same authors, our data revealed
higher MEC values of the Flavi complex (geometric mean
= 19.49 μg/ml).
Isolates identified as belonging to Nigri complex showed
significantly higher MIC values to ITC, similar to studies
by Howard et al.45 , who found 52% of isolates from Ni-
gri complex with ITC MIC > 8 μg/ml, as well to other
authors.21,46,47
Interestingly, antifungal susceptibility patterns of iso-
lates belonging to Circumdati complex revealed high MICs
to ITC (geometric mean = 3.67 μg/ml) but especially high
to AMB (geometric mean = 6.17 μg/ml). Reduced suscepti-
bility to AMB was also noted by other authors.22,48 Because
of the high prevalence of isolates from Circumdati complex
in the hospital environment, their ability to grow at 37o C,18
and their reduced susceptibility to several antifungals, this
complex is probably underestimated as a potentially dan-
gerous pathogen.
15
With respect to Versicolores complex, our data were
concordant with other authors,21,43 with several isolates
that belong to this complex having reduced susceptibility to
AMB and azoles. Significant differences were found, with
Versicolores having higher ITC and PCZ MIC values than
do the Fumigati and Flavi sections and AMB values higher
than the Nigri complex.
The present study highlights local differences in the sus-
ceptibility patterns to the different antifungals and rein-
forces the utility of routine susceptibility testing in order to
monitor the rate of resistance in clinical and environmental
strains and its potential impact on initial antifungal choices
Downloaded from http://mmy.oxfordjournals.org/ at Universidad Nacional Autonoma de Mexico on May 27, 2016
and therapeutic outcome. Our data reveal high MICs in dif-
ferent species-sections. The large number of non-fumigatus
strains and the large number of isolates collected from di-
verse environments may influence our overall susceptibility
pattern. Noteworthy were the significant differences in the
susceptibilities between clinical and environmental strains,
among different environmental niches and also different
species-sections. Although there were few cryptic species
found among the Fumigati section isolates, species identifi-
cation of isolates is important since different species within
the same complex may display different susceptibility pro-
files. Thus, further molecular analysis will be performed
aiming to detect cryptic species among the other species
sections and to study resistance mechanisms and their dis-
tribution.
Acknowledgements
Raquel Sabino was financially supported by a fellowship from
Fundação para a Ciência e Tecnologia (FCT) Portugal (contract
SFRH/BPD/72775/2010).
The authors thank Vicki Chen for her technical assistance.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and the writing of the paper.
Supplementary Material
Supplementary material is available at Medical Mycology online
(http://www.mmy.oxfordjournals.org/).
References
1. Lin S-J, Schranz J, Teutsch SM. “Aspergillosis case-fatality rate:
systematic review of the literature.” Clin Infect Dis 2001; 32:
358–366.
2. van der Linden JW, Snelders E, Kampinga GA et al. “Clinical
implications of azole resistance in Aspergillus fumigatus, the
16
Netherlands, 2007–2009.” Emerg Infect Dis 2011; 17: 1846–
1854.
3. Pfaller M, Boyken L, Hollis R et al. “Use of epidemiological cut-
off values to examine 9-year trends in susceptibility of Candida
species to anidulafungin, caspofungin, and micafungin.” J Clin
Microbiol 2011; 49: 624–629.
4. Alanio A, Cordonnier C, Bretagne S. “Azole resistance in As-
pergillus fumigatus—current epidemiology and future perspec-
tives.” Curr Fungal Infect Rep 2011; 5: 168–178.
5. Espinel-Ingroff A, Fothergill A, Fuller J et al. “Wild-type MIC
distributions and epidemiological cutoff values for the tria-
zoles and six Aspergillus spp. for the CLSI broth microdilu-
tion method (M38–A2 document).” J Clin Microbiol 2010; 48:
3251–3257.
6. Espinel-Ingroff A, Fothergill A, Fuller J et al. “Wild-type MIC
distributions and epidemiological cutoff values for caspofungin
and Aspergillus spp. for the CLSI broth microdilution method
(M38-A2 document).” Antimicrob Agents Chemother 2011; 55:
2855–2859.
7. Lass-Florl C, Kofler G, Kropshofer G et al. “In-vitro testing of
susceptibility to amphotericin B is a reliable predictor of clini-
cal outcome in invasive aspergillosis.” J Antimicrob Chemother
1998; 42: 497–502.
8. Warnock DW, Arthington-Skaggs BA, Li RK. “Antifungal drug
susceptibility testing and resistance in Aspergillus.” Drug Resist
Update 1999; 2: 326–334.
9. Mortensen KL, Mellado E, Lass-Flörl C et al. “Environmen-
tal study of azole-resistant Aspergillus fumigatus and other as-
pergilli in Austria, Denmark, and Spain.” Antimicrob Agents
Chemother 2010; 54: 4545–4549.
