Invest Clin 64(4): 471 - 481, 2023 https://doi.org/10.54817/IC.v64n4a4

Corresponding author: Xiomara Moreno Calderón. Microbiology Department, Bacteriology Facultad de Medicina,

Escuela de Bioanálisis. Universidad Central de Venezuela. Instituto Médico La Floresta. Caracas, Venezuela. Tel.:

+584143722227. Email: xmorenoc1356@gmail.com

Antifungal susceptibility of Aspergillus

genus determined by the Etest® method:

eleven years of experience at the Instituto

Médico La Floresta. Caracas, Venezuela.

Xiomara Moreno Calderón1,2, Carolina Macero Estévez1 and Débora Oliveira Oliveira1

1Microbiology Department. Instituto Médico La Floresta. Caracas, Venezuela.

2Microbiology Department. Bacteriology. Facultad de Medicina, Escuela de Bioanálisis.

Universidad Central de Venezuela. Caracas, Venezuela.

Keywords: susceptibility; Aspergillus spp; cryptic species; antifungals; Etest diffusion

method; minimal inhibitory concentration.

Abstract. This research aimed to determine the susceptibility of Aspergil-

lus spp. to four antifungal agents using the Etest® method in several clinical

samples (respiratory samples, soft tissue, otic tissue, and ocular tissue, among

others) from a private health center in Venezuela. Thirty-three strains were

evaluated: 11 Aspergillus section Flavi, eight Aspergillus section Fumigati, six

Aspergillus section Nigri, four Aspergillus section Terrei, and four Aspergillus

spp. A 0.5 McFarland standard suspension of a 5-day culture of each Aspergillus

strain was prepared on Potato Dextrose agar and then inoculated on Sabouraud

agar plates with 2% glucose. Voriconazole (VCZ), amphotericin B (AMB), caspo-

fungin (CAS), and posaconazole (PCZ) were tested. Minimal inhibitory concen-

trations (MIC) in μg/mL were determined after 24 and 48 hours of incubation

at 35 °C and th range (R), geometric mean (GM), MIC50, and MIC90 were calcu-

lated. The results for the 33 Aspergillus spp. tested after 24 h were the follow-

ing: VCZ (R = 0.031- 16; GM = 0.145; MIC50 = 0.125 and MIC90 = 0.5), AMB (R

= 0.031-16; GM = 0.644; MIC50 = 0.5 and MIC90 = 8), CAS (R = 0.031-16; GM

= 0.1076; MIC50 = 0.063 and MIC90 = 1), PCZ (R =0.031 - 0.5; GM = 0.0755;

MIC50 = 0.063 and MIC90 = 0.25). This investigation allowed assessing the an-

tifungal susceptibility profiles of Aspergillus spp. isolated from clinical samples

by the Etest® method, which is practical, reproducible and easy to perform in

microbiology laboratories.

472 Moreno et al.

Investigación Clínica 64(4): 2023

Susceptibilidad a los antifúngicos del género Aspergillus

determinada por el método Etest®: once años de experiencia

en el Instituto Médico La Floresta. Caracas, Venezuela.

Invest Clin 2023; 64 (4): 471 – 481

Palabras clave: susceptibilidad; Aspergillus spp.; especies crípticas; antifúngicos;

método de difusión Etest; concentración mínima inhibitoria.

Resumen. El objetivo de esta investigación fue determinar la susceptibili-

dad de Aspergillus spp., a cuatro antifúngicos mediante el método de Etest®,

en aislados clínicos (muestras respiratorias, partes blandas, óticas, y oculares,

