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Revista de Salud Pública

Print version ISSN 0124-0064

Rev. salud pública vol.19 no.6 Bogotá Nov./Dec. 2017

http://dx.doi.org/10.15446/rsap.v19n6.54654 

Artículos/Investigación

In vitro antifungal susceptibility of clinical isolates of Fusarium from Colombia

Susceptibilidad antifúngica in vitro de aislamientos clínicos de Fusarium de Colombia

Adelaida Gaviria-Rivera1  , Alejandra Giraldo-López2  , Luz E. Cano-Restrepo3 

1 AG: Ing. Agrónoma. Ph. D. Biological Sciences. Escuela de Biociencias. Facultad de Ciencias. Universidad Nacional de Colombia, Medellín, Colombia. amgavirr@unal.edu.co

2 AG: Bact., M. Sc. Biotecnología. Ph. D. Ciencias Médicas Básicas. Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands. a.giraldo@westerdijkinstitute.ni

3 LC: Técnica de Laboratorio Clínico. Licenciatura en Bacteriología y Laboratorio Clínico. Doctorado en Ciencias. Escuela de Bacteriología y Laboratorio Clínico, Universidad de Antioquia. Grupo de Micología Médica y Experimental, CIB-U de A-UPB. Corporación para Investigaciones Biológicas. Escuela de Microbiología, U de A. Escuela de la Salud, UPB. Medellín, Colombia. lcano@cib.org.co; luz.Cano@udea.edu.co

ABSTRACT

Objective

The aim of the present study was to evaluate the antifungal susceptibilities of isolates of Fusarium to amphotericin B, itraconazole and voriconazole.

Methods

The susceptibility of 44 isolates of Fusarium was tested by the E-test methodology.

Results

All the isolates were resistant to itraconazole, and 89 % and 54,5 % were resistant to amphotericin B and voriconazole, respectively.

Discussion

The results confirm the high level of resistance reported, regardless of the species or the strain of Fusarium involved. The high MICs level observed are worrying and suggest that new drugs are needed.

Key words: Fusarium; amphotericin B; voriconazole (source: MeSH, NLM)

RESUMEN

Objetivo

Evaluar la susceptibilidad antifúngica in vitro de aislamientos de Fusarium a los antimicóticos amfotericina B, itraconazol y voriconazol.

Métodos

La susceptibilidad de 44 aislamientos clínicos de Fusarium fue evaluada por el método de difusión en disco, E-test.

Resultados

Todos los aislamientos fueron resistentes al itraconazol, y 89 % y 54,5 % fueron resistentes a la amfotericina B y al voriconazol, respectivamente.

Discusión

Los resultados confirman el alto nivel de resistencia reportado, independiente de la especie o la cepa de Fusarium involucrada. Los valores tan altos de MICs son preocupantes y sugieren la necesidad de evaluar nuevos medicamentos.

Palabras-clave: Fusarium; anfotericina B; voriconazol (fuente: DeCS, BIREME)

Fungi of the genus Fusarium are primarily plant pathogens and saprobes that cause a broad spectrum of infections in humans; including superficial, local, invasive, and disseminated infections, in immunologically deficient humans 1. After aspergillosis, disseminated fusariosis is the second most common cause of invasive infection by filamentous fungi in patients with hematologic malignancies or those undergoing transplants of hematopoietic progenitors; its high mortality rate and the lack of an optimal management protocol have raised increasing interest in this mycosis 2.

The most frequent species causing fusariosis are F. solani, F. oxysporum, and F. verticillioides1,3. Although less frequent, several other species also cause human infections. Some of these species are F. chlamydosporum, F. dimerum, F. incarna-tum and also the following species that are included into the Gibberella fujikuroi species complex: F. napiforme, F. nygamai, F. proliferatum, and F. sacchari4. F. temperatum has been recently reported as an agent of keratitis 5. However, the relevance of one species could change depending on the geographic area and the kind of infection involved. In the North of Italy, F. verticillioides was the most frequent isolated species from deep-seated infections and, F. solani was the most frequent isolated species from superficial infections 1. In Brazil, strains of F. solani have represented the 88 % of a total of 41 isolates involved in Fusarium keratitis 3, and in Bogotá (Colombia), F. solani, F. oxysporum and F. verticillioides represented the 64,9; 32,8; and 2,3 % respectively, from a total of 137 patient with onychomy-cosis by Fusarium6.

