Characterization and in vitro antifungal susceptibility profiles of fusarium Spp. clinical isolates

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Characterization and in vitro antifungal susceptibility profiles of Fusarium spp. 1

clinical isolates. 2

Guevara M1, Sopo L2, Cepero de García MC1, De Bedout C3, Cano L3, García AM3, Motta, 3

A4, Cárdenas M1, Amezquita A1, Cano-Lira J5, Guarro J5, Restrepo S1, Celis A1* 4

5

1. Laboratorio de Micología y Fitopatología, Departamento de Ciencias Biológicas 6

Universidad de Los Andes, Bogotá, Colombia. 7

2. Laboratorio Especializado de Micología Médica (LEMM), Bogotá, Colombia. 8

3. Unidad de Biología Celular y Molecular, Corporación Para Investigaciones Biológicas 9

(CIB), Medellín, Colombia. 10

4. Universidad El Bosque, Bogotá, Colombia; Hospital Simón Bolívar, Bogotá, Colombia. 11

5. Unitat de Microbiologia, Facultat de Medicina, Universitat Rovira i Virgili, Reus, Spain. 12

13 *

Corresponding author: Celis Adriana, M.Sc. 14

Address: Carrera 1 No 18A-10. Building J Laboratory 204. 15

Telephone number: 57 (1) 3394949 Ext 3757 16

17

Institution: Laboratorio de Micología y Fitopatología, Departamento de Ciencias 18

Biológicas Universidad de Los Andes, Bogotá, Colombia. 19

E-mail: acelis@uniandes.edu.co 20

21 22

No conflict of interest declared. 23

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ABSTRACT 1

As a human opportunistic pathogen Fusarium causes a wide spectrum of infections 2

such as onychomycosis and keratomycosis in healthy individuals. Besides, it can cause 3

invasive or disseminated infections in immunosuppressed patients. The prevalence of 4

onychomycosis infections by Fusarium spp. could reach 6%. Furthermore, in disseminated 5

infections the percentage of mortality caused by this pathogen is around 70% to 100%. It 6

has been suggested that these rates could be associated with a remarkable therapeutic 7

failure caused by a high resistance to antifungal compounds and the difficulty in identifying 8

Fusarium as a human pathogen. In Colombia, there are few epidemiological studies of 9

Fusarium associated with onychomycosis and other infections. Positive isolates of 10

Fusarium spp. were morphologically characterized and genotypically analyzed. 11

Phylogenetic analyses were conducted to improve the identification and characterization of 12

the circulating strains. Once the strains were properly identified, we performed an 13

epidemiological study to determine the risk factors associated to onychomycosis. Then we 14

determined the antifungal susceptibility/resistance spectra of all the Fusarium isolates. This 15

study revealed that Bayesian analysis is a robust method that can be used for the precise 16

identification of circulating species of Fusarium. In addition, the main risk factor associated 17

to onychomycosis was the pedicure. Finally, we observed a high resistance of circulating 18

Fusarium species against antifungal compounds therapeutically used for systemic and 19

superficial infections and AMB is the most effective drug in vitro susceptibility of 20

Fusarium spp associated to human infections, without difference in MIC between 21

Fursarium solani species complex (FSSC) and Fusarium oxysporum species complex 22

(FOSC). 23

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Our results suggest the importance of finding alternative treatments to fusariosis 1

that could be included in health schemes in Colombia. This is the first study in Colombia 2

that suggests a robust method for accurately identify circulating species of Fusarium, 3

allowing the performance of epidemiological surveillance. 4

Keywords: Fusarium, onychomycosis, human infections, pedicure. 5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

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INTRODUCTION 1

Fusarium spp. are halohyphomycetes belonging to the order Hypocreales 2

(ascomycetes) (Hibbett et al., 2007). Members of the genus Fusarium are filamentous fungi 3

that have been reported as common soil saprophytes and as plant pathogens that are 4

worldwide distributed (P. E. Nelson, Dignani, & Anaissie, 1994) and have been associated 5

with different teleomorphs like: Gibberella, Nectria, Neocosmospora, Haematonectria,

6

Cyanonectria, Geejayessia and Albonectria (Schroers, Gräfenhan, Nirenberg, & Seifert, 7

2011). 8

In the last years they have been repeatedly reported as common causes of infections 9

in humans and animals (Nucci & Anaissie, 2002; Pereira et al., 2013; Scheel et al., 2013). 10

In humans, Fusarium spp. cause a wide spectrum of infections and the severity of the 11

disease is related to host immunity (Nucci & Anaissie, 2007). In healthy individuals the 12

most common infections are superficial ones: onychomycosis and keratomycosis (Chang et 13

al., 2006; Guilhermetti, Takahachi, Shinobu, & Svidzinski, 2007). Although Fusarium nail 14

infections are less common than onychomycoses caused by dermatophytes, the prevalence 15

of Fusarium spp. onychomycoses could reach 6% (Guilhermetti et al., 2007; Moreno & 16

Arenas, 2010). 17

In immunosuppressed patients Fusarium spp. can cause invasive or disseminated 18

infections and in recent years they have been considered as emerging pathogens because of 19

the increased incidence of infections (Jossi, Ambrosioni, Macedo-Vinas, & Garbino, 2010; 20

Nucci & Anaissie, 2007; Pereira et al., 2013). The percentage of mortality caused by this 21

pathogen is between 70% to 100% caused by challenging clinical forms of fusariosis in 22

immunocompromised patients (Nucci et al., 2003, 2004). Previous studies showed that 23

prolonged neutropenia, haematological malignancy, solid organ transplant or bone marrow

(5)

and prolonged treatment with corticosteroids are the main risk factors in invasive fungal

1

infections, while in superficial fungal infections trauma is reported as the main associated

2

factor (Anaissie et al., 2001; Boutati & Anaissie, 1997; Castro López et al., 2009; Nucci &

3

Anaissie, 2007; Nucci et al., 2003, 2004).

4

The most frequently isolated Fusarium species from human infections are:

5

Fusarium solani (~50%), followed by Fusarium oxysporum (~20%), Fusarium moniliforme 6

(~10%) and Fusarium verticillioides (~10%) (Nucci & Anaissie, 2007; Tortorano et al.,

7

2008). However, the isolation of Fusarium species associated to human infections may vary

8

around the world. In Colombia Fusarium solani has been the most frequently isolated

9

species from onychomycosis, followed by Fusarium oxysporum and F. verticilloides 10

(Castro López et al., 2009). In Switzerland and Israel, F. oxysporum has been the main

11

isolated species, while in Brazil the most prevalent species isolated from superficial

12

mycosis changed from Fusarium oxysporum to Fusarium solani between 2007 and 2013,

13

showing a transition in the epidemiology of this fungus and highlighting the importance of

14

a continuous surveillance of this pathogen along with a robust identification protocol of the

15

species (Guilhermetti et al., 2007; Ninet et al., 2005; Nir-paz et al., 2004; Scheel et al.,

16

2013).