entre otras) provenientes de un centro de salud privado en Venezuela. Se eva-

luaron 33 cepas: 11 Aspergillus sección Flavi, ocho Aspergillus sección Fumi-

gati, seis Aspergillus sección Nigri, cuatro Aspergillus sección Terrei y cuatro

Aspergillus spp. Se preparó una suspensión al 0,5 MacFarland a partir de culti-

vos de 5 días de incubación de cada cepa de Aspergillus en agar Papa Dextrosa,

que se inocularon posteriormente en placas de agar Sabouraud con glucosa

al 2%. Los antifúngicos ensayados fueron: voriconazol (VCZ), anfotericina B

(AMB), caspofungina (CAS) y posaconazol (PCZ). Posterior a la incubación a

35 °C, se determinó la Concentración Mínima Inhibitoria en μg/mL (CMI) para

cada antifúngico a las 24 y 48 h. Se calculó el rango (R), media geométrica

(MG), CMI50 y CMI90. Los resultados a las 24 h para las 33 cepas de Aspergillus

fueron: VO (R = 0,031- 16; MG = 0,145; CMI50 = 0,125 y CMI90 = 0,5), AB (R

= 0,031-16; MG = 0,644; MIC50 = 0,5 y MIC90 = 8), CS (R = 0,031-16; MG =

0,1076; MIC50 = 0,063 y MIC90 = 1), PO (R =0,031 - 0,5; MG = 0,0755; MIC50

= 0,063 y MIC90 = 0,25). Esta investigación permitió valorar los perfiles de sus-

ceptibilidad antifúngica en aislamientos clínicos de Aspergillus spp., mediante

el método de Etest®, el cual es práctico, reproducible y fácil de realizar en los

laboratorios de microbiología.

Received: 15-01-2023 Accepted: 18-05-2023

INTRODUCTION

There has been a recent increase in epi-

demiological changes in filamentous fungi

that cause diseases related to cryptic Asper-

gillus species. These species comprised 10

to 15% of Aspergillus isolates in epidemio-

logical inquiries from Spain and the United

States, particularly as the cause of invasive

aspergillosis (IA) 1-3. They are referred to as

“cryptic” due to being sister species whose

morphological distinction is rather complex,

as they exhibit different phenotypic and ge-

notypic characteristics1.

Molecular studies have shown how the

conventional identification method, based

on morphological characteristics, is limited

when it comes to differentiating Aspergillus

species, as evidenced by the fact that such

methodologies could only use one species or

section (such as Fumigati, Flavi, Nidulantes,

Usti, and Terrei) to identify morphologically

identical species that could be separated

through molecular methods 4.

Antifungal susceptibility by Etest method in Aspergillus Genus 473

Vol. 64(4): 471 - 481, 2023

The Aspergillus species most frequently

isolated in a clinical context are A. fumiga-

tus, A. flavus, A. niger, and A. terreus. The

members of the Fumigati section, consisting

of A. fumigatus sensu stricto and its cryptic

species, are the most commonly isolated

from clinical specimens and often from envi-

ronmental sources. Furthermore, resistance

to azoles has increased among clinical sam-

ples of the Fumigati section 5.

The prophylaxis and treatment of inva-

sive aspergillosis are controversial due to its

increasing morbidity and mortality 6. While

voriconazole (VCZ) is the drug of choice, isa-

vuconazole (ISZ) can be used against Asper-

gillus spp., and is considered the most effec-

tive by European guidelines 7,8. Posaconazole

(PCZ) is recommended for primary antifungal

prophylaxis during induction chemotherapy,

immunosuppressive therapy for graft-versus-

host disease after hematopoietic stem cell

transplantation (HSCT), and salvage therapy

for refractory IA 1–5. Lipid formulations of am-

photericin B (AMB) and echinocandins are

an alternative to azoles in aspergillosis treat-

ment 9. However, epidemiological changes,

including cryptic Aspergillus species’ resis-

tance to azoles, are of growing concern 4.

This study evaluated the levels of azoles

(VCZ, PCZ), echinocandins (CAS), and am-

photericin B susceptibility in Aspergillus

species found in human samples using the

Etest® gradient diffusion method.

MATERIAL AND METHODS

Aspergillus isolates

Clinical isolates of Aspergillus spp. were

collected during 11 years (2011-2021) from

patient samples processed in the Instituto

Médico La Floresta microbiology laboratory

in Caracas, Venezuela. Each clinical sample

came from a different patient. The age, gen-

der, and underlying disease of each patient

were recorded. The isolates were preserved

in distilled water with glycerol until the mo-

ment of the study. The different Aspergillus

species’ identification was based on the cri-

teria by De Hoog et al. 10 and Klich et al. 11,

assessing macro and microscopic aspects

from subcultures on Sabouraud Dextrose

Agar (SDA-Oxoid, USA), Mycosel Agar (Ox-

oid, USA), and Potato Dextrose Agar (PDA-

Oxoid, USA), incubated in a temperature

range between 20-30 °C.

In vitro susceptibility using the gradient

diffusion method Etest®

A subculture on PDA agar of each As-

pergillus spp. isolates were made and in-

cubated for five days to prepare a conidia

suspension in 0.85% sterile saline solution.