Fusarium is one of the most genetically heterogeneous fungi groups. Many species of this genus, that were identified -based on morphological characters- proved to be species complexes with little to no morphological differences, rather than single species 7. Many species, as F. solani, F. oxysporum, F. verticillioides, F. chlamydos-porum and F. dimerum represent complexes of species 4.

The huge genetic diversity of Fusarium, somehow is reflected in the susceptibility patrons to antifungals. Controversial results of susceptibility to antifungal and a high level of resistance are reported. Some species are less sensible than others, or strains of the same species have different levels of susceptibility to the same product 1,7. The F. solani species complex is one of the group with the poorest response, in vitro and in vivo to different drugs, as well as one of the most heterogeneous genetically speaking 1,8,9. The F. fujikuroi species complex showed resistance patterns species-specific 10.

The triazoles represent the frontline drugs for the treatment of mould diseases; nevertheless, emerging moulds (including Fusarium spp.,) may be less susceptible or resistant to these antifungals 11. Polyenes and azole compounds are routinely applied chemotherapy to fungal keratitis 12. Amphotericin b and voriconazole are the preferred drugs of choice for treatment of deep and disseminated infections, although some Fusarium species are not susceptible to them 7. However; good results have also been found, with better activity of the amphotericin B than the voriconazole 1,3,13 or voriconazole with better activity than amphotericin B 1,14.

Therefore, taking into account that the data of anti-fungal susceptibility of Fusarium spp are conflicting and could depend on the species, strain, kind of fusariosis and the antifungal drug 13, we have studied the susceptibility of 44 clinical isolates of Fusarium to amphotericin b, itraconazole and voriconazole by the E-test methodology. The results showed that all the Fusarium isolates were resistance to the itraconazole and 89 % of them to amphotericin b, too. Voriconazole had a moderate activity; only 15, 9 % of the isolates were sensible. These suggest that others antifungals should be considered.

METHODOLOGY

Isolates

The isolates were recovered from patients at the Corporación para Investigaciones Biológicas (CIB) in Medellin (Colombia) since 2004 to 2006. A total of 44 Fusarium isolates, from toenails (n=35), hand nails (n=2), skin (n=4) and cornea (n=I) were evaluated. These were identified as Fusarium spp, by the direct exam in Chinese ink and KOH at 20 %, and by their macroscopic and microscopic morphological features after they were cultured in the media Sabouraud, potato dextrose agar (PDA) and Mycosel at 23 °c for one to three weeks. The identity to the specie level of 35 isolates was determined by partial sequence of the transduction elongation factor gene (TE-FIa), in another work 15. All the isolates were preserved in sterile water at room temperature in darkness.

Antifungal susceptibility

The in vitro activity of amphotericin b, itraconazole and voriconazole was evaluated against 44 isolates of Fusarium, by the disk diffusion test according to the methods provided in CLSI M38-A 16,17.

The isolates were sub-cultured on PDA plates and incubated at 25 °c for seven days. Each colony was recovered with 10 mL of distilled water into a glass sterile tube; and after sedimented for 20 min., the upper part of each tube was collected in a new sterile tube. The suspensions were adjusted to a transmittance of 68-70 % at 530 nm, with distilled water, corresponding to an inoculum of 106 UFC/mL. A volume of 200 μl of each inoculum was added onto plates with 16 mL of RPMI medium supplemented with 1,5 % of agar, 2 % of glucose, at pH 7, and 0,165 m of buffer MOPS (Morpholine propane sulfonic acid, AES laboratory, Paris, France). The inoculum was allowed to dry for 15-30 minutes.

The E-test method was performed by following the instructions of the manufacturer (Etest®-AB Biomérieux). The antifungal agents were tested in concentrations than ranged from 256 to 0,016 μg/mL; two strips with the antifungal concentration, were placed in opposite direction on the inoculum. These were cultured at 28 °c. The mics that produced inhibition of growth were read after 48 hours, by visual examination; mics were recorded as the lowest drug concentration where the border of the inhibition ellipse intersects with the scale on the plastic antifungal strip. Candida krusei ATCC 6 258 was included as a quality control strain 18.

