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. 2024 Mar 26;62(4):myae029. doi: 10.1093/mmy/myae029

Comparison of the antifungal activity of the pyrimidine analogs flucytosine and carmofur against human-pathogenic dematiaceous fungi

Rowena Alves Coelho 1,✉, Fernando Almeida-Silva 2, Maria Helena Galdino Figueiredo-Carvalho 3, Vanessa Brito de Souza Rabello 4, Gabriela Rodrigues de Souza 5, Maria Cristina da Silva Lourenço 6, Marcio L Rodrigues 7,8, Rodrigo Almeida-Paes 9
PMCID: PMC11008743  PMID: 38533658

Abstract

Chromoblastomycosis (CBM) and pheohyphomycosis (PHM) are the most common implantation mycoses caused by dematiaceous fungi. In the past, flucytosine (5-FC) has been used to treat CBM, but development of resistance is common. Carmofur belongs to the same class as 5-FC and has in vitro inhibitory activity against the main agents of CBM and PHM. The aim of this study was to compare the action of these two pyrimidine analog drugs against CBM and PHM agents. The minimum inhibitory concentration (MIC) and the selectivity index based on cytotoxicity tests of these two drugs against some agents of these mycoses were determined, with carmofur presenting a higher selectivity index than 5-FC. Carmofur demonstrated here synergistic interactions with itraconazole and amphotericin B against Exophiala heteromorpha, Fonsecaea pedrosoi, Fonsecaea monophora, and Fonsecaea nubica strains. Additionally, carmofur plus itraconazole demonstrated here synergism against a Phialophora verrucosa strain. To evaluate the development of carmofur resistance, passages in culture medium containing subinhibitory concentrations of this pyrimidine analog were carried out, followed by in vitro susceptibility tests. Exophiala dermatitidis quickly developed resistance, whereas F. pedrosoi took seven passages in carmofur-supplemented medium to develop resistance. Moreover, resistance was permanent in E. dermatitidis but transient in F. pedrosoi. Hence, carmofur has exhibited certain advantages, albeit accompanied by limitations such as the development of resistance, which was expected as with 5-FC. This underscores its therapeutic potential in combination with other drugs, emphasizing the need for a meticulous evaluation of its application in the fight against dematiaceous fungi.

Keywords: Chromoblastomycosis, pheohyphomycosis, antifungal susceptibility, drug repositioning, carmofur

Introduction

Dematiaceous fungi are commonly found in nature, especially in decaying organic matter. Some thermotolerant species of these fungi, that is, able of growing at temperatures close to 37 ºC, can cause mycoses when they are accidentally implanted in the skin through a traumatic injury. Chromoblastomycosis (CBM) and phaeohyphomycosis (PHM) are the most common mycoses caused within this context.1–4

CBM is usually limited to the subcutaneous tissue and is characterized by the presence of muriform bodies within host tissues. PHM, on the other hand, is characterized by the presence of toruloid dematiaceous hyphae in infected tissues. Unlike CBM, PHM is not limited to the skin or adjacent subcutaneous tissues and is associated with a wide range of inflammatory responses. Non-cutaneous PHM can involve any organ or system, but is most common in the sinuses, lungs, and brain.5

Both CBM and PHM can be caused by several species of dematiaceous fungi. The genera Fonsecaea, Cladophialophora, Rhinocladiella, Phialophora, and Exophiala can cause CBM.6 In PHM the most frequent agents are Bipolaris spp., Exophiala spp., and Phialophora verrucosa.5,7 The most common agent of CBM is Fonsecaea pedrosoi,8 while one of the most common PHM agents is Exophiala dermatitidis.9