10. Lass-Flörl C, Griff K, Mayr A et al. “Epidemiology and outcome
of infections due to Aspergillus terreus: 10-year single centre
experience.” B J Haematol 2005; 131: 201–207.
11. Tortorano AM, Prigitano A, Dho G et al. “In vitro activity of
amphotericin B against Aspergillus terreus isolates from different
countries.” J Chemother 2008; 20: 756–757.
12. Kontoyiannis DP, Lewis RE. “Antifungal drug resistance of
pathogenic fungi.” Lancet 2002; 359: 1135–1144.
13. Howard SJ. “Multi-resistant aspergillosis due to cryptic species.”
Mycopathologia 2014; 178: 435–439.
14. Alastruey-Izquierdo A, Alcazar-Fuoli L, Cuenca-Estrella M.
“Antifungal susceptibility profile of cryptic species of As-
pergillus.” Mycopathologia 2014; 178: 427–433.
15. Alastruey-Izquierdo A, Mellado E, Pelaez T et al. “Population-
based survey of filamentous fungi and antifungal resistance in
Spain (FILPOP Study).” Antimicrob Agents Chemother 2013;
57: 3380–3387.
16. Araujo R, Pina-Vaz C, Rodrigues AG. “Susceptibility of envi-
ronmental versus clinical strains of pathogenic Aspergillus.” Int
J Antimicrob Agents 2007; 29: 108–111.
17. Sabino R, Verı́ssimo C, Parada H et al. “Molecular screen-
ing of 246 Portuguese Aspergillus isolates among different
clinical and environmental sources.” Med Mycol 2014; 52:
519–529.
18. Clinical Laboratory Standard Institute. “Reference method for
broth dilution antifungal susceptibility testing of filamentous
fungi.” Approved standard. M38-A2. 2008; Clinical Laboratory
Standard Institute, Wayne, PA, USA.
Medical Mycology, 2016, Vol. 00, No. 00
19. Daniel DD, Cross Chad L. Biostatistics: A Foundation for Anal-
ysis in the Health Sciences, 10th edn. New York: John Wiley and
Sons, 2014.
20. Lockhart SR, Frade JP, Etienne KA et al. “Azole resistance in As-
pergillus fumigatus isolates from the ARTEMIS global surveil-
lance study is primarily due to the TR/L98H mutation in the
cyp51A gene.” Antimicrob Agents Chemother 2011; 55: 4465–
4468.
21. Gomez-Lopez A, Garcia-Effron G, Mellado E et al. “In vitro
activities of three licensed antifungal agents against Spanish clin-
ical isolates of Aspergillus spp.” Antimicrob Agents Chemother
2003; 47: 3085–3088.
22. Howard SJ, Webster I, Moore CB et al. “Multi-azole resistance
in Aspergillus fumigatus.” Int J Antimicrob Agents 2006; 28:
Downloaded from http://mmy.oxfordjournals.org/ at Universidad Nacional Autonoma de Mexico on May 27, 2016
450–453.
23. Chen J, Li H, Li R et al. “Mutations in the cyp51A gene and
susceptibility to itraconazole in Aspergillus fumigatus serially
isolated from a patient with lung aspergilloma.” J Antimicrob
Chemother 2005; 55: 31–37.
24. Mann PA, Parmegiani RM, Wei SQ et al. “Mutations in
Aspergillus fumigatus resulting in reduced susceptibility to
posaconazole appear to be restricted to a single amino acid in
the cytochrome P450 14alpha-demethylase.” Antimicrob Agents
Chemother 2003; 47: 577–581.
25. Diaz-Guerra TM, Mellado E, Cuenca-Estrella M et al.
“A point mutation in the 14alpha-sterol demethylase
gene cyp51A contributes to itraconazole resistance in As-
pergillus fumigatus.” Antimicrob Agents Chemother 2003; 47:
1120–1124.
26. Nascimento AM, Goldman GH, Park S et al. “Multiple re-
sistance mechanisms among Aspergillus fumigatus mutants
with high-level resistance to itraconazole.” Antimicrob Agents
Chemother 2003; 47: 1719–1726.
27. Lionakis MS, Lewis RE, Torres HA et al. “Increased fre-
quency of non-fumigatus Aspergillus species in amphotericin
B or triazole pre-exposed cancer patients with positive cul-
tures for aspergilli.” Diagn Microbiol Infect Dis 2005; 52:
15–20.
28. Arikan S, Sancak B, Alp S et al. “Comparative in vitro activities
of posaconazole, voriconazole, itraconazole, and amphotericin
B against Aspergillus and Rhizopus, and synergy testing for Rhi-
zopus.” Med Mycol 2008; 46: 567–573.
29. Messer SA, Jones RN, Fritsche TR. “International surveillance
of Candida spp. and Aspergillus spp.: report from the SENTRY
Antimicrobial Surveillance Program (2003).” J Clin Microbiol
2006; 44: 1782–1787.