RESULTS

The results showed that all the 44 isolates of Fusarium evaluated, except the control (C. krusei) were resistant to itraconazole; 39 of the isolates (representing the 89 %) were also resistant to amphotericin B; the others five were intermediate or sensible dose-dependent (two of F. oxys-porum and two of F. solani, and the other isolate -63 946- was not identified) (Table 1).

Table 1 Antifungal susceptibilities of clinical isolates of Fusarium to amphotericin B, itraconazole and voriconazole by the E-test method 

The voriconazole was the only antifungal that showed moderate activity, with seven isolates (representing the 15,9 %) sensible to the product (with mics of less than 1 μg/mL); five of them were identified as F. oxysporum (the two remaining were not identified); 13 isolates (29,6 %) were sensible dose-dependent; six of F. oxysporum; three of F. solani and one of F. incarnatum (56 665); the three remaining were not identified. The others 24 isolates (representing the 54,5 %) were resistant to voriconazole; 10 of F. oxysporum; 10 of F. solani, and 4 that were no identified. All the isolates resistant to voriconazole were also resistant to amphotericin b and itraconazole. All of them were taken from nails, except for the isolate 56 988 of F. solani, which was taken from the cornea (Table 1). It is important to highlight the number of isolates with MICS higher than 32 μg/m: 44 (all the isolates), 36 and four to itraconazole, amphotericin b and voriconazole, respectively.

DISCUSSION

Fusarium spp., is a well-known opportunistic fungal agent that can cause important infections in immuno-compromised patients. It is also one of the main myco-toxigenic fungi 19. Fusarium is the leading pathogen of fungal keratitis in most of the studies worldwide, particularly in tropical regions 3; its ability to form biofilm was suggested as a contributing factor in recent outbreaks 9. Fusarium spp. have frequently been isolated from patients with onychomycosis, mainly of the specie F. oxysporum6.

The typical profile of the antifungal susceptibility of Fusarium spp. is the resistance to most antifungal agents. Due to the susceptibility pattern of Fusarium spp., the antifungal therapy options are limited 20. Additionally, information on epidemiology, antifungal susceptibilities and correlation with clinical outcomes is lacking, and such information is useful from a prognostic, diagnostic and therapeutic viewpoint 3. On the other hand, mics break points are not available for mold testing, therefore the isolates has been grouped as susceptible (MIC or MEC,<Iμg/mL), intermediate (MIC or MEC, 2 μg/mL) and resistant (MIC or MEC, >4 μg/mL), based on reported in vitro data obtained with large numbers of isolates 17,21. The levels for the antifungal that we evaluated were: <1, 1-2, >2 μg/mL for sensible, sensible dose-dependent and resistant, respectively.

In our work, the epidemiological data showed that, from the 44 clinical isolates, 84 % were involved in on-ychomycosis and 70 % were taken from females, which suggest that the generalized practice of manicure and pedicure in Colombia could be contributing to the dispersion of Fusarium spp. The most prevalent specie was F. oxysporum, with 60 % (21 of 35 isolates previously identified), followed by F. solani with 37 % and F. incarnatum with one isolate, in agreement with previous reports 6.

The data of susceptibility of Fusarium spp., to antifungal drugs are conflicting 13; different works have shown that the susceptibility is species-related, with F. solani having the highest MICS values 1,8,9, or strain-related as those biofilm producers, over all of F. solani3. The susceptibility to the same antifungal is variable. It seems that itraconazole has a poor activity against Fusarium spp., as we found in our work. In some cases, amphotericin B has shown better activity than voriconazole 1,3,13, or in some others, voriconazole is better than amphotericin B 14, in agreement with our work.

A better efficacy of the amphotericin b than itraconazole against strains of different clades of F. solani has been reported 8. Similarly, the amphotericin b has been shown as the most active drug against F. solani, while voriconazole and posaconazole were active against other Fusarium species 1. Strains of F. solani that produce biofilms has lower susceptibility, mainly for amphotericin B, which seems to be related with a worse clinical outcomes for F. solani compared with other Fusarium species 3.

In a study made in the United States of America it was found that, from the isolates involved in keratitis, the species of F. solani were the most common, followed by F. oxysporum species; and more strains of F. solani formed biofilm than strains of F. oxysporum,, and the ability to form biofilm varied by strain and clade type 9. None of the isolates of F. solani of our work was sensitive to voriconazole; instead, there was of F. oxysporum, although the isolates came from patient with onychomycosis mainly.