The conidia concentration was determined

by a Neubauer counting chamber (Hausser

Scientific, Horsham, PA, USA) and standard-

ized at 1 – 5 x 106 CFU/mL (Densimat™

bioMérieux, France) at 530 nm 12-14. Plates

containing Müeller-Hinton Agar, 2% glu-

cose with Methylene blue, were inoculated,

streaked in three directions, and left to dry

for 15 minutes. Etest® strips of each anti-

fungal (AB bioMérieux, France); VCZ, PCZ

(0.002-32 μg/ mL), AMB, and caspofungin

(CAS=0.016-256 μg/ mL) were placed ac-

cording to the manufacturer’s instructions.

Each plate was incubated at 35 °C. MIC was

measured at 24 h, with a maximum time of

48 h, in case the lecture was not possible at

the stipulated time.

Criteria for interpreting the minimum

inhibitory concentration

The MIC was defined as the lowest drug

concentration at which the border of the el-

liptical inhibition zone intercepted the scale

on the antifungal strip. To compare the MICs

obtained during this study with the epide-

miological cut-off values (ECVs) established

by the Clinical and Laboratory Standards In-

stitute (CLSI, M61 document, 2017), they

were placed between two sequential dilu-

tions taken to the subsequent higher dilu-

tion from the reference method. The values

on the strip’s upper end were taken to the

highest concentration allowed, while those

on the lower end were left unchanged. Ac-

474 Moreno et al.

Investigación Clínica 64(4): 2023

cording to de CLSI, ECVs in wild and non-

wild isolates are classified based on the fol-

lowing MICs: VCZ: A. fumigatus=1 μg/mL;

A. flavus, A. niger, and A. terrreus=2 μg/mL.

PCZ: A. flavus=0.5 μg/mL; A. niger=2 μg/

mL, A. terreus=1 μg/mL. AMB: A, flavus and

A. terreus=4 μg/mL; A. fumigatus, A. niger,

and A. versicolor=2 μg/mL. CAS: A. flavus

and A. fumigatus=0.5 μg/mL; A. niger=0.25

μg/mL, and A. terreus=0.12 μg/mL15.

Statistical analysis

A database was created in Excel® 2010.

The data was analyzed through percentages

and central tendency measures: ranges, geo-

metric mean (GM), mode (Mo), and median

(Mdn) for each antifungal. The MIC values

that inhibited 50% (MIC50) and 90% (MIC90)

of the isolates were also calculated.

Quality control

American Type Culture Collection

(ATCC®) control strains were used in or-

der to evaluate the susceptibility tests: A.

fumigatus ATCC® 204305, Candida krusei

ATCC® 6258, and Candida parapsilosis

ATCC® 22019.

RESULTS

The strains analyzed came from 33 pa-

tients, 18 female and 15 male, aged between

2-76 years and an average of 56 years. Thir-

ty-three Aspergillus spp. isolates were iden-

tified, mostly from lower respiratory tract

samples (17;51.5%), followed by isolates

obtained from soft tissue (6;18.2%), ear dis-

charge (4;12.12%), corneal ulcer scraping

(2;6.06%), and one of each one from nasal

septum, peritoneal fluid, bone marrow, and

nail (1;3.03%). Table 1 shows Aspergillus

species identified through phenotypic tests,

isolation place, underlying disease, and MICs

for each tested antifungal.

According to the ECV of CLSI, the re-

sults showed that 97% of Aspergillus isolates

tested against VCZ were categorized as wild

strains, while for PCZ, all the isolates were

categorized as 100% wild strains. However,

for AMB, 18.2% of isolates were wild strains.

Fig. 1 (A, B, C, D) shows the graphical

distribution of each Aspergillus spp. against an-

tifungals with their respective MICs. Table 2 de-

scribes the in vitro activity according to MICs,

CMI50 and CIM90.

DISCUSSION

Although it was found that the resis-

tance of Aspergillus spp. tested in this study

was low, without involving Aspergillus spe-

cies with intrinsic resistance to some anti-

fungals; it is necessary to be cautious when

discussing susceptibility patterns in these

species of filamentous fungi. The aim is to

highlight the importance of monitoring re-

sistance at local, national and international

levels while investigating emerging resis-

tance mechanisms 6.

