Antifungal Susceptibility Patterns of Opportunistic Fungi in the Genera Verruconis and Ochroconis

Abstract

Species of Verruconis and species of Ochroconis are dematiaceous fungi generally found in the environment but having the ability to infect humans, dogs, cats, poultry, and fish. This study presents the antifungal susceptibility patterns of these fungi at the species level. Forty strains originating from clinical and environmental sources were phylogenetically identified at the species level by using sequences of the ribosomal DNA internal transcribed spacer (rDNA ITS). In vitro antifungal susceptibility testing was performed against eight antifungals, using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. The geometric mean MICs for amphotericin B (AMB), flucytosine (5FC), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), and posaconazole (POS) and minimum effective concentrations (MECs) for caspofungin (CAS) and anidulafungin (AFG) across the Ochroconis and Verruconis species were as follows, in increasing order. For Verruconis species, the values (μg/ml) were as follows: AFG, 0.04; POS, 0.25; ITC, 0.37; AMB, 0.50; CAS, 0.65; VRC, 0.96; 5FC, 10.45; and FLC, 47.25. For Ochroconis species, the values (μg/ml) were as follows: AFG, 0.06; POS, 0.11; CAS, 0.67; VRC, 2.76; ITC, 3.94; AMB, 5.68; 5FC, 34.48; and FLC, 61.33. Antifungal susceptibility of Ochroconis and Verruconis was linked with phylogenetic distance and thermotolerance. Echinocandins and POS showed the greatest in vitro activity, providing possible treatment options for Ochroconis and Verruconis infections.

INTRODUCTION

Recently, by combined molecular phylogeny, morphology, and ecology, the taxonomy of the Ochroconis lineage was revised (1). Two genera were recognized: Ochroconis and Verruconis. Within melanized filamentous fungi, members of Ochroconis and Verruconis are morphologically exceptional by having sympodial conidiogenesis with rhexolytic conidial dehiscence (2). However, both genera are melanized, oligotrophic, and regularly encountered in indoor environments, in soil, or in heated habitats, and some species have the ability to cause superficial, cutaneous, and systemic infections in immunocompromised patients (3–6).

Verruconis species are thermophilic, with Verruconis gallopava occurring in hot environments, such as thermal soils, broiler house litter, hot springs, and self-heated waste (1). Pathology in Verruconis is restricted to V. gallopava, which is the main agent of human brain infections and is responsible for encephalitis in poultry and wild birds (7–15), dogs (16), and cats (17). In contrast, Ochroconis species are mesophilic saprobes, with an optimum growth temperature between 15 and 30°C and an inability to grow at 37°C, which occasionally infect cold-blooded vertebrates (1, 18). Only a single infection was noted in a warm-blooded animal, i.e., a subcutaneous lesion in a cat (19), while the first subcutaneous human infection due to Ochroconis tshawytschae was recently reported (20).

Despite significant medical and veterinary importance, little is known regarding the species-specific antifungal susceptibility profiles of Verruconis and Ochroconis species. The polyene agents exert their antifungal activity via binding to ergosterol in the fungal cell membrane. This disrupts cell permeability and results in rapid cell death. Flucytosine exerts antifungal activity via inhibition of both DNA synthesis and protein synthesis in the fungal cell. Azole agents exert their antifungal activity by blocking the demethylation of lanosterol, thereby inhibiting ergosterol synthesis. The mechanism of activity of the echinocandins is inhibition of the production of (1,3)-β-d-glucan, an essential component in the fungal cell wall (21). We therefore investigated the in vitro susceptibilities of a large collection of clinical and environmental isolates of thermophilic and mesophilic species to eight antifungal drugs.

(Some of these results were presented at the 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Denver, CO, 10 to 13 September 2013.)

MATERIALS AND METHODS

Fungal strains.