There are no standard therapies for infections caused by dematiaceous fungi, regardless of the causative agent. To the best of our knowledge, there are no specific clinical trials focusing on the treatment of CBM or PHM. However, much more experience has been accumulated with itraconazole than with any other drug alone, through some case reports and case series using this azole drug for treatment.10,11 Flucytosine (5-FC) has been used to treat CBM in the past, but its use in monotherapy is limited due to the frequent development of resistance.12–14 In addition, 5-FC usually presents high values of minimum inhibitory concentration (MIC) to CBM and PHM agents. In combination with amphotericin B, 5-FC can be used to treat mycoses such as cryptococcosis,15 candidiasis,16 chromoblastomycosis,17 phaeohyphomycosis,18 and aspergillosis.19 5-FC also plays an important role for the treatment of colorectal cancer.20

5-FC is a pyrimidine analog, synthetic compound without intrinsic antifungal ability that, after uptake by susceptible fungal cells, it is converted to 5-fluorouracil (5-FU), which is then converted to metabolites that inhibit fungal RNA and DNA synthesis.20 A notable constraint is its limited availability, as it is not widely accessible in many parts of the world.21 Another drawback is the potential for severe side effects and toxicity in patients treated with this pyrimidine analog. Clinical manifestations of 5-FU toxicity include fever, fatigue, mucositis, stomatitis, nausea, vomiting, and diarrhea.22 Studies aiming to identify 5-FU derivatives with less toxicity to the host and more active against malignancies led to carmofur (1-hexylcarbamoyl-5-fluorouracil) synthesis. It has been used in the treatment of patients with colorectal cancer for over 30 years.23,24 Its hexylcarbamoyl structure makes carmofur more lipophilic than 5-FU, which facilitates its transport through the cell membrane and provides a greater therapeutic index.24 Carmofur is one of the 727 drugs present in the NIH Clinical Collection, a screening library useful for the discovery of new targets for marketed drugs. The in vitro inhibitory activity of carmofur against Fonsecaea pedrosoi and Exophiala dermatitidis, two important dematiaceous fungi that cause human infections, was recently demonstrated in a study conducted with this drug library.25

In order to increase the therapeutical options to CBM and PHM, the aim of this study was to compare the in vitro activity of these two pyrimidine analogs, 5-FC and carmofur, against some CBM and PHM agents.

Methods

Strains and growth conditions

This study used eight strains obtained from the Fiocruz Pathogenic Fungi Collection (CFP): Fonsecaea pedrosoi (CFP 00791), Cladophialophora carrionii (CFP 00910), Phialophora verrucosa (CFP 00937), Fonsecaea monophora (CFP 00911), Fonsecaea nubica (CFP 00912), Rhinocladiela similis (CFP 00790), Exophiala heteromorpha (CFP 01088), and Exophiala dermatitidis (CFP 01087). Strains were maintained on potato dextrose agar (PDA) (Sigma Chemical Corporation, St. Louis, MO, USA). Cultures were incubated for 7 days at 30°C until their use in the assays.

Determination of minimum inhibitory concentration

Minimum inhibitory concentration values (MICs) were determined using the methods proposed by the European Committee for Antimicrobial Susceptibility Testing (EUCAST) with minor modifications. Carmofur and 5-FC (both from Sigma Chemical Corporation, St. Louis, MO, USA) were serially diluted (0.15625 to 80 µM) in RPMI 1640 powder with L-glutamine and without sodium bicarbonate (Sigma Chemical Corporation, St. Louis, MO, USA), 10.4 g/l buffered with 0.165 M [morpholino] propanesulfonic acid (MOPS) (Vetec Química Fina, Ltda, RJ, Brazil), pH 7.0 containing 2% glucose, in plates of 96 wells. Fungal inocula were prepared following the EUCAST protocol.26 Plates were incubated at 35°C for 72–96 h. The MIC was determined by visual reading as the lowest concentration capable of inhibiting 100% of fungal growth when compared to the growth control well, without antifungal drug. Aspergillus flavus and Aspergillus fumigatus reference strains were used as controls when testing the traditional antifungal drugs described in the EUCAST protocol.