17

Until recently the identification of the Fusarium species was based on phenotypic

18

characters (Guarro, Nucci, Akiti, & Gené, 2000). However, numerous studies indicate that

19

morphology alone may not be sufficient for species determination and the characterization

20

of the intraspecies complex due to the variability in the interpretation of these methods

21

(Balajee et al., 2009; Boutati & Anaissie, 1997; Esnakula, Summers, & Naab, 2013; Healy

22

et al., 2005; O’Donnell et al., 2009). Therefore, the identification of 33 to 50% of Fusarium 23

isolates was erroneous (Healy et al., 2005). Previous studies indicated that differences in

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the susceptibility profiles among different species of Fusarium can be related to treatment

1

failure. That is why it has been repeatedly suggested that the identification of the isolations

2

to the species level is important for epidemiological purposes and clinical treatments(Azor,

3

Gene, Cano, & Guarro, 2007; Diekema et al., 2003; O’Donnell et al., 2007). The 4

sequencing and analysis of multiple loci have been implemented for a better and more 5

accurate identification of Fusarium spp. (Balajee et al., 2009; O’Donnell et al., 2010, 6

2013). Sequences such as the nuclear ribosomal internal transcribed spacer region (ITS1, 7

5.8S rRNA, and ITS2), elongation factor 1α (EF-1α), β-tubulin (β-TUB), and RNA 8

polymerase II second largest subunit (RPB2) have been used alone or in combination 9

allowing the identification the presence of multiple cryptic species and defining Fusarium

10

species complexes (Azor et al., 2009; Balajee et al., 2009; O’Donnell et al., 2004, 2010). 11

Based on molecular identification of the strains, it has been established that the 12

clinically important Fusarium species complexes are: F. solani species complex (FSSC), F.

13

oxysporum species complex (FOSC), Gibberella (Fusarium) fujikuroi species complex 14

(GFSC), F. incarnatum - F. equiseti species complex (FIESC), F. sambucinum species 15

complex (FSAMSC), F. tricinctum species complex (FTSC), F. chlamydosporum species 16

complex (FCSC) and F. dimerum species complex (FDSC) (Guarro, 2013). In the United 17

States, species clasified in these four complexes account for approximately 85% of the 18

fusariosis (O’Donnell et al., 2009; Zhang et al., 2006). In Colombia there are few studies of 19

molecular epidemiology that include disseminated and superficial infections caused by 20

Fusarium spp. 21

In recent years, the antifungal susceptibility testing of mycelial fungi in vitro, have 22

gained importance due to the increased incidence of fungal infections caused by molds in 23

immunocompromised and immunocompetent patients (Alastruey-Izquierdo, Cuenca-24

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Estrella, Monzón, Mellado, & Rodríguez-Tudela, 2008; Pfaller, 2012). These tests are often 1

used to select active compounds for controlling a microorganism, but some authors suggest 2

that detection of resistance is most important (Pfaller, 2012). The Fusarium genus has been 3

associated with high resistance to many of the antifungal compounds used to 4

treat infections (Alastruey-Izquierdo et al., 2008; Cuenca-estrella et al., 2006).The only 5

drugs that have shown some in vitro activity are posaconazole, amphotericin B and 6

voriconazole, although these drugs are used for the treatment of disseminated infections. 7

The drugs most frequently used to treat onychomycosis are fluconazole and itraconazole 8

but, the have shown high minimum inhibitory concentrations (MICs) values in in vitro

9

susceptibility tests (Azor et al., 2007; Perfect et al., 2003). Additionally, some studies 10

indicated that an association between high MICs and poor response to antifungal treatment 11

exists (Perfect, 2005). Therefore, establishing the susceptibility profile of Fusarium spp. 12

could assist the epidemiological surveillance of resistance in circulating strains in the 13

population and in the selection of the best antifungal therapy. 14

The goals of this study were to determine the frequency of Fusarium species 15

complexes isolated from patients with onychomycosis and other infections in Colombia, to 16

identify onycomychosis-associated risk factors and to analyze the susceptibility profile in

17

vitro of antifungal compounds against the circulating species of Fusarium in the study 18

population. 19

20

MATERIALS AND METHODS 21

Samples collection 22

Samples were obtained from patients with one of the following kind of infections: 23

onycomychosis, keratomycosis, systemic infections and other infections. The samples were 24

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collected and processed between 2010 and 2012 by the Corporación de Investigaciones 1

Biológicas (CIB) Medellín, Colombia and Laboratorio Especializado de Micología Médica 2

(LEMM) Bogotá, Colombia. 3

All patients were informed about the sampling procedure, which was then 4

performed after all patients had signed the informed consent form. An epidemiological 5

clinical survey was conducted to assess variables such as age, gender, sports practice, nail 6

trauma, bath barefoot and pedicure. The Ethics Committee of the Universidad de los Andes 7

approved the protocol and the investigations were conducted according to the Declaration 8

of Helsinki Principles. 9

10

Phenotypic Characterization 11

The isolates were recovered on potato dextrose agar PDA (Oxoid). Subsequently, 12

we performed monosporic cultures (Choi, Hyde, & Ho, 1999; Hyde, 2001). Colony 13

morphology, color and texture were determined from strains grown at 25ºC on PDA. All 14

isolates were also grown on carnation leaf agar (agar 1.7%, KCl 0.4% and sterile carnation 15

leaf pieces) and incubated at 25 °C for 8 days. The identification was carried out based on 16

macroscopical and microscopical observations using features previously described (Leslie, 17

Summerell, & Bullock, 2006; P. E. Nelson et al., 1994; P. Nelson, Toussoun, & Marasas, 18

1983). 19

20

Molecular Characterization 21

Fungal isolates were grown in 50 ml SDY medium (2% peptone, 4% dextrose, 2% 22

yeast extract) and incubated with shaking at 25°C for 8 days. Mycelia were harvested by 23

filtration and lyophilized. DNA was extracted using a hexadecyltri-methylammonium 24

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bromide-CTAB (Sigma Chemical Co., St. Louis, MO) method as previously described 1

(O’Donnell, Cigelnik, & Nirenberg, 1998). All samples were treated with RNase (RNase A; 2

Sigma, St. Louis, Mo.) for 1 h at 37°C. DNA concentration was measured using a 3

Nanodrop ND-1000 (Thermo Scientific, Wilmington, DE, USA). 4

PCR was performed to amplify the ITS1-5.8S-ITS2 rDNA regions using the primers 5

ITS1 and ITS4 (White, Bruns, Lee, & Taylor, 1990), the DNA dependent RNA polymerase 6

II largest subunit (RPB2) using the primers RPB2-5F   and RPB2-7R (Liu, Whelen, & 7

Hall, 1999) and the translation elongation factor 1 alpha (EF-1α) using the primers EF-1H 8

and EF-2T (O’Donnell et al., 2004). All PCRs were performed in a final volume of 25 µl. 9

Each reaction for ITS and EF-1α contained 1X buffer, 2 mmol l−1 MgCl2, 0.2 mmol l−1 10

dNTPs, 0.2 mmol l−1 of each primer 1 unit of Taq and 100-400 ng of the DNA sample. For 11