Aspergillus flavus was the most frequent

Aspergillus species isolated in this study, fol-

lowed by A. fumigatus. This result is not com-

parable to that reported in the international

literature, according to which A. fumigatus

is the most identified species 1,4,14,16,17. Sus-

ceptibility tests showed that 94% of Asper-

gillus species tested against VCZ had MICs

lower than 1 μg/mL compared to the ECVs

reported by CLSI, where these species were

categorized as wild strains. However, one of

the isolates MIC showed ≥16 μg/mL, which

could be attributed to the fact that the Fu-

migati section contains A. lentulus, which

has been observed to be intrinsically resis-

tant to VCZ 18. The molecular techniques

corroborating this description were not fea-

sible for this study. These results were simi-

lar to those reported by Castanheira et al.17,

who also obtained MICs90 of 0.5 μg/mL in A.

fumigatus, A. terreus, and A. niger against

VCZ, as well as to those obtained by Espinell-

Ingroff et al. 12. As is the case for most azoles,

VCZ acts on 14-α-sterol demethylase, and on

24-methylene dihydrolanosterol demethyl-

ase, another enzyme from the ergosterol bio-

synthetic pathway.

Antifungal susceptibility by Etest method in Aspergillus Genus 475

Vol. 64(4): 471 - 481, 2023

Table 1

Epidemiological, clinical characteristics and in vitro susceptibility to antifungal

agents tested in Aspergillus spp. Isolates.

Nº Type of sample Aspergillus Age Gender Diagnosis VCZ

(ug/

mL)

AMB

(ug/

mL)

CAS

(ug/

mL)

PCZ

(ug/

mL)

1 Sputum A. fumigatus 68 M Lung cancer 0.064 0.5 0.125 0.031

2 Sputum A. niger 62 F Bile duct cancer 0.031 0.063 0.015 0.031

3 Sputum A. terreus 62 M Bile duct cancer 0.031 8 0.015 0.015

4 Nasal septum A. versicolor 59 M Lung cancer 0.5 0.5 1 0.250

5 Sputum A. terreus 63 M Lung cancer 0.25 4 0.125 0.500

6 Sputum A. fumigatus 59 F COPD 0.125 0.5 0.250 0.064

7 Sputum A. fumigatus 70 F Pneumonía 0.060 0.125 0.015 0.064

8Ear discharge

canal

A. flavus 45 F Otitis media 0.064 0.5 0.064 0.125

9 Sputum A. fumigatus 66 F COPD 0.250 0.064 0.015 0.064

10 Foot discharge A. terreus 69 F Breast cancer 0.125 8 0.031 0.031

11 Bronchoalveolar

lavage

A. fumigatus 57 F Aspergilloma ≥16 ≥16 0.064 0.5

12 Ear discharge

canal

A. flavus 2 M Otitis media 0.125 1 0.015 0.064

13 Jaw discharge A. fumigatus 76 F Reconstructive

surgery

0.250 0.5 0.031 0.250

14 Ear discharge

canal

A. niger 68 M Otitis media 0.064 ≥16 0.064 0.031

15 Thigh discharge A. penicellioides 52 M Trauma 0.250 2 0.031 0.063

16 Sputum A. flavus 68 F COPD 0.250 2 ≥16 0.250

17 Sputum A. nidulans 68 F COPD 0.064 0.125 ≥16 0.031

18 Sputum A. niger 68 F Breast cancer 0.031 1 0.125 0.031

19 Corneal ulcer A. flavus 39 M Keratitis 0.125 2 0.015 0.063

20 Ear discharge

canal

A. niger 37 M Otitis media 0.125 1 0.063 0.031

21 Peritoneal fluid A. penicellioides 49 F Renal

insufficiency

0.250 1 0.5 0.031

22 Bronchoalveolar

lavage

A. flavus 58 M COPD 0.25 1 0.015 0.031

23 Sputum A. flavus 57 F Colon cancer 0.015 0.250 0.015 0.063

24 Endotracheal

discharge

A. flavus 63 M Lung cancer 0.015 0.125 0.031 0.063

25 Bronchial

discharge

A. flavus 53 M Pneumonia 0.063 0.5 0.015 0.125

26 Bone marrow A. fumigatus 58 F Lymphoid

leukemia

0.5 0.5 0.015 0.125

476 Moreno et al.

Investigación Clínica 64(4): 2023

This mechanism of action could explain

the effectiveness of this antifungal compared

to other azoles 19. These drugs, VCZ in par-

ticular, are the first line of prophylaxis and

treatment for fungal infections, although

fluconazole is inactive against filamentous

fungi 20.