Determination of the minimum fungicidal concentration

The minimum fungicidal concentration (MFC) was determined by transferring an aliquot of 5 μl from each well without fungal growth of the microdilution plates used for the MIC determination, as described above, in Sabouraud 2% glucose agar (Sigma Chemical Corporation). The MFC was determined as the lowest drug concentration capable of preventing fungal growth on the medium after 5 days of incubation at 35 ˚C. Classification of the drug as fungistatic or fungicidal was done after determining the MFC/MIC ratio. When this ratio ranges from 1 to 2, the substance is considered fungicidal against the pathogen and if the ratio is higher than 2, the mode of action is probably fungistatic.27,28

Analysis of synergism with itraconazole, terbinafine, and amphotericin B

In order to evaluate the type of interaction between carmofur and the antifungal drugs most used in the treatment of CBM and PHM (itraconazole, terbinafine, and amphotericin B), a checkerboard assay was performed. Drugs were purchased from Sigma Chemical Corporation (St. Louis, MO, USA). The fractional inhibitory concentration index (FICI) was determined for each drug combination and fungal species. FICI defines the type of interaction between the drugs in combination as follows: synergism if FICI ≤ 0.5; indifference if FICI > 0.5 and ≤ 4 and antagonism if FICI > 4.29 FICI was obtained following the formula: (A/MIC(a)) + (B/MIC(b)) = FICI, where: A = MIC of the drug (a) in combination; MIC(a) = MIC of drug (a) alone; B = MIC of drug (b) in combination; MIC (b) = MIC of drug (b) alone.30,31 The test was performed using the checkerboard method, in which two drugs were applied in a single 96-well plate, resulting in a plate format that contains different concentrations of the drug combination in each well. Drug dilutions were prepared following the methodology proposed by EUCAST, starting from a stock solution of the substance/drug 100 × concentrated, according to the methodology for determining the MIC.26 In this assay, carmofur concentrations ranged from 0.078 to 40 μM, while the other antifungals ranged from 0.125 to 8.0 μM, with the serial dilutions of the different drugs performed following the protocol similar to the previous one. Strains presenting synergistic combinations with carmofur were additionally tested with 5-FC, to see if the synergisms seen with carmofur would be shared with other pyrimidine analogs. The experiments were the same as those previously described, only replacing carmofur with 5-FC.

Cytotoxicity assessment and selectivity index (SI) determination

VERO cells (ATCC CCL-81, a kidney tissue derived from a normal adult African green monkey) were cultivated in 199 medium with Earle's salts supplemented with 100 U/ml of penicillin, 100 µg/ml of streptomycin (Cultilab LTDA, Brazil), and 10% fetal bovine serum (FBS, Cultilab LTDA, Brazil) in an incubator at 37°C with 5% CO2. Cells were subcultured in 25 or 75 cm2 culture flasks once a week and the culture medium was also changed once a week. The cells used in the experiments were from passages 10 to 29.32 Cytotoxicity assays to assess the percentage of cell viability were performed in 96-well plates with 5 × 10 4 cells/well that were exposed to treatment solutions for 24 h at 37°C in an incubator with 5% CO2. MTT formazan powder (Sigma-Aldrich®, USA) was used and a 10% Tween 80 solution was the positive control.33 Results were read at 492 nm on a Thermo Scientific® Multiskan microplate spectrophotometer reader and expressed as % cell viability in the culture medium after the addition of a 10% Tween 80 solution. Initially, cells were treated with a range of concentrations of carmofur and 5-FC (3.125–100 µg/ml) to determine No Observed Effect Concentrations (NOEC) and CC50 (concentration that inhibits 50% of growth). Controls received only the culture medium with or without 1% DMSO. The SI was calculated using the formula: SI = CC50 (µM)/MIC (µM). The higher the ratio obtained, the more selective the substance is against the pathogen.