RPB2 conditions were PCR amplifications employed Platinum Taq DNA polymerase 12

(Invitrogen Life Technologies, Carlsbad, CA) 13

Amplification conditions for ITS were as follows: 95 °C for 5 min, followed by 35 14

cycles consisting of 95 °C for 1 min, 56 °C for 1 min and 72 °C for 2 min and final 15

elongation step at 72 °C for 5 min. EF-1α conditions were 94 °C for 5 min s, followed by 16

35 cycles consisting of 95 °C for 30 s, 55 °C for 1 min and 72 °C for 1 min and a final 17

elongation step of 72 °C for 7 min and for RPB2 the conditions were 94 °C for 90 s, 18

followed by 40 cycles consisting of 94 °C for 30 s, 58 °C for 90 s and 72 °C for 2 min, 19

finalized with an extension for final 5 min at 72 °C. Aliquots (2 ul) of each PCR product 20

were visualized in 1% (w/v) agarose gel electrophoresis in 1x TBE buffer, stained with 21

GelRedTM and revealed by UV. 22

PCR products were purified and sequenced using an ABI3730xl DNA analyzer 23

(Applied Biosystems) and an ABI3500 Genetic analyzer (PE Applied Biosystems). 24

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Sequence assembly and editing were performed manually using Geneious software v.4.8.5 1

(http://www.geneious.com). The nucleotide sequences obtained in this study were 2

submitted to GenBank under the accession numbers XXX –XXX for XXX gene, etc. 3

4

Phylogenetic reconstruction 5

To perform phylogenetic inference, data were aligned using the MUSCLE algorithm 6

(Edgar, 2004). A Bayesian inference analysis was performed for the phylogenetic 7

reconstruction using MrBayes 3.2.1(Ronquist & Huelsenbeck, 2003), with 10 million 8

MCMC generations, with 2 chains (1 cold chain and 1 heated chains), two runs, model 9

specification per matrix (ITS- K80+G; RPB2- TrNef+G; EF-1α- TrN+G) and a burnin of 10

1% of the total run. 11

12

Antifungal susceptibility testing 13

A total of 35 clinical isolates were evaluated for susceptibility to voriconazole (VRC; 14

Sigma-Aldrich, Madrid, Spain) itraconazole (ITZ; Europack Farma, Bogotá, Colombia), 15

amphotericin B (AMB; Aldrich, Madrid, Spain), posaconazole (POS; Sigma-16

Aldrich, Madrid, Spain) and fluconazole (FLU; Europack Farma, Bogotá, Colombia). The 17

broth microdilution test was performed according to the methods provided in Clinical and 18

Laboratory Standards Institute (CLSI) document: “Standard for the susceptibility testing of 19

filamentous fungi M38-A”. MIC for the QC reference isolates Candida krusei ATCC 6258, 20

Candida parasilopsis ATCC 22019 and Paecilomyces variotii ATCC 3630 were within 21

established ranges (Clinical and Laboratory Standards Institute, 2008; Espinel-Ingroff et al., 22

2007) 23

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For VRC, POS and AMB, the MIC was determined to be the lowest concentration 1

resulting in a 90% reduction in growth compared to what was seen for the drug-free control 2

tube, while the MIC for FLZ and ITZ was the drug concentration necessary to inhibit a 3

50% in growth compared to that for the control (Clinical and Laboratory Standards 4

Institute, 2008; Espinel-Ingroff et al., 2007). 5

6

Statistical Analyses 7

To understand the epidemiological factors that might be associated to the presence

8

of clinically relevant species of Fusarium in patients with onychomycosis a binomial

9

logistic regression was performed. Statistical analyses were performed using data from 10

patients with onychomycosis and epidemiological data from patients with direct and 11

negative culture. 12

The explanatory power of each of the predictor variables was evaluated with a 13

likelihood test and the Wald statistic (Buse, 1982). Statistical analyses were performed 14

with the R software (http://www.r-project.org). P-values ≥0.05 were considered significant. 15

16

RESULTS 17

A robust method for identifying circulating species of Fusarium in the study 18

population. 19

A total of 89 Fusarium spp isolates were obtained. The results of phylogenetic 20

analysis of RPB2 showed with high bootstrap support values that the 89 isolates were 21

grouped in two species complexes: Fusarium solani species complex (FSSC) and Fusarium

22

oxysporum species complex (FOSC) with a frequency of 55% and 45% respectively (Figure 23

1). 24

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In order to improve the resolution of the phylogenetic reconstruction and to 1

discriminate among the phylogenetic species of Fusarium species complexes we performed 2

a Bayesian analysis. The concatenated analysis of rDNA and EF-1α confirmed the finding 3

of the two complexes FOSC and FSSC and the high posterior probabilities suggested that 4

this approach provides a better resolution for the discrimination of the Fusarium species 5

complex associated to human infections. 6

Additionally, the Bayesian analysis showed that most of the isolates classified as 7

Fusarium oxysporum species complex (FOSC) were distributed into the genotypes FOSC-3 8

and FOSC-4 (Figure 2). The isolates in the Fusarium solani species complex (FSSC) were 9

distributed into the genotypes FSSC-1, FSSC-6, FSSC-2, FSSC-16 and FSSC-3+4 (Figure 10

2). The results of the phylogenetic analyses of the three genes, rDNA, EF-1α and RPB2 11

supported the results obtained from the phenotypic characterization of the strains where two 12

distinct morphotypes could be identified. 13

14

Recipients of pedicure treatments have a less probability of acquiring onycomychosis. 15

Of the 89 isolates from patients with ages ranging from 15 to 87, 80 were obtained 16

from onychomycosis, 5 from keratomycosis, 2 from systemic infections and 2 from other 17

infections (Table S1). In addition, epidemiological data of 68 patients without 18

onychomycosis were gathered. The frequency of FOC and FSSC, identified with the 19

phylogenetic analysis, associated to patients with onychomycosis were 48,1% and 51,9 20

respectively. 21

Onycomychosis frequency was higher in women (74%) than in men (26%) according 22

to the total population. However, we did not find significant evidence that the development 23

of onychomycosis caused by FSSC or FOSC depend on the age or gender of the patient (p= 24

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0.6037 and 0.2919, respectively). Other variables like sport practice, nail trauma and 1

bathing barefoot did not show a significant difference neither (p>0.05) between the species 2

of Fusarium. However, people who received pedicure treatments had a less probability of 3

acquiring onycomychosis caused by FSSC or FOSC (p=0.0156; z-value= -2.418; N=142). 4

Pedicure was the only variable that showed a significant difference between infected and 5

uninfected patients. 6

7

AMB is the most effective drug in vitro susceptibility of Fusarium spp associated to 8

human infections. 9

Results of the broth microdilution test showed that most of the isolates had high

10

values of minimum inhibitory concentration (MIC) for the five antifungals evaluated.