PCZ is one of the last triazoles effec-

tive against various filamentous fungi, even

Mucorales. Therefore, it has become the

antifungal of choice in primary and salvage

prophylaxis, especially for oncohematology

patients 21. The mean of the MIC (0.063

μg/mL), the MIC50 (0.063 μg/mL), and the

Fig. 1. Distribution of the different minimum inhibitory concentrations (MIC) obtained for Aspergillus spp.

isolates (n=33), compared by antifungal agents tested. A) voriconazole; B) amphotericin B; C) cas-

pofungin; and D) posaconazole.

Nº Type of sample Aspergillus Age Gender Diagnosis VCZ

(ug/

mL)

AMB

(ug/

mL)

CAS

(ug/

mL)

PCZ

(ug/

mL)

27 Finger discharge A. niger 61 F Diabetes 0.063 0.125 0.015 0.031

28 Ankle tissue A. terreus 42 F Trauma 0.250 16 0.250 0.063

29 Nail A. flavus 72 F Diabetes 0.5 1 1 0.250

30 Sputum A. flavus 68 M Lung cancer/

COVID

0.5 1 0.063 0.250

31 Leg ulcer A. flavus 81 F Colon cancer 0.250 0.015 1 0.015

32 Sputum A. fumigatus 18 M Idiopathic

hepatitis

1 0.5 0.25 0.063

33 Corneal ulcer A. niger 35 M Keratitis 0.031 2 0.063 0.250

Table 1

CONTINUATION

VCZ: voriconazole; AMB: amphotericin B; CAS: caspofungin; PCZ: posaconazole, COPD: chronic obstructive pul-

monary disease.

Antifungal susceptibility by Etest method in Aspergillus Genus 477

Vol. 64(4): 471 - 481, 2023

MIC90 (0.25 μg/mL) obtained through this

research shows the excellent in vitro activity

of this triazole when compared to the ECVs

reported by CLSI. The most frequently ob-

tained MICs were 0.031 μg/mL and 0.063

μg/mL, although MICs for PCZ were rela-

tively low. These results are similar to those

obtained by Build et al. 22, confirming this

drug’s effectiveness in the tested isolates.

However, that study suggests that high doses

of PCZ could be used to treat azole-resistant

Aspergillus spp. isolates.

Several studies have reported about

the resistance of A. fumigatus to azoles.

This is probably due to cross-resistance be-

tween triazoles used in agriculture 14,17,23,24.

These resistances are transmitted to hu-

mans through food and water consumption

9. Most of them are mediated by the cyp51A

gene. Depending on the specific mutation,

one or even all triazoles can be resistant 4.

Resistance rates vary widely among medical

centers worldwide, reporting high rates or

rates of 1% or less 23-25. MICs varied between

Aspergillus species against AMB. Fortu-

nately, resistance to this antifungal is very

rare. Even so, the MIC was above the ECV

reported by the CLSI in six of the Aspergil-

lus species isolates. Four A. terreus isolates

showed MICs ≥ 4 μg/mL, while MICs of

both one A. niger isolate and one A. fumiga-

tus were ≥ 2 μg/mL

The Fumigati section susceptibility pro-

file is not consistent because this section

contains A. lentulus and A. fumigatiaffinis,

which have high MICs for azoles and AMB 26.

Despite this, it should be noted that data ob-

tained from the Fumigati section regarding

MICs were two dilutions lower than those

reported by Denardi et al.9 (Brazil) and Cas-

tanheira et al. 17 (global study).

Aspergillus terreus is known to be intrin-

sically resistant to AMB, but this depends on

the cryptic species within the Terrei section

16. Despite testing a few isolates, this study’s

A. terreus MICs results are comparable to

those reported in the literature. Aspergil-

lus terreus has emerged as an opportunistic

pathogen, capable of causing pulmonary

aspergillosis, onychomycosis, and fungal

keratitis, among other diseases; it has also

garnered attention due to its natural in vitro

and in vivo resistance 19.

Amphotericin B is the antifungal of

choice to treat severe fungal infections.

Most hospitals or healthcare services com-

monly use it. The selective pressure in these

environments could contribute to the emer-

gence of resistant phenotypes. Resistance to

AMB is most likely associated with low levels

of ergosterol in the cell membrane, which re-

duces the effectiveness of the drug because

of mutations in the Erg3 gene that inacti-

vate 5,6 sterol desaturase, an enzyme that

functions as a step in the sterol biosynthet-

ic pathway, creating dysfunctional sterols.