Development of carmofur resistance

Two isolates, Fonsecaea pedrosoi (CFP 00791) and Exophiala dermatitidis (CFP 01087), were used to verify if successive weekly subcultures in PDA medium supplemented with ½ MIC of carmofur (test) would induce carmofur resistance compared to the test performed with the strain subcultured in PDA medium without antifungal supplementation (control). Carmofur susceptibility test was performed for up to nine consecutive weeks in order to monitor MIC increases in strains maintained at subinhibitory concentrations of carmofur. When resistance was detected, the isolate previously subcultured in PDA medium containing ½ MIC of carmofur was subcultured in PDA medium without antifungal drugs for five weeks, to determine whether the resistance was transient or permanent. In this case, after the weekly subcultures in medium without antifungal agent, the in vitro susceptibility test to carmofur was repeated and the MIC compared with that yielded with the original strain and with the strain that emergence of resistance was detected. Finally, the strains with resistance induced by carmofur had their MIC determined against the antifungals amphotericin B, itraconazole and terbinafine, following the EUCAST protocol as described above, in order to verify cross-resistance with the main drugs used in the treatment of CBM and PHM.

Results

Carmofur has broad antifungal activity against dematiaceous fungi

The antifungal drug 5-FC showed MIC values above 80 µM for all tested isolates. Since growth inhibition was not verified by 5-FC up to the highest concentration tested (80 µM), the analysis of its fungicidal activity was not performed. Carmofur demonstrated here MIC values varying between 0.156 and 20 µM against different species and after MFC determination, it was observed that carmofur was fungicidal for F. pedrosoi. The values of MFC and MIC of carmofur against the different studied species are presented in Table 1.

Table 1.

In vitro antifungal activity of carmofur against eight dematiaceous fungi.

Strains MIC (µM) MFC (µM) MFC:MIC Ratio
Cladophialophora carrionii 1.25 40 32
Phialophora verrucosa 1.25 >80 >64
Exophiala dermatitidis 5 80 16
Exophiala heteromorpha 20 >80 >4
Fonsecaea pedrosoi 5 5 1
Fonsecaea monophora 5 80 16
Fonsecaea nubica 0.15625 40 256
Rhinocladiella similis 5 >80 >16
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Legend: MIC: minimum inhibitory concentration; MFC: minimum fungicidal concentration; MFC/MIC ratio - when this ratio ranges from 1 to 2, the substance is considered fungicidal against the pathogen and if the ratio is higher than 2, the mode of action is probably fungistatic.27,28

Carmofur has synergism with antifungal drugs

Supplementary Table details the results obtained from the three antifungal drugs combinations with carmofur. Carmofur and itraconazole combination produced synergistic interactions (FICI ≤ 0.50) against P. verrucosa, E. heteromorpha, F. pedrosoi, F. monophora, and F. nubica strains, while indifferent interactions were observed for the other species. The combination of carmofur with amphotericin B produced synergism against E. heteromorpha, F. pedrosoi, F. monophora, and F. nubica strains and indifferent interactions to the other species. The combination of carmofur with terbinafine showed indifferent interactions for all tested isolates. The interaction of 5-FC with itraconazole against P. verrucosa, E. heteromorpha, F. pedrosoi, F. monophora, and F. nubica was evaluated and synergism (FICI = 0.49) occurred only with F. pedrosoi, being indifferent to the other strains. The interaction of 5-FC with amphotericin B was also synergic with F. pedrosoi (FICI = 0.50) and indifferent with other strains, despite some FICI values were very close to synergism (range 0.51–0.53) (Figure 1).

Figure 1.

Figure 1.

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In vitro interactions of carmofur and flucytosine with amphotericin B, itraconazole and terbinafine against eight species of dematiaceous fungi that can cause chromoblastomycosis and/or phaeohyphomycosis. The violin plots represent the distribution, central tendency, and variability of the fractional inhibitory concentration index of the indicated antifungal combinations. Circles represent FICI values of each species. Their colors represent the species evaluated. Drug interactions ranges are indicated by the background color of the graph as follows: green, synergism; white, indifference; red, antagonism. 5FC, flucytosine; AMB, amphotericin B; CAR, carmofur; ITR, itraconazole; TRB, terbinafine.