11

Results of the in vitro susceptibility test are shown in Table 1. In general, AMB was the

12

most effective drug, while ITZ, VRC, POS and FLZ showed high values of MIC. In

13

addition there was no difference in MIC between Fursarium solani species complex 14

(FSSC) and Fusarium oxysporum species complex (FOSC). 15

16

DISCUSSION 17

This study is the first epidemiological approach of Fusarium species complexes 18

associated to human infections in Colombia. Our results showed that FOSC and FSSC were 19

the species complexes most frequently isolated from human infections within the study 20

population. In addition, in the in vitro susceptibility tests we confirmed their high resistance 21

to antifungal compounds that are frequently used to treat systemic infections and 22

onychomycosis, Previous studies have reported the isolation of Fusarium solani, Fusarium

23

oxysporum and Fusarium verticillioides associated to onycomychosis in Colombia (Alvarez 24

(14)

& Caicedo, 2007; Bueno et al., 2010; Castro López et al., 2009). However, despite the 1

importance of these studies, the phylogenetics species within FOSC and FSSC complexes 2

as previously reported by O’Donnell were not used to describe their findings. 3

Using a Bayesian analysis of the concatenated rDNA and EF-1α genes we propose a 4

highly robust approach to identify the circulating species of Fusarium in the population of 5

study and to better characterize the phylogenetic relationships among them. Previous 6

studies indicated that EF-1α, rDNA and RPB2 are reliable for the identification of clinical 7

isolates (Balajee et al., 2009; Guarro, 2013; O’Donnell et al., 2008, 2010). Nevertheless, in 8

these previous studies the supports of some clades were sometimes low (O’Donnell et al., 9

2013). We therefore recommend the use of Bayesian analyses as O'Donnell, et al. very 10

recently reported in 2013 including species of medical and agricultural importance within 11

the genus Fusarium (O’Donnell et al., 2013). 12

Our Bayesian phylogenetic analysis of concatenated rDNA and EF-1α showed that 13

the Colombian isolates were nested within the genotypes of FSSC and FOSC and that they 14

are associated to previously reported clinical isolates (Guarro, 2013). Genotypes FSSC-1, 15

FSSC-6, FSSC-2, FSSC-16 and FSSC-3+4 were associated mainly to FSSC. Fusarium

16

solani has been one of the most frequently isolated fungus from invasive mycoses in

17

immunocompromised and immunosuppressed patients (O’Donnell et al., 2008; Zhang et

18

al., 2006). Colombian isolates obtained from disseminated infections were placed in the

19

FSSC genotype 2. Our results showed high levels of diversity of FSSC associated to human

20

infections in Colombia, supporting previous reports that suggested a wide diversity of fungi

21

within this species complex (Migheli et al., 2010). Regarding the FOSC, little is known

22

about the molecular epidemiology of clinically relevant members of this group, even

23

though several previous reports suggested that this kind of isolates are grouped within the

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3rd and 4th clade of the complex. Most of the Colombian isolates included in the FOSC

1

were classified within the 3rd clade, which comprises the widest diversity of genotypes

2

associated to human mycoses (O’Donnell et al., 2004). However, five isolates identified as

3

members of the FOSC were grouped within the 4th clade described previously by

4

O’Donnell et al (2009). Studies that employ multiple-loci analysis should be implemented

5

as one of the standard approaches for identifying and characterizing the genetic diversity of

6

the Fusarium genus (Guarro, 2013; Migheli et al., 2010; O’Donnell et al., 2004, 2009,

7

2013).

8

Previous studies of onychomycoses epidemiology included variables like: sports 9

practice, nail direct trauma, hyperhidrosis and use of tight shoes as risk factors associated to 10

this infection (Elewski, 1998). Additionally, a higher prevalence of onychomicosis has been 11

reported in females than in males, what may be explained by the fact that women assist 12

more frequently to dermatologic appointments (Escobar & Carmona-Fonseca, 2003). Our 13

data suggest that people who receive pedicure treatments are less likely to acquire 14

onychomycosis. These findings are not in agreement with previously studies where 15

pedicure was found to be a risk factor to develop onychomycosis (Castro López et al., 16

2009). Three different aspects could explain these results: first, the binomial logistic 17

regression is a robust method that allows a better analysis of epidemiological factors 18

associated to onychomycosis; second, cosmetic treatments like pedicure can potentially 19

reduce the number of patients with a clinical diagnosis and a positive culture os Fusarium; 20

and third but not less important, the use of sterile tools to performed the pedicure treatments 21

can reduce the chances of aquiring onychomycosis caused by Fusarium in the pedicure 22

practice.

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Fusarium spp. have shown high resistance to antifungal compounds in the in vitro

1

antifungal susceptibility tests (Azor et al., 2007; Córdoba et al., 2008; Tortorano et al., 2

2008). In our study, we observed poor in vitro activity of azoles drugs against most of the 3

isolates in the FOSC and FSSC, in agreement with previous reports where azoles showed 4

poor activity against Fusarium species (Alastruey-Izquierdo et al., 2008). On the other 5

hand, amphotericin B was the drug that showed the lowest MICs. Previous studies reported 6

that amphotericin B has an efficacy in approximately 30–45% of systemic infections by 7

Fusarium spp. (Perfect, 2005). This polyene remains the antifungal drug of choice for the 8

treatment in immunocompromised patients (Nucci & Anaissie, 2007; Nucci et al., 2004; 9

Perfect, 2005). However, nephrotoxicity reported is some cases have limited its 10

implementation (Walsh et al., 1998). In addition, monitoring the resistance to compounds 11

as voriconazole and posaconazole is important because these drugs are used for the 12

treatment of fusariosis in refractory patients, as a second choice treatment or salvage 13

therapies (Perfect et al., 2003; Ruíz-Cendoya, Mariné, Rodríguez, & Guarro, 2009). 14

Fluconazole is the only antifungal compound approved by the Public health system in 15

Colombia (POS; Plan obligatorio de salud) to treat onychomycosis. Nevertheless, our 16

results showed that this was the drug with the highest MIC values. We therefore strongly 17

suggest that new therapeutic schemes for onychomycosis by Fusarium in Colombia are 18

urgently needed. 19

20

ACKNOWLEDGEMENTS 21

This work was supported by the Faculty of Sciences at Universidad de los Andes

22

and the Departamento Administrativo de Ciencia, Tecnología e Innovación (Colciencias) 23

agreement Nº RC 268-2010. 24

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REFERENCES 1

Alastruey-Izquierdo, A., Cuenca-Estrella, M., Monzón, A., Mellado, E., & Rodríguez-2

Tudela, J. L. (2008). Antifungal susceptibility profile of clinical Fusarium spp. isolates 3

identified by molecular methods. The Journal of antimicrobial chemotherapy, 61(4), 4

805–9. doi:10.1093/jac/dkn022 5

Alvarez, M. I., & Caicedo, L. D. (2007). Medically important fungi found in hallux nails of 6

university students from Cali, Colombia. Mycopathologia, 163(6), 321–5. 7

doi:10.1007/s11046-007-9016-9 8

Anaissie, E. J., Kuchar, R. T., Rex, J. H., Francesconi, a, Kasai, M., Müller, F. M., … 9