30. Misra R, Malik A, Singhal S. “Comparison of the activities of
amphotericin B, itraconazole, and voriconazole against clini-
cal and environmental isolates of Aspergillus species.” Indian
J Pathol Microbiol 2011; 54: 112–116.
31. Mortensen KL, Johansen HK, Fuursted K et al. “A prospective
survey of Aspergillus spp. in respiratory tract samples: preva-
lence, clinical impact and antifungal susceptibility.” Eur J Clin
Microbiol Infect Dis 2011; 30: 1355–1363.
32. Pinto E, Lago M, Branco L et al. “Evaluation of Etest performed
in Mueller-Hinton agar supplemented with glucose for antifun-
gal susceptibility testing of clinical isolates of filamentous fungi.”
Mycopathologia 2014; 177: 157–166.
Sabino et al.
33. Snelders E, van der Lee H, Kuijpers J et al. “Emergence of azole
resistance in Aspergillus fumigatus and spread of a single resis-
tance mechanism.” PLoS Med 2008; 5: e219.
34. Verweij PE, Snelders E, Kema GH. “Azole resistance in As-
pergillus fumigatus: a side-effect of environmental fungicide
use?” Lancet Infect Dis 2009; 9: 789–795.
35. Howard SJ, Cesar D, Anderson MJ et al. “Frequency and evolu-
tion of azole resistance in Aspergillus fumigatus associated with
treatment failure.” Emerg Infect Dis 2009; 15: 1068–1076.
36. Teixeira AB, Silva M, Lyra L et al. “Antifungal susceptibility
and pathogenic potential of environmental isolated filamentous
fungi compared with colonizing agents in immunocompromised
patients.” Mycopathologia 2005; 160: 129–135.
37. Rodriguez-Tudela Jl, Alcazar-Fuoli l, Mellado E. “Epidemiolog-
ical cutoffs and cross-resistance to azole drugs in Aspergillus fu-
migatus.” Antimicrob Agents Chemother 2008; 52: 2468–2472.
38. Mayr A, Lass-Flörl C. “Epidemiology and antifungal resistance
in invasive aspergillosis according to primary disease: review of
the literature.” Eur J Med Res 2011; 16: 153–157.
39. Varga J, Houbraken J, Van Der Lee HA et al. “Aspergillus cali-
doustus sp. nov., causative agent of human infections previously
assigned to Aspergillus ustus.” Eukaryot Cell 2008; 7: 630–638.
40. Alastruey-Izquierdo A, Cuesta I, Houbraken J et al. “In vitro
activity of nine antifungal agents against clinical isolates of As-
pergillus calidoustus.” Med Mycol 2010; 48: 97–102.
41. Guinea J, Recio S, Pelaez T et al. “Clinical isolates of As-
pergillus species remain fully susceptible to voriconazole in the
post-voriconazole era.” Antimicrob Agents Chemother 2008;
52: 3444–3446.
17
42. Arabatzis M, Kambouris M, Kyprianou M et al. “Polypha-
sic identification and susceptibility to seven antifungals of
102 Aspergillus isolates recovered from immunocompromised
hosts in Greece.” Antimicrob Agents Chemother 2011; 55:
3025–3030.
43. Baddley JW, Marr KA, Andes DR et al. “Patterns of susceptibil-
ity of Aspergillus isolates recovered from patients enrolled in the
Transplant-Associated Infection Surveillance Network.” J Clin
Microbiol 2009; 47: 3271–3275.
44. European Centre for Disease Prevention and Control. “Risk as-
sessment on the impact of environmental usage of triazoles on
the development and spread of resistance to medical triazoles in
Aspergillus species.” Technical report. 2013; European Centre
for Disease Prevention and Control, Stockholm, Sweden.
Downloaded from http://mmy.oxfordjournals.org/ at Universidad Nacional Autonoma de Mexico on May 27, 2016
45. Howard SJ, Harrison E, Bowyer P et al. “Cryptic species and
azole resistance in the Aspergillus niger complex.” Antimicrob
Agents Chemother 2011; 55: 4802–4809.
46. Saurav K, Kannabiran K. “In vitro susceptibility pattern and
distribution of Aspergillusspp. in hospitalized patients with
chronic pulmonary infection.” Br J Pharmacol Tox 2010; 1:
45–49.
47. Shi J-Y, Xu Y-C, Shi Y et al. “In vitro susceptibility testing
of Aspergillusspp. against voriconazole, itraconazole, posacona-
zole, amphotericin B and caspofungin.” Chin Med J 2010; 123:
2706–2709.
48. Garcı́a-Martos P, Garcı́a-Agudo L, Gutiérrez-Calzada J et al. “In
vitro activity of amphotericin B, itraconazole and voriconazole
against 20 species of Aspergillus using the Sensititre microdilu-
tion method.” Enferm Infecc Microbiol Clin 2005; 23: 15–18.