Cytotoxicity assessment

The CC50 values found were >100 µg/ml (>388.71µM) for carmofur and >100 µg/ml (>775.19 µM) for 5-FC. Cell viability data are shown in Figure 2. The data obtained demonstrate a better selectivity of carmofur, which presented higher SI values for all strains tested (Table 3).

Figure 2.

Figure 2.

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Cell viability after treatment with different concentrations of Carmofur and flucytosine using monkey kidney epithelial cells. Results are expressed as the % of total cell viability (spectrophotometric readings at 492 nm) in the culture medium after addition of a 5 mg/ml MTT solution (positive response). The height of the histogram bar is the mean ± SEM of three independent experiments. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Table 3.

Carmofur and 5-flucytosine Selectivity Index.

Strains Selectivity Index
Carmofur 5-FC
Cladophialophora carrionii 310.96 9.69
Phialophora verrucosa 310.96 9.69
Exophiala dermatitidis 77.74 9.69
Exophiala heteromorpha 19.43 9.69
Fonsecaea pedrosoi 77.74 9.69
Fonsecaea monophora 77.74 9.69
Fonsecaea nubica 2,487.74 9.69
Rhinocladiella similis 77.74 9.69
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Evaluation of carmofur resistance development

Exophiala dermatitidis showed resistance to carmofur after two passages in medium containing subinhibitory concentrations of this drug (2.5 µM). Fonsecaea pedrosoi, on the other hand, showed resistance after seven passages in the presence of a subinhibitory concentration of carmofur (Figure 3). It is noteworthy that when these strains were subcultured in a carmofur-free medium (control), they consistently exhibited no variations in MIC values across all tested time points. After five passages of these resistant strains in PDA without antifungal supplementation, we observed that E. dermatitidis maintained resistance (MIC > 80 µM) to carmofur (permanent resistance), however F. pedrosoi returned to the MIC value of 5 µM (transient resistance) (Table 4). Finally, the strains with resistance induced by carmofur showed unchanged MIC values against the antifungals amphotericin B, itraconazole, and terbinafine (previously demonstrated in Table 2), suggesting that cross-resistance between carmofur and other antifungals is unlikely.

Figure 3.

Figure 3.

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Temporal evolution of carmofur resistance development in Fonsecaea pedrosoi and Exophiala dermatitidis strains exposed to subinhibitory concentrations (2.5 µM) of carmofur. The graph is divided into two sections, illustrating weekly passages in carmofur-supplemented medium (blue background) and in drug-free medium (green blackground).

Table 4.

Carmofur MIC values of dematiaceous fungi weekly subcultured on potato dextrose agar (PDA) supplemented or not with subinhibitory (½ MIC) concentration of the pyrimidine analog.

Strains MIC Carmofur (µM)
Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9
Exophiala dermatitidis (Control) 5 5 5 5 NT NT NT NT NT NT
Exophiala dermatitidis (½ MIC) 5 5 ≥80 ≥80 NT NT NT NT NT NT
Fonsecaea pedrosoi (Control) 5 5 5 5 5 5 5 5 5 5
Fonsecaea pedrosoi (½ MIC) 5 5 5 5 5 5 5 80 80 80
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NT: not tested.

Table 2.

Combination of 5-FC plus itraconazole and amphotericin B.

Strains FICI
5-FC + itraconazole		 FICI
5-FC + amphotericin B
Phialophora verrucosa 1.25 (I) NT
Exophiala heteromorpha 1 (I) 0.51 (I)
Fonsecaea pedrosoi 0.49 (S) 0.50 (S)
Fonsecaea monophora 0.51 (I) 1 (I)
Fonsecaea nubica 0.62 (I) 0.53 (I)
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FICI: fractional inhibitory concentration index; (S): synergistic combination; (I): indifferent combination; NT: not tested.