Walsh, T. J. (2001). Fusariosis associated with pathogenic fusarium species 10

colonization of a hospital water system: a new paradigm for the epidemiology of 11

opportunistic mold infections. Clinical infectious diseases : an official publication of 12

the Infectious Diseases Society of America, 33(11), 1871–8. doi:10.1086/324501 13

Azor, M., Gene, J., Cano, J., & Guarro, J. (2007). Universal In Vitro Antifungal Resistance 14

of Genetic Clades of the Fusarium solani Species Complex. Antimicrobial Agents and

15

Chemotherapy, 51(4), 1500–1503. doi:10.1128/AAC.01618-06 16

Azor, M., Gene, J., Cano, J., Manikandan, P., Venkatapathy, N., & Guarro, J. (2009). Less-17

Frequent Fusarium Species of Clinical Interest: Correlation between Morphological 18

and Molecular Identification and Antifungal Susceptibility. Journal of Clinical

19

Microbiology, 47(5), 1463–1468. doi:10.1128/JCM.02467-08 20

Balajee, S. a, Borman, a M., Brandt, M. E., Cano, J., Cuenca-Estrella, M., Dannaoui, E., 21

Wickes, B. L. (2009). Sequence-based identification of Aspergillus, fusarium, and 22

mucorales species in the clinical mycology laboratory: where are we and where should 23

we go from here? Journal of clinical microbiology, 47(4), 877–84. 24

doi:10.1128/JCM.01685-08 25

Boutati, E. I., & Anaissie, E. J. (1997). Fusarium, a significant emerging pathogen in 26

patients with hematologic malignancy: ten years’ experience at a cancer center and 27

implications for management. Blood, 90(3), 999–1008. Retrieved from 28

http://www.ncbi.nlm.nih.gov/pubmed/9242529 29

Bueno, J. G., Martinez, C., Zapata, B., Sanclemente, G., Gallego, M., & Mesa, a C. (2010). 30

In vitro activity of fluconazole, itraconazole, voriconazole and terbinafine against 31

fungi causing onychomycosis. Clinical and experimental dermatology, 35(6), 658–63. 32

doi:10.1111/j.1365-2230.2009.03698.x 33

Buse, a. (1982, August). The Likelihood Ratio, Wald, and Lagrange Multiplier Tests: An 34

Expository Note. The American Statistician. doi:10.2307/2683166 35

(18)

Castro López, N., Casas, C., Sopo, L., Rojas, A., Del Portillo, P., Cepero de García, M. C., 1

& Restrepo, S. (2009). Fusarium species detected in onychomycosis in Colombia. 2

Mycoses, 52(4), 350–6. doi:10.1111/j.1439-0507.2008.01619.x 3

Chang, D. C., Grant, G. B., Donnell, K. O., Wannemuehler, K. A., Noble-wang, J., Rao, C. 4

Y., … Park, B. J. (2006). Multistate Outbreak of Fusarium of a Contact Lens Solution. 5

The journal of the American Medical Association JAMA., 296(8), 953–963. 6

Choi, Y., Hyde, K. D., & Ho, W. W. H. (1999). Single spore isolation of fungi. 7

Clinical and Laboratory Standards Institute. (2008). Reference Method for Broth Dilution 8

Antifungal Susceptibility Testing of Filamentous Fungi  ; Approved Standard — 9

Second Edition. Clinical and Laboratory Standards Institute, Wayne, PA.

10

Córdoba, S., Rodero, L., Vivot, W., Abrantes, R., Davel, G., & Vitale, R. G. (2008). In 11

vitro interactions of antifungal agents against clinical isolates of Fusarium spp. 12

International journal of antimicrobial agents, 31(2), 171–4. 13

doi:10.1016/j.ijantimicag.2007.09.005 14

Cuenca-estrella, M., Gomez-lopez, A., Buitrago, M. J., Monzon, A., Luis, J., Mellado, E., 15

& Rodriguez-tudela, J. L. (2006). Head-to-Head Comparison of the Activities of 16

Currently Available Antifungal Agents against 3 , 378 Spanish Clinical Isolates of 17

Yeasts and Filamentous Fungi Head-to-Head Comparison of the Activities of 18

Currently Available Antifungal Agents against 3 , 378. doi:10.1128/AAC.50.3.917 19

Diekema, D. J., Messer, S. A., Hollis, R. J., Jones, R. N., Pfaller, A., & Pfaller, M. A. 20

(2003). Activities of Caspofungin , Itraconazole , Voriconazole , and Amphotericin B 21

against 448 Recent Clinical Isolates of Filamentous Fungi Activities of Caspofungin , 22

Itraconazole , Posaconazole , Ravuconazole , Voriconazole , and Amphotericin B 23

against 448 R. doi:10.1128/JCM.41.8.3623 24

Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high 25

throughput. Nucleic acids research, 32(5), 1792–7. doi:10.1093/nar/gkh340 26

Elewski, B. E. (1998). Onychomycosis: pathogenesis, diagnosis, and management. Clinical

27

microbiology reviews, 11(3), 415–29. Retrieved from 28

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=88888&tool=pmcentrez&r 29

endertype=abstract 30

Escobar, L., & Carmona-Fonseca, J. (2003). Onicomicosis por hongos ambientales no 31

dermatofíticos, 6–10. 32

Esnakula, A. K., Summers, I., & Naab, T. J. (2013). Case Report Fatal Disseminated 33

Fusarium Infection in a Human Immunodeficiency Virus Positive Patient, 2013, 3–8. 34

Espinel-Ingroff, a., Fothergill, a., Ghannoum, M., Manavathu, E., Ostrosky-Zeichner, L., 35

Pfaller, M. a., … Walsh, T. J. (2007). Quality Control and Reference Guidelines for 36

(19)

CLSI Broth Microdilution Method (M38-A Document) for Susceptibility Testing of 1

Anidulafungin against Molds. Journal of Clinical Microbiology, 45(7), 2180–2182. 2

doi:10.1128/JCM.00399-07 3

Guarro, J. (2013). Fusariosis, a complex infection caused by a high diversity of fungal 4

species refractory to treatment. European journal of clinical microbiology & infectious

5

diseases : official publication of the European Society of Clinical Microbiology. 6

doi:10.1007/s10096-013-1924-7 7

Guarro, J., Nucci, M., Akiti, T., & Gené, J. (2000). Mixed infection caused by two species 8

of Fusarium in a human immunodeficiency virus-positive patient. Journal of clinical

9

microbiology, 38(9), 3460–2. Retrieved from 10

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=87407&tool=pmcentrez&r 11

endertype=abstract 12

Guilhermetti, E., Takahachi, G., Shinobu, C. S., & Svidzinski, T. I. E. (2007). Fusarium 13

spp. as agents of onychomycosis in immunocompetent hosts. International journal of

14

dermatology, 46(8), 822–6. doi:10.1111/j.1365-4632.2007.03120.x 15

Healy, M., Reece, K., Walton, D., Huong, J., Frye, S., Raad, I. I., & Kontoyiannis, D. P. 16

(2005). Use of the DiversiLab System for Species and Strain Differentiation of 17