Strains used in this study are listed in Table 1, with origin, identification number, and clinical data for each isolate. In total, 40 strains from clinical and environmental sources were used. Lyophilized fungal strains were obtained from the reference collection of the CBS-KNAW Fungal Biodiversity Centre (CBS, Utrecht, The Netherlands) and selected according to their historical pathogenicity. In addition, the representative type species of saprophytic strains were used for environmental isolates of both genera (Table 1). All isolates were cultured on malt extract agar (MEA) at 24°C for 14 days. Morphological identifications were confirmed by sequence-based analysis of the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) region, as described previously (1). Briefly, sequences were edited using the SeqMan tool of Lasergene software (DNAStar Inc., Madison, WI) and then aligned interactively using Ward's averaging in the BioNumerics package v. 4.61 (Applied Maths, Kortrijk, Belgium). The ITS sequences were finally aligned with the program MUSCLE (www.ebi.ac.uk/Tools/msa/muscle), and the aligned sequences were adjusted using BioEdit v. 7.0.5.2. The ITS data set was then analyzed by use of MEGA5 software (22), in which the Tamura three-parameter model with gamma distribution (T92+G) was searched as the best model. The maximum likelihood (ML) heuristic method with 1,000-replicate bootstrapping and the maximum parsimony (MP) method with 1,000-replicate bootstrapping were performed for tree reconstructions and phylogeny tests. To strongly confirm the analyses, the ML method with the approximate likelihood ratio test (aLRT) was also performed with PhyML (23). Trees were viewed and edited with TreeView v. 1.6.6, FigTree v. 1.1.2, and MEGA5.

TABLE 1.

Isolation data for examined strains of Ochroconis and Verruconis spp.

