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INTRODUCTION
There has been a recent increase in epidemiological changes in filamentous fungi that cause diseases related to cryptic Aspergillus species. These species comprised 10 to 15% of Aspergillus isolates in epidemiological 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 genotypic 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.
The Aspergillus species most frequently isolated in a clinical context are A. fumigatus, 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 environmental sources. Furthermore, resistance to azoles has increased among clinical samples of the Fumigati section 5.
The prophylaxis and treatment of invasive aspergillosis are controversial due to its increasing morbidity and mortality 6. While voriconazole (VCZ) is the drug of choice, isavuconazole (ISZ) can be used against Aspergillus spp., and is considered the most effective 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 amphotericin B (AMB) and echinocandins are an alternative to azoles in aspergillosis treatment 9. However, epidemiological changes, including cryptic Aspergillus species’ resistance to azoles, are of growing concern 4.
This study evaluated the levels of azoles (VCZ, PCZ), echinocandins (CAS), and amphotericin 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, gender, and underlying disease of each patient were recorded. The isolates were preserved in distilled water with glycerol until the moment of the study. The different Aspergillus species’ identification was based on the criteria 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 (Oxoid, 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 Aspergillus spp. isolates were made and incubated 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 standardized at 1 - 5 x 106 CFU/mL (Densimat™ bioMérieux, France) at 530 nm 12-14. Plates containing Müeller-Hinton Agar, 2% glucose with Methylene blue, were inoculated, streaked in three directions, and left to dry for 15 minutes. Etest® strips of each antifungal (AB bioMérieux, France); VCZ, PCZ (0.002-32 μg/mL), AMB, and caspofungin (CAS=0.016-256 μg/mL) were placed according 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 elliptical inhibition zone intercepted the scale on the antifungal strip. To compare the MICs obtained during this study with the epidemiological cut-off values (ECVs) established by the Clinical and Laboratory Standards Institute (CLSI, M61 document, 2017), they were placed between two sequential dilutions taken to the subsequent higher dilution 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. According to de CLSI, ECVs in wild and non- wild isolates are classified based on the following 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, geometric 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.
RESULTS
The strains analyzed came from 33 patients, 18 female and 15 male, aged between 2-76 years and an average of 56 years. Thirty-three Aspergillus spp. isolates were identified, mostly from lower respiratory tract samples (17;51.5%), followed by isolates obtained from soft tissue (6;18.2%), ear discharge (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.
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 |
| 8 | Ear 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 |
| 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 |
VCZ: voriconazole; AMB: amphotericin B; CAS: caspofungin; PCZ: posaconazole, COPD: chronic obstructive pulmonary disease.
According to the ECV of CLSI, the results 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 antifungals with their respective MICs. Table 2 describes the in vitro activity according to MICs, CMI50 and CIM90.
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) caspofungin; and D) posaconazole.
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.
DISCUSSION
Although it was found that the resistance of Aspergillus spp. tested in this study was low, without involving Aspergillus species with intrinsic resistance to some antifungals; 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 resistance at local, national and international levels while investigating emerging resistance mechanisms 6.
Aspergillus flavus was the most frequent Aspergillus species isolated in this study, followed by A. fumigatus. This result is not comparable to that reported in the international literature, according to which A. fumigatus is the most identified species 1,4,14,16,17. Susceptibility tests showed that 94% of Aspergillus 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 Fumigati section contains A. lentulus, which has been observed to be intrinsically resistant to VCZ 18. The molecular techniques corroborating this description were not feasible for this study. These results were similar 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 EspinellIngroff et al.12. As is the case for most azoles, VCZ acts on 14-α-sterol demethylase, and on 24-methylene dihydrolanosterol demethylase, another enzyme from the ergosterol biosynthetic pathway.
This mechanism of action could explain the effectiveness of this antifungal compared to other azoles 19. These drugs, VCZ in particular, 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 effective 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 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 obtained MICs were 0.031 μg/mL and 0.063 μg/mL, although MICs for PCZ were relatively 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 between triazoles used in agriculture 14,17,23,24. These resistances are transmitted to humans 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. Fortunately, resistance to this antifungal is very rare. Even so, the MIC was above the ECV reported by the CLSI in six of the Aspergillus species isolates. Four A. terreus isolates showed MICs ≥ 4 μg/mL, while MICs of both one A. niger isolate and one A. fumigatus were ≥ 2 μg/mL.
The Fumigati section susceptibility profile 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 obtained from the Fumigati section regarding MICs were two dilutions lower than those reported by Denardi et al.9 (Brazil) and Castanheira et al. 17 (global study).
Aspergillus terreus is known to be intrinsically 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. Aspergillus 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 commonly use it. The selective pressure in these environments could contribute to the emergence of resistant phenotypes. Resistance to AMB is most likely associated with low levels of ergosterol in the cell membrane, which reduces the effectiveness of the drug because of mutations in the Erg3 gene that inactivate 5,6 sterol desaturase, an enzyme that functions as a step in the sterol biosynthetic pathway, creating dysfunctional sterols. There are also Aspergillus species capable of producing enzymes with reducing activity, decreasing the oxidative stress of AMB in fungal metabolism 28,29.
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 reported similar results, the number of isolates tested from these species was not significant enough to obtain sufficient data to draw more informed conclusions 7-29.
Echinocandins are one of the new antifungals used for aspergillosis treatment. These molecules inhibit the synthesis of β-(1,3)-d-glucan synthase, indirectly affecting β-(1,3)-d-glucan incorporation into fungal cell walls. Caspofungin is used successfully 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) regardless of the methodology used. In the treatment of aspergillosis, echinocandins are focused mainly on the wall of the apical region of the Aspergillus hyphae, ignoring 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. nidulans, showed MIC ≥ 16 μg/mL, categorizing them as non-wild. Resistance to echinocandins 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 resistance with an in vitro rate of around 40% against at least one antifungal 6,13. The resistance rate against azoles, polyenes, and echinocandins varies by region, hence the importance of getting global epidemiological data. Furthermore, MICs from environmental and clinical samples of azole-resistant Aspergillus species should be compared to understand this antifungal resistance phenomenon. In order to determine a precise ECV that could improve the use of clinical cut-off points for Aspergillus species, it seems imperative to obtain both more epidemiological and more semiotic data (clinical and molecular), which includes the treatment of IA caused by resistant strains to different antifungal drugs 1,17,23.
We ratify the need to identify the different 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 provided clinical guidelines for treating infections caused by Aspergillus species isolated in Venezuela.