Discussion

Currently, the drug repurposing strategy is widely used in the search for new antifungal agents,34–37 since developing new drugs is time-consuming and expensive,38 especially for neglected diseases, such as CBM.39 A screening of the NIH clinical collection recently revealed that some drugs have off-label antifungal activity against F. pedrosoi. One of them is carmofur, a pyrimidine analog derived from 5-FU, an antineoplastic agent that has been used in the treatment of breast and colorectal cancer.20 The antifungal drug 5-FC, another pyrimidine analog, is known to have high MIC values for CBM agents,40 which corroborates the results of the present study with eight different species of human pathogenic black fungi. In addition, for Cryptococcus neoformans, another pathogenic fungus, 5-FC also exhibits high MICs. Nevertheless, when combined with amphotericin B, this combination has shown to be successful in inhibiting fungal growth.41 Therefore, we decided to compare antifungal activities of 5-FC and carmofur against dematiaceous fungi, particularly in combination with drugs currently used to treat CBM or PHM.

Comparing these two drugs, carmofur showed better antifungal activity (with lowest MIC values and highest SI values) against the same agents, including fungicidal action against the main CBM agent, F. pedrosoi. Carmofur demonstrated here synergism with itraconazole and amphotericin B against E. heteromorpha, F. pedrosoi, F. monophora, and F. nubica strains. Additionally, carmofur and itraconazole demonstrated synergism against a P. verrucosa strain, an important PHM agent. However, 5-FC demonstrated here synergism with itraconazole and amphotericin B exclusivelly against F. pedrosoi, suggesting that carmofur may surpass 5-FC in terms of its potential for drug combination against a variety of dematiaceous fungi. In addition, carmofur demonstrated here higher SI values than 5-FC. This suggests that carmofur may be more effective at killing fungi while causing less harm to human cells. This enhanced selectivity is an additional advantage of carmofur, as it opens the possibility of using smaller doses of this drug, potentially leading to fewer side effects. Indeed, a phase II trial study with carmofur revealed that patients with pancreatic carcinoma receiving 300–500 mg per square meter of body surface presented moderate toxicity.42 Giving doses lower than that to patients infected with dematiaceous fungi would diminish this toxic effect. Studies are necessary to address this.

In vitro studies with strains, as well as clinical trials with humans have shown that 5-FC can be used in combination with amphotericin B to treat several mycoses, including CBM.20 Additionally, the combination of itraconazole and 5-FC has been shown to be effective in a group of patients, even in severe forms of subcutaneous mycoses.43 Pharmacological data with a small number of patients showed that these two drugs have an additive effect against fungi, where 5-FC suppresses DNA synthesis and itraconazole acts on the cell membrane, inhibiting ergosterol synthesis.44 Some studies have suggested that combined therapy with these two drugs may be an option in severe cases of CBM, despite the insufficient number of cases for a detailed comparison.44,45 Our results corroborate these studies and suggest that the use of carmofur, replacing the 5-FC in the treatment of severe cases of CBM in combination with itraconazole or amphotericin B, may provide better results.

Monotherapy with 5-FC is limited due to frequent development of resistance.12–14 In our study, carmofur, from the same class as 5-FC, showed lower MIC values against the CBM agents tested. However, it also evolved resistance, akin to 5-FC. Although resistance to 5-FC is well known, the genetic basis of resistance regulation remains an enigma. It has been shown that resistance to 5-FC can result from the loss or mutation of any of the enzymes involved in the activation of cytosine permease, cytosine deaminase, or uracil phosphoribosyltransferase or from increased production of pyrimidines.46 We observed that resistance to carmofur was maintained in E. dermatitidis, a common PHM agent, after antifungal removal, but was transient in F. pedrosoi, the main CBM agent. Furthermore, it was demonstrated experimentally that this resistance was not shared with other classes of antifungal drugs (itraconazole, terbinafine, and amphotericin B), as expected. Therefore, as already occurs with 5-FC12,47 we also advise against the use of carmofur as a monotherapy for CBM and PHM.