CBS ID	Species	Other collection no.	GenBank accession no.	Source or origin	Geography (city, state, country)	Yr of isolationa	Reference(s)
CBS 125817	V. calidifluminalis	IFM 54739	AB385699	Hot spring effluent	Kanakawa, Hakone, Japan	2004	47
CBS 125818 (type strain)	V. calidifluminalis	IFM 54738	AB385698	Hot spring effluent	Kanakawa, Hakone, Japan	2004	47
CBS 118.91	V. gallopava	CDC B-4954	HQ667551	Human	Atlanta, GA, USA	1991	48
CBS 166.85	V. gallopava	dH 14821	HQ667554	Environment	France	1985	27
CBS 265.97	V. gallopava	dH 14836	HQ667555	Australorp chick	Brisbane, Queensland, Australia	1990	14
CBS 437.64 (type strain)	V. gallopava	ATCC 16027, CDC 45-492-62, MUCL 6683	HQ667553	Turkey (Meleagris gallopavo)	Bishopville, SC, USA	1964	7
CBS 547.81	V. gallopava		HQ667560	Environment	Christchurch, New Zealand	1981	27
CBS 862.95	V. gallopava	ATCC 60633, CDC B-4224		Human	South Carolina	1990	49
CBS 863.95	V. gallopava	CDC B-5637	HQ667548	Human, bronchial aspirate	USA	1996	27
CBS 865.95	V. gallopava	CDC B-4872	HQ667549	Human, mine worker, sputum	Johannesburg, South Africa	1989	27
CBS 866.95	V. gallopava	CDC B-4767		Human	Mobile, AL, USA	1996	27
CBS 867.95	V. gallopava	CDC B-4682	HQ667561	Human, sputum	Salisbury, MD, USA	1996	27
CBS 100437	V. gallopava	ATCC 48169, IMI 241149	HQ667556	Broiler chicken	Scotland, UK	1998	27
CBS 116660	V. gallopava	CDC B-5813	HQ667557	Human, bronchoalveolar lavage fluid	USA	ND	27
CBS 116646	V. gallopava	IMI 308437	HQ667559	Human, sputum	Western Australia		27
CBS 119640	V. gallopava	dH 14079, NCPF 7122		Human	Australia	ND	27
CBS 119641	V. gallopava	NCPF 2923, dH 4078	HQ667547	Human, sputum	UK	ND	27
CBS 119642	V. gallopava	dH 14077, NCPF 2221	HQ667550	Chick	ND	ND	27
CBS 119922	V. gallopava	dH 14073		Human, L3 puncture	The Netherlands
CBS 120153	V. gallopava	dH13131, RKI 579/00		Human	Germany
CBS 729.95 (type strain)	O. mirabilis	dH 14850	KF156029	Regulator of diver	Haarlem, The Netherlands	1995	50
CBS 124.65	O. mirabilis	MUCL 6479	HQ667532	Human	India	1965
CBS 102468	O. mirabilis		HQ667533	Human	Nijmegen, The Netherlands	2000
CBS 113948	O. mirabilis	dH 13215	HQ667530	Human	Haarlem, The Netherlands
CBS 118685	O. mirabilis		HQ667529	Human, 8-year-old girl	Sweden
CBS 123268	O. mirabilis	dH 17473	HQ667526	Human	Denmark
CBS 124210	O. mirabilis	dH 17059	KF156028	Human	Denmark
CBS 135920	O. mirabilis	dH 22275	KF156033	Human	Thailand	2011
CBS 100438 (type strain)	O. tshawytschae	dH 10758, dH 14814, ATCC 9915	HQ667562	Fish (Chinook salmon)	USA	1946	51
CBS 129970	O. tshawytschae	CMCC(f)D.31a	JN974456	Human	Nanjing, China	2011	52
CBS 100486	O. constricta	NJM 9471	KF156026	Fish (devil stinger)	Kagoshima, Japan	1995	53
CBS 131913	O. constricta	dH22432	KF156025	Human	Thailand	2011
CBS 211.53 (type strain)	O. constricta	ATCC 11419, DAOM 28282, IMI 051380, MUCL 9896	HQ667519	Soil	Ancaster, Ontario, Canada	1952
CBS 135766	Ochroconis sp.	UIIII09		Fish	Sweden	2012
CBS 475.80 (type strain)	O. cordanae	dH 14825	KF156022	Dead leaf (Palmae)	Colombia	1979
CBS 116655 (type strain)	O. humicola	dH 13739, IMI 110131, UAMH 10241	HQ667521	Peat soil	Ontario, Canada	1962	54
CBS 510.71 (type strain)	O. minima	dH 14792, ATCC 22631, IMI 082933	HQ667522	Rhizosphere	Samaru, Zaria, Nigeria	1967	55
CBS 239.78 (type strain)	O. gamsii	dH 14835, CBS H-7440	KF156019	Plant leaf	Sri Lanka	1973	56
CBS 383.81 (type strain)	O. verrucosa	IMI 211655	KF156015	Soil	Kerala, Kolkata, India	1981	57
CBS 284.64 (type strain)	O. anellii	IHEM 4516, IMI 089069, MUCL 9473	FR832477	Stalactites	Bari, Italy	1962	58
CBS 131815 (type strain)	O. lascauxensis	CMFISB 1862, LX A1	FR832474	Black stains	Montignac, Lascaux Cave, France	2008	59

^aND, not determined.

In vitro antifungal susceptibility testing.

In vitro antifungal susceptibility testing was performed using a broth microdilution format against eight antifungal compounds according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (24). The final concentrations of the antifungal agents ranged from 0.016 to 16 μg/ml for amphotericin B (AMB), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), posaconazole (POS), caspofungin (CAS), and anidulafungin (AFG). Flucytosine (5FC) was assayed over a 2-fold concentration range from 0.064 to 64 μg/ml. Reading of results was performed using a reading mirror and a microtitration plate spectrophotometric reader (Anthos htIII; Anthos Labtec Instruments, Salzburg, Austria).