Fusarium Species Isolates Use of the DiversiLab System for Species and Strain 18

Differentiation of Fusarium Species Isolates. doi:10.1128/JCM.43.10.5278 19

Hibbett, D. S., Binder, M., Bischoff, J. F., Blackwell, M., Cannon, P. F., Eriksson, O. E., … 20

Lücking, R. (2007). A higher-level phylogenetic classification of the Fungi. 21

Mycological research, 111(Pt 5), 509–47. doi:10.1016/j.mycres.2007.03.004 22

Hyde, K. (2001). Australasian mycologist, 20(2). 23

Jossi, M., Ambrosioni, J., Macedo-Vinas, M., & Garbino, J. (2010). Invasive fusariosis 24

with prolonged fungemia in a patient with acute lymphoblastic leukemia: case report 25

and review of the literature. International journal of infectious diseases : IJID : official 26

publication of the International Society for Infectious Diseases, 14(4), e354–6. 27

doi:10.1016/j.ijid.2009.05.004 28

Leslie, J. F., Summerell, B. A., & Bullock, S. (2006). The Fusarium laboratory manual

29

(Vol. 2). Wiley Online Library. 30

Liu, Y. J., Whelen, S., & Hall, B. D. (1999). Phylogenetic relationships among 31

ascomycetes: evidence from an RNA polymerse II subunit. Molecular biology and

32

evolution, 16(12), 1799–808. 33

Migheli, Q., Balmas, V., Harak, H., Sanna, S., Scherm, B., Aoki, T., & O’Donnell, K. 34

(2010). Molecular phylogenetic diversity of dermatologic and other human pathogenic 35

fusarial isolates from hospitals in northern and central Italy. Journal of clinical

36

microbiology, 48(4), 1076–84. doi:10.1128/JCM.01765-09 37

(20)

Moreno, G., & Arenas, R. (2010). Other fungi causing onychomycosis. Clinics in

1

dermatology, 28(2), 160–3. doi:10.1016/j.clindermatol.2009.12.009 2

Nelson, P. E., Dignani, M. C., & Anaissie, E. J. (1994). Taxonomy, biology, and clinical 3

aspects of Fusarium species. Clinical microbiology reviews, 7(4), 479–504. 4

Nelson, P., Toussoun, T. A., & Marasas, W. (1983). Fusarium species: An illustrated 5

manual for identification. Pennsylvania State University Press, University Park. 6

Ninet, B. atrice, Jan, I., Bontems, O., Léchenne, B., Jousson, O., Lew, D., … Monod, M. 7

(2005). Molecular Identification of≥ i> Fusarium≥/i> Species in Onychomycoses. 8

Dermatology, 210(1), 21–25. 9

Nir-paz, R., Strahilevitz, J., Shapiro, M., Goldschmied-reouven, A., Yarden, O., Keller, N., 10

… Polacheck, I. (2004). Clinical and Epidemiological Aspects of Infections Caused by 11

Fusarium Species  : a Collaborative Study from Israel Clinical and Epidemiological 12

Aspects of Infections Caused by Fusarium Species  : a Collaborative Study from 13

Israel. doi:10.1128/JCM.42.8.3456 14

Nucci, M., & Anaissie, E. (2002). Cutaneous infection by Fusarium species in healthy and 15

immunocompromised hosts: implications for diagnosis and management. Clinical

16

infectious diseases : an official publication of the Infectious Diseases Society of

17

America, 35(8), 909–20. doi:10.1086/342328 18

Nucci, M., & Anaissie, E. (2007). Fusarium infections in immunocompromised patients. 19

Clinical microbiology reviews, 20(4), 695–704. doi:10.1128/CMR.00014-07 20

Nucci, M., Anaissie, E. J., Queiroz-Telles, F., Martins, C. a, Trabasso, P., Solza, C., Souza, 21

C. a. (2003). Outcome predictors of 84 patients with hematologic malignancies and 22

Fusarium infection. Cancer, 98(2), 315–9. doi:10.1002/cncr.11510 23

Nucci, M., Marr, K. a, Queiroz-Telles, F., Martins, C. a, Trabasso, P., Costa, S., … 24

Anaissie, E. (2004). Fusarium infection in hematopoietic stem cell transplant 25

recipients. Clinical infectious diseases : an official publication of the Infectious 26

Diseases Society of America, 38(9), 1237–42. doi:10.1086/383319 27

O’Donnell, K., Cigelnik, E., & Nirenberg, H. I. (1998). Molecular systematics and 28

phylogeography of the Gibberella fujikuroi species complex. Mycologia, 90(3), 465– 29

493. 30

O’Donnell, K., Rooney, A. P., Proctor, R. H., Brown, D. W., McCormick, S. P., Ward, T. 31

J., … Geiser, D. M. (2013). Phylogenetic analyses of RPB1 and RPB2 support a 32

middle Cretaceous origin for a clade comprising all agriculturally and medically 33

important fusaria. Fungal Genetics and Biology, 52, 20–31. 34

doi:10.1016/j.fgb.2012.12.004 35

(21)

O’Donnell, K., Sarver, B. a J., Brandt, M., Chang, D. C., Noble-Wang, J., Park, B. J., … 1

Ward, T. J. (2007). Phylogenetic diversity and microsphere array-based genotyping of 2

human pathogenic Fusaria, including isolates from the multistate contact lens-3

associated U.S. keratitis outbreaks of 2005 and 2006. Journal of clinical microbiology, 4

45(7), 2235–48. doi:10.1128/JCM.00533-07 5

O’Donnell, K., Sutton, D. a, Fothergill, A., McCarthy, D., Rinaldi, M. G., Brandt, M. E., 6

Geiser, D. M. (2008). Molecular phylogenetic diversity, multilocus haplotype 7

nomenclature, and in vitro antifungal resistance within the Fusarium solani species 8

complex. Journal of clinical microbiology, 46(8), 2477–90. doi:10.1128/JCM.02371-9

07 10

O’Donnell, K., Sutton, D. a, Rinaldi, M. G., Gueidan, C., Crous, P. W., & Geiser, D. M. 11

(2009). Novel multilocus sequence typing scheme reveals high genetic diversity of 12

human pathogenic members of the Fusarium incarnatum-F. equiseti and F. 13

chlamydosporum species complexes within the United States. Journal of clinical

14

microbiology, 47(12), 3851–61. doi:10.1128/JCM.01616-09 15

O’Donnell, K., Sutton, D. a, Rinaldi, M. G., Sarver, B. a J., Balajee, S. A., Schroers, H.-J., 16

… Geiser, D. M. (2010). Internet-accessible DNA sequence database for identifying 17

fusaria from human and animal infections. Journal of clinical microbiology, 48(10), 18

3708–18. doi:10.1128/JCM.00989-10 19

O’Donnell, K., Sutton, D. A., Rinaldi, M. G., Magnon, K. C., Cox, P. A., Revankar, S. G., 20