There are also Aspergillus species capable

of producing enzymes with reducing activ-

ity, decreasing the oxidative stress of AMB in

fungal metabolism 28,29.

Table 2

Activity of antifungal agents tested by the E-Test® gradient diffusion method

against Aspergillus spp. (n=33)

Antifúngicos Range Mean Mode Median MIC50 MIC90

Voriconazole 0.015-16 0.015 0.25 0.125 0.125 0.5

Anphotericin B 0.015-16 0.1732 0.5 1 0.5 8

Caspofungin 0.015-16 0.0848 0.015 0.063 0.063 1

Posaconazole 0.015-0.5 0.0723 0.031 0.063 0.063 0.25

MIC50: minimal inhibitory concentration that inhibited the growth of 50% of the isolates; MIC90: minimal inhibitory

concentration that inhibited the growth of 90% of the isolates. MIC: μg/mL.

478 Moreno et al.

Investigación Clínica 64(4): 2023

In this study, other isolates, such as A.

niger and A. nidulans, were categorized as

non-wild-type or AMB-resistant strains. In

any case, although other studies have re-

ported similar results, the number of iso-

lates tested from these species was not sig-

nificant enough to obtain sufficient data to

draw more informed conclusions 7-29.

Echinocandins are one of the new an-

tifungals used for aspergillosis treatment.

These molecules inhibit the synthesis of

β-(1,3)-d-glucan synthase, indirectly affect-

ing β-(1,3)-d-glucan incorporation into fun-

gal cell walls. Caspofungin is used success-

fully in salvage therapy against IA. During

this study, 94% of Aspergillus species were

resistant against CAS, and showed mean,

MIC50, and MIC90 values of 0.063 μg/mL,

0.063 μg/mL, and 1 μg/mL, respectively,

when compared to the ECVs reported by

CLSI. The GM of the Aspergillus spp. against

CAS (0.063 μg/mL) is a lower dilution than

that of Denardi et al. 9 (0.078 μg/mL) re-

gardless of the methodology used. In the

treatment of aspergillosis, echinocandins

are focused mainly on the wall of the api-

cal region of the Aspergillus hyphae, ignor-

ing the rest of the fungal structures. The

activity of this group of antifungals thus

affects the growth rate of the fungus but

leaves other physiological aspects intact 30.

Two other isolates, A. flavus and A. nidu-

lans, showed MIC ≥ 16 μg/mL, categoriz-

ing them as non-wild. Resistance to echino-

candins is not common among Aspergillus

species; however, some recent reports of

resistance to CAS 30,31 are consistent with

our findings.

These cryptic species are significant

mainly because they can display intrinsic re-

sistance with an in vitro rate of around 40%

against at least one antifungal 6, 13. The resis-

tance rate against azoles, polyenes, and echi-

nocandins varies by region, hence the impor-

tance of getting global epidemiological data.

Furthermore, MICs from environmental and

clinical samples of azole-resistant Aspergil-

lus species should be compared to under-

stand this antifungal resistance phenom-

enon. In order to determine a precise ECV

that could improve the use of clinical cut-off

points for Aspergillus species, it seems im-

perative to obtain both more epidemiologi-

cal and more semiotic data (clinical and mo-

lecular), which includes the treatment of IA

caused by resistant strains to different anti-

fungal drugs 1,17,23.

We ratify the need to identify the differ-

ent species in each section using molecular

techniques and include susceptibility tests.

In this study, the Etest® agar strip diffusion

method proved to help obtain ECV-guided

MICs established by CLSI. These MICs pro-

vided clinical guidelines for treating infec-

tions caused by Aspergillus species isolated

in Venezuela.

Conflict of interest

The authors declare they have no con-

flicts of interest.

Funding

The Instituto Médico La Floresta Mi-

crobiology Department entirely financed the

study.

ORCID number of authors

•Xiomara Moreno Calderón (XM):

0000-0002-5924-6158

• Carolina Macero Estévez (CM)

0000-0002-7620-7580

• Débora Oliveira Oliveira (DO):

0000-0003-3279-1591

Authors Contribution

XM study conceptualization and de-

sign; research; analysis and interpretation

of results; preparation, writing, review and

editing of the final manuscript. CM and DO

research; analysis, interpretation of results

and final manuscript editing.

Antifungal susceptibility by Etest method in Aspergillus Genus 479

Vol. 64(4): 471 - 481, 2023

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