Ochroconis isolates were incubated at 25°C, and Verruconis isolates were incubated at 37°C. Agitation of plates was not used. The MICs of AMB, FLC, 5FC, ITC, VRC, and POS were determined visually with an inverted mirror by comparison of growth in the wells containing the drug and that of the drug-free control. The minimum effective concentrations (MECs) of CAS and AFG were read with a plate microscope (Olympus SZX9; Olympus Nederland, Zoeterwoude, The Netherlands) at a magnification of ×25 to ×50. The MEC was defined as the lowest concentration at which abnormal, short, and branched hyphal clusters were observed, in contrast to the long, unbranched hyphal elements that were seen in the growth control well.

Paecilomyces variotii (ATCC 22319), Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258) were used as quality controls in all experiments. The ranges and geometric means (GM) of the MICs and MECs were determined for each species and drug after 48 to 168 h of incubation. Furthermore, the MIC₅₀s and MIC₉₀s for the isolates were calculated by use of the criteria for MIC determinations described above. The MIC₅₀ and MIC₉₀ values were calculated for those species with 10 or more isolates. If the MIC values of the replicates were different, the GM values of the replicates were used for comparison with other isolates. All experiments were performed in three independent replicates with each strain on different days.

Statistical analysis.

Data analyses were performed by using GraphPad Prism, version 5.0, for Windows (GraphPad Software, San Diego, CA). MIC/MEC distributions between isolates were compared by using the Mann-Whitney-Wilcoxon test. Statistical significance was defined as having a P value of ≤0.05 (two-tailed).

RESULTS

All strains were identified to the species level by sequence-based analysis and tested against eight antifungal compounds. Two genera have been recognized based on molecular phylogeny, viz., Verruconis and Ochroconis. In Fig. 1, data are summarized for relevant species, displaying their mutual phylogenetic distances, interspecies variability according to temperature tolerance, and antifungal susceptibility profiles per species. GM MICs, MIC ranges, and MIC₅₀ and MIC₉₀ distributions for eight antifungal agents are summarized in Table 2.

FIG 1.

MEGA5 maximum likelihood tree created from ITS sequences of Ochroconis and Verruconis isolates. The geometric mean susceptibility profiles of eight antifungals against each species have been incorporated into the figure. Numbers on branches are percent bootstrap values obtained from ML, aLRT, and MP analyses. Type strains are highlighted by a “T.” ND, not determined.

TABLE 2.

Geometric mean MICs, MIC ranges, MIC₅₀s, and MIC₉₀s obtained by susceptibility testing of antimycotic agents against Ochroconis and Verruconis spp.

Organism (n) and drug	MIC or MEC (mg/liter)a
	Range	50%	90%	Geometric mean
All thermotolerant strains (Verruconis spp.) (n = 20)
Amphotericin B	0.125–4	0.25	0.5	0.50
Flucytosine	0.5–64	4	32	10.45
Fluconazole	1–>64	64	>64	47.25
Itraconazole	<0.016–4	0.125	0.5	0.37
Voriconazole	0.063–2	1	2	0.96
Posaconazole	<0.016–4	0.031	0.125	0.25
Caspofungin	0.25–1	0.5	1	0.65
Anidulafungin	0.016–0.125	0.031	0.063	0.04
All mesophilic strains (Ochroconis spp.) (n = 20)
Amphotericin B	0.25->16	2	>16	5.68
Flucytosine	0.125->64	16	>64	34.48
Fluconazole	8->64	64	>64	61.33
Itraconazole	<0.016–>16	0.25	>16	3.94
Voriconazole	0.125–8	2	8	2.76
Posaconazole	<0.016–0.25	0.063	0.25	0.11
Caspofungin	0.063–2	0.5	1	0.67
Anidulafungin	0.016–0.25	0.031	0.125	0.06
V. gallopava (n = 18)
Amphotericin B	0.125–4	0.25	0.5	0.54
Flucytosine	0.5–64	4	32	11.53
Fluconazole	4->64	64	>64	52.22
Itraconazole	0.016–4	0.125	0.5	0.40
Voriconazole	0.5–2	1	2	1.06
Posaconazole	<0