Francesconi, A. (2004). Genetic Diversity of Human Pathogenic Members of the 21

Fusarium oxysporum Complex Inferred from Multilocus DNA Sequence Data and 22

Amplified Fragment Length Polymorphism Analyses  : Evidence for the Recent 23

Dispersion of a Geographically Widespread Clonal Linea. 24

doi:10.1128/JCM.42.11.5109 25

Pereira, G. H., de Angelis, D. A., Brasil, R. A., dos Anjos Martins, M., de Matos Castro e 26

Silva, D., Szeszs, M. W., & de Souza Carvalho Melhem, M. (2013). Disseminated 27

amphotericin-resistant fusariosis in acute leukemia patients: report of two cases. 28

Mycopathologia, 175(1-2), 107–14. doi:10.1007/s11046-012-9585-0 29

Perfect, J. R. (2005). Treatment of non-Aspergillus moulds in immunocompromised 30

patients, with amphotericin B lipid complex. Clinical infectious diseases : an official 31

publication of the Infectious Diseases Society of America, 40 Suppl 6(Suppl 6), S401– 32

8. doi:10.1086/429331 33

Perfect, J. R., Marr, K. a, Walsh, T. J., Greenberg, R. N., DuPont, B., de la Torre-Cisneros, 34

J., … Johnson, E. (2003). Voriconazole treatment for less-common, emerging, or 35

refractory fungal infections. Clinical infectious diseases : an official publication of the 36

Infectious Diseases Society of America, 36(9), 1122–31. doi:10.1086/374557 37

(22)

Pfaller, M. a. (2012). Antifungal drug resistance: mechanisms, epidemiology, and 1

consequences for treatment. The American journal of medicine, 125(1 Suppl), S3–13. 2

doi:10.1016/j.amjmed.2011.11.001 3

Ronquist, F., & Huelsenbeck, J. P. (2003). MrBayes 3: Bayesian phylogenetic inference 4

under mixed models. Bioinformatics, 19(12), 1572–1574. 5

doi:10.1093/bioinformatics/btg180 6

Ruíz-Cendoya, M., Mariné, M., Rodríguez, M. M., & Guarro, J. (2009). Interactions 7

between triazoles and amphotericin B in treatment of disseminated murine infection 8

by Fusarium oxysporum. Antimicrobial agents and chemotherapy, 53(4), 1705–8. 9

doi:10.1128/AAC.01606-08 10

Scheel, C. M., Hurst, S. F., Barreiros, G., Akiti, T., Nucci, M., & Balajee, S. A. (2013). 11

Molecular analyses of Fusarium isolates recovered from a cluster of invasive mold 12

infections in a Brazilian hospital. BMC infectious diseases, 13, 49. doi:10.1186/1471-13

2334-13-49 14

Schroers, H.-J., Gräfenhan, T., Nirenberg, H. I., & Seifert, K. a. (2011). A revision of 15

Cyanonectria and Geejayessia gen. nov., and related species with Fusarium-like 16

anamorphs. Studies in mycology, 68, 115–38. doi:10.3114/sim.2011.68.05 17

Tortorano, A. M., Prigitano, A., Dho, G., Esposto, M. C., Gianni, C., Grancini, A., … 18

Viviani, M. A. (2008). Species distribution and in vitro antifungal susceptibility 19

patterns of 75 clinical isolates of Fusarium spp. from northern Italy. Antimicrobial

20

agents and chemotherapy, 52(7), 2683–5. doi:10.1128/AAC.00272-08 21

Walsh, T. J., Hiemenz, J. W., Seibel, N. L., Perfect, J. R., Horwith, G., Lee, L., … 22

Anaissie, E. J. (1998). Amphotericin B lipid complex for invasive fungal infections: 23

analysis of safety and efficacy in 556 cases. Clinical infectious diseases : an official

24

publication of the Infectious Diseases Society of America, 26(6), 1383–96. Retrieved 25

from http://www.ncbi.nlm.nih.gov/pubmed/9636868 26

White, T., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of 27

fungal ribosomal RNA genes for phylogenetics. In M. Innis, D. Gelfand, J. Shinsky, & 28

T. White (Eds.), PCR Protocols: A Guide to Methods and Applications (pp. 315–322). 29

San Diego: Academic Press. 30

Zhang, N., O’Donnell, K., Sutton, D. a, Nalim, F. A., Summerbell, R. C., Padhye, A. a, & 31

Geiser, D. M. (2006). Members of the Fusarium solani species complex that cause 32

infections in both humans and plants are common in the environment. Journal of

33

clinical microbiology, 44(6), 2186–90. doi:10.1128/JCM.00120-06 34

35 36 37

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Figure 1. Phylogenetic reconstruction of the Fusarium species complexes using the DNA 18

dependent RNA polymerase II largest subunit (RPB2) gene and Bayesian inference (BI) of 19

isolates circulating in Colombia. We included medically relevant complexes. Support 20

values are above branches, and represent bootstrap values for posterior probabilities. 21

Cordyceps confragosa strain CBS 101247 was used as the outgroup. 22

23

Figure 2. Phylogenetic diversity of human isolates within the Fusarium oxysporum species 24

complex (FOSC) and Fusarium solani species complex using Bayesian inference (BI) 25

analysis of the combined EF-1α & rDNA sequence data. Support values are above 26

branches, and represent bootstrap values for posterior probabilies. *Accession numbers of 27

sequences retrieved from GenBank. Cordyceps confragosa strain CBS 101247 was used as 28

the outgroup. 29

(24)

Table 1. MIC90 values for voriconazole (VRC), posaconazole (POS), itraconzazole (ITZ)

and amphotericin B (AMB) and MIC50 for fluconazole (FLZ) (µg/mL).

ISOLATE BROTH MICRODILUTION MIC (Ug/ml)

AB FLZ POS VOR ITZ

121480 1 ≥32 ≥16 ≥16 ≥16

121647 2 ≥32 ≥16 1 ≥16

121988 2 ≥32 ≥16 2 ≥16

121584 ≥16 ≥32 ≥16 ≥16 ≥16

122181 ≥16 ≥32 ≥16 ≥16 ≥16

110751 2 ≥32 ≥16 ≥16 ≥16

CIB_35 2 ≥32 ≥16 1 ≥16

110107 3 ≥32 ≥16 4 ≥16

110787 3 ≥32 ≥16 2 ≥16

CIB_10 ≥16 ≥32 ≥16 ≥16 ≥16

122066 3 ≥32 ≥16 ≥16 ≥16

122165 1 ≥32 ≥16 ≥16 ≥16

122098 1 ≥32 ≥16 ≥16 ≥16

121995 1 ≥32 ≥16 ≥16 ≥16

121885 1 ≥32 ≥16 ≥16 ≥16

121984 1 ≥32 ≥16 ≥16 ≥16

121398 1 ≥32 ≥16 ≥16 ≥16

122008 1 ≥32 ≥16 ≥16 ≥16

121472 1 ≥32 ≥16 ≥16 ≥16

121875 1 ≥32 ≥16 ≥16 ≥16

122160 3 ≥32 ≥16 ≥16 ≥16

122060 ≥16 ≥32 ≥16 ≥16 ≥16

121988 3 ≥32 ≥16 ≥16 ≥16

110248 3 ≥32 ≥16 ≥16 ≥16

110157 5 ≥32 ≥16 ≥16 ≥16

CIB_4 ≥16 ≥32 ≥16 ≥16 ≥16

CIB_6 ≥16 ≥32 ≥16 ≥16 ≥16

CIB_28 ≥16 ≥32 ≥16 ≥16 ≥16

122162 ≥16 ≥32 ≥16 ≥16 ≥16

110751 ≥16 ≥32 ≥16 ≥16 ≥16

CIB_2 ≥16 ≥32 ≥16 ≥16 ≥16

CIB_5 5 ≥32 ≥16 ≥16 ≥16

CIB_29 3 ≥32 ≥16 ≥16 ≥16

CIB_15 5 ≥32 ≥16 ≥16 ≥16

(25)

Table 1S. Clinical isolates of Fusarium species complexes used in the phylogenetic

analysis.

Species

complex Isolate Source

Molecular identification GenBank accession number

ITS RPB2

Fusarium oxysporum

species complex

(FOSC)

110197 Human Toenail xxxxx xxxxx xxxxx

CIB 4 Human Toenail xxxxx xxxxx xxxxx

(26)

CIB 31 Abdominal

drainage xxxxx xxxxx xxxxx

CIB 24 Secretion leg xxxxx xxxxx xxxxx

Fusarium solani species complex

(FSSC)

CIB 9 Cornea xxxxx xxxxx xxxxx

CIB 20 Biopsy foot xxxxx xxxxx xxxxx

(27)

CIB 28 Blood culture xxxxx xxxxx xxxxx

CIB 29 Pleural fluid xxxxx xxxxx xxxxx

(28)

121995

CIB_15

111306

110357

110262

110206

110283

110819

110946

121398

122050

122060

122165

CIB_13

CIB_31

CIB_5

CIB_7

110787

CIB_16

FOSC 3-a 43455_NRRL*

FOSC 3-b 43431_NRRL*

FOSC 3-a 43521_NRRL*

CIB_23

110791

CIB_17

CIB_11

FOSC 3f 43668_NRRL*

110414

121988

FOSC 3-d 43466_NRRL*

110751

121480

FOSC 4b 43504_NRRL*

CIB_12

FOSC 4a 43454_NRRL*

1220

FOSC 4c 43539_NRRL*

121470

121377

110347

110358

110197

110248

FOSC ST-63

FOSC 3-c 43442_NRRL*

110231

121584

FOSC 3-e 43655_NRRL*

111009

FOSC ST-27

FOSC ST-2

F. fujikuroi 13566_NRRL*

F. proliferatum 22944_NRRL*

F. sacchari 13999_NRRL*

F.mangiferae 25226_NRRL*

F. xylarioides 25486_NRRL*

121607

7

F. verticillioides 20956_NRRL*

F. thapsinum 22045_NRRL*

F.guittiforme 22945_NRRL*

F. circinatum 25331_NRRL*

F. subglutinans 22016_NRRL*

FIESC 26-a 26417_NRRL*

FIESC 15-a 32175_NRRL*

F. equiseti 20697_NRRL*

F. lacertarum 20423_NRRL*

F. sciripi 9-b 13402_NRRL*

JX171584

JX171601

JX171614

JX171608

JX171643

122004

EF470161

EU329635

JX171610

110676

121371

EF470093

EU329568

EU329629

CIB_9

DQ790572

EU329528

EU329531

JX171655

110739

EU329535

EU329556

EF469980

EU329521

DQ790561

EF469959

110157

CIB_10

121647

110404

CIB_8

121617

121472

121340

110184

110107

110029

111347

110562

EU329551

122008

EU329549

EU329609

110266

EU329538

110148

EF470004

EU329534

JX171653

EF470005

EU329527

EU329577

EU329554

DQ790594

DQ790566

Cordyceps confragosa

strain CBS 101247 (OUTGROUP)

0,99 0,92 0,99

Fusarium fujikuroi

species complex

Fusarium incarnatum-

equiseti

species complex

Fusarium solani

species complex

(FSSC)

Fusarium oxysporum

species complex

(FOSC)

0,99

(29)

CIB_20

CIB_11

CIB_5 CIB_12

FOSC 3-a 43455_NRRL* FOSC 3-d 43466_NRRL* FSSC-1D 32304_NRRL*

121340

FOSC 4-c 43539_NRRL*

-122066

110206 FSSC- 6B 22782_NRRL*

122160

FSSC- 3+4+O 28559_NRRL* CIB_29 110791 121398 111374 121480 CIB_8

FSSC- 16B 32434_NRRL*

CIB_24 CIB_22 110946 110739 110107 CIB_30 CIB_23 CIB_28

FSSC- 16C 34123_NRRL*

111009 110197 CIB_18

FSSC- 2A 22641_NRRL*

121584 FSSC- 6A 43489_NRRL*

111420

FSSC-1C 43812_NRRL*

122203

FSSC- 2B 43373_NRRL*

110358 CIB_15 122200 CIB_25 121607 CIB_16 110404 121885 122050 CIB_4 FSSC- 3+4+M 43537_NRRL*

CIB_3

110787 110157

CIB_17 CIB_6

FOSC 3-f 43668_NRRL*

FOSC 3-a 43521_NRRL* 122314

FOSC 3-b 43431_NRRL* CIB_19

CIB_26 110562

122162

FSSC- 5G 25388_NRRL*

FSSC- 3+4+C 43536_NRRL* 122008 110148 CIB_35484 110248 CIB_10 121371

FSSC- 3+4+UU 32743_NRRL*

110751 CIB_9

FSSC- 3+4+F 22781_NRRL* FSSC- 3+4+Q 28563_NRRL*

121995 CIB_14

FSSC- 3+4+Y 32331_NRRL* 122181 FSSC-1E 32856_NRRL* 110347 110472 121875 121472

FOSC 3-c 43442_NRRL* 110676

FSSC- 3+4+H 25456_NRRL* 110184

121377 110029

FSSC- 3+4+A 43441_NRRL* 121647 122098 110231 110819 FSSC-6C 22792_NRRL* 121984

122060 121927

111306 FSSC- 5H 28679_NRRL*

FSSC- 3+4+M 28555_NRRL*

FSSC- 3+4+E 43529_NRRL* 110266

122004

CIB_31 121988

FOSC 4-a 43454_NRRL*

122165

CIB_13 CIB_2

121617

FOSC 4-b 43504_NRRL*

110262 121470 110357 110283 CIB_7 110414

FOSC 3-e 43655_NRRL* FSSC- 3+4+G 22938_NRRL*

1 , 0,95 1 1 1 0,94 1 1 0,84 0,98 0,81 0,95 , , 1 0,97

Especies del

complejo de

Fusarium

solani

(FSSC)

Especies del

complejo de

Fusarium

oxysporum

(FOSC)

Cordyceps confragosa strain CBS 101247 Genotipo 1 Genotipo 6 Genotipo 2 Genotipo 16 Genotipo 5 Genotipo 3+4 Genotipo 4 Genotipo 3