Voriconazole has been used to treat fungal infections in immunocompromised patients, including those caused by Fusarium spp 6. In our work, voriconazole was the best of the three antifungals evaluated against Fusarium spp., although only the 15, 9 % and 29 %, 6 % of the isolates was sensible and sensible dose-dependent, respectively to the product. As we have said, all the sensitive isolates belong to F. oxysporum and, from the 13 isolates (29,6 %) sensibles dose-dependent, six were of F. oxysporum, three of F. solani and one of F. incarnatum (the remaining three were not identified), which suggest that F. solani strains are less sensible. However, equal number of strains (ten) of F. oxysporum and F. solani were found resistant to voriconazole. Similarly, in another study made in Colombia with 137 patients with on-ychomycosis by Fusarium spp., the highest MICS values with voriconazole were of the isolates of F. solani, followed by F. oxysporum and F. verticillioides; 83.9 % and 66.7 % of the F. solani and F. oxysporum isolates were resistant to voriconazole, respectively 6.

Fusarium spp. show higher MICS value compared to other genus 6. In a study made in Colombia, the in vitro activity of fluconazole, itraconazole, voriconazole and terbinafine against fungi causing onychomycosis by species of Candida, Fusarium, Fusicoccum dimidiatum, Scytalidium hyalinum and dermatophytes showed that the Fusarium species had the highest MIC values, with all the antifungal agents, compared to the other fungal genera 22. In agreement with our work, they found the highest MICS values with itraconazole to F. solani and F. oxysporum, while voriconazole showed lower values but, contrary to our results, the isolates of F. oxysporum were less sensible than those of F. solani (between 2-i6 and 2-8 μg/mL, respectively) 22. Also, they reported more species of Fusarium: six isolates of F. oxysporum, two of F. solani, one of F. proliferatum, one F. dimidiatum, and one of F. nygamai22; however, the differences between the methods for identification of isolates between these two works should be considered.

A few works have compared numerous antifungal products against Fusarium spp. The antifungal susceptibilities from a strain collection of 48 isolates of Fusarium, belonging to the less-common Fusarium species of clinical interest, F. chlamydosporum, F. dimerum, F. incarnatum, F. napiforme, F. nygamai, F. proliferatum, and F. sacchari was evaluated against ii antifungal drugs (including amphotericin B, itraconazole and voriconazole) 13. Terbinafine was the most active drug against all the species tested with the exception of F. incarnatum, for which amphotericin b was the most active; amphotericin b was the second most active drug and, voriconazole although showed poor activity against all the tested strains. It was the third most active antifungal drug 13.

In Brazil, the in vitro susceptibility of isolates of F. napiforme responsible for a disseminated fusariosis were evaluated against amphotericin b, itraconazole, voriconazole, micafungin, 5-flucytosine, miconazole and fluconazole. The isolates were resistant to amphotericin B, with mic ranging from 2 to 4 μg/mL; the azoles were the most active against all the tested isolates 14.

In summary, the in vitro and in vivo activity against Fusarium species is not predictable. The unsatisfactory susceptibility profiles in vitro can be attributed to several factors, including the species of Fusarium, the strain, and the kind of antifungal drug. In vivo other factors are affecting too, as the kind of fusariosis and the underlying disease of the patient. Therefore, the choice of the anti-fungal should be determined on a case-by-case basis, depending on the species and susceptibilities performed at an experienced center, whenever feasible to obtain 22.

As it has been said "...despite of the methodological advance for determining antifungal susceptibility for fungi, the interpretation of the results and determination of how best to use these results continue to cause considerable confusion" 21, seems to be the best interpretation of the susceptibility of Fusarium. Therefore, categorical conclusions are impossible, but for our local area in Colombia, itraconazole should not be used for the treatment of fusariosis; nor amphotericin b, since any of the isolates was sensible to it. Voriconazole could be used but a test is always required *

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Funding: The study was funding by COLCIENCIAS, the Universidad Nacional de Colombia (code 20101007679) and la Corporación para Investigaciones Biológicas.

Received: December 14, 2015; Revised: October 22, 2016; Accepted: March 07, 2017

Conflict of interest:

None.

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