The treatment of cryptococcal meningitis, as recommended by several guidelines, involves the use of initial combination therapy of amphotericin B and 5-FC.48,49 Furthermore, in vitro studies with 5-FC and other antifungals, including amphotericin B, suggest that the ability to overcome 5-FC resistance may depend on the mechanism of resistance.50,51 If resistance is due to a defective cytosine permease, it can be overcome by a drug such as amphotericin B, which facilitates cellular uptake of flucytosine.51 Furthermore, it has been demonstrated that in vitro synergism between amphotericin B and 5-FC can occur even when there is evidence of flucytosine monoresistance.41,51 These findings suggest that similar strategies could be used in the context of CBM treatment. For example, carmofur could be used as an induction treatment to accelerate the healing process, and then removed from the therapeutic scheme in the consolidation phase. Even if the isolate develops resistance during the induction phase, this resistance will not be shared with other drugs used in the consolidation phase of CBM treatment. Future studies with an in vivo model are necessary in order to determine the ideal dose of carmofur to treat CBM and PHM and then advance to clinical trials.

The in vitro results of this study suggest that carmofur could be considered as an alternative for the treatment of diseases caused by dematiaceous fungi, especially CBM, combined with amphotericin B or itraconazole in the induction phase of the treatment. In conclusion, carmofur may be a better option than 5-FC in combination therapy and could be a future addition to the therapeutic arsenal for CBM.

Supplementary Material

myae029_Supplemental_File
myae029_supplemental_file.docx (14.6KB, docx)

Acknowledgement

We thank Luna Sobrino Joffe for technical support in screening with the NIH Clinical collection library, and Marcos Vinicius Santos for his help in creating the graphics.

Contributor Information

Rowena Alves Coelho, Mycology Laboratory, National Institute of Infectious Diseases Evandro Chagas, INI/Fiocruz, Rio de Janeiro, Brazil.

Fernando Almeida-Silva, Mycology Laboratory, National Institute of Infectious Diseases Evandro Chagas, INI/Fiocruz, Rio de Janeiro, Brazil.

Maria Helena Galdino Figueiredo-Carvalho, Mycology Laboratory, National Institute of Infectious Diseases Evandro Chagas, INI/Fiocruz, Rio de Janeiro, Brazil.

Vanessa Brito de Souza Rabello, Mycology Laboratory, National Institute of Infectious Diseases Evandro Chagas, INI/Fiocruz, Rio de Janeiro, Brazil.

Gabriela Rodrigues de Souza, RPT 11B Bioassay Platform, National Institute of Infectious Diseases Evandro Chagas, INI/Fiocruz, Rio de Janeiro, Brazil.

Maria Cristina da Silva Lourenço, RPT 11B Bioassay Platform, National Institute of Infectious Diseases Evandro Chagas, INI/Fiocruz, Rio de Janeiro, Brazil.

Marcio L Rodrigues, Carlos Chagas Institute, Fiocruz, Paraná, Brazil; Institute of Microbiology, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.

Rodrigo Almeida-Paes, Mycology Laboratory, National Institute of Infectious Diseases Evandro Chagas, INI/Fiocruz, Rio de Janeiro, Brazil.

Author contributions

Rowena Alves Coelho (Conceptualization, Data curation, Investigation, Methodology, Writing – original draft), Fernando Almeida-Silva (Conceptualization, Data curation, Methodology, Writing – review & editing), Maria Helena Galdino Figueiredo-Carvalho (Data curation, Methodology, Visualization, Writing – review & editing), Vanessa Brito de Souza Rabello (Methodology), Gabriela Rodrigues de Souza (Methodology, Visualization), Maria Cristina da Silva Lourenço (Methodology, Validation, Visualization), Marcio L. Rodrigues (Funding acquisition, Methodology, Resources, Writing – review & editing), and Rodrigo Almeida-Paes (Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing).

Conflict of interest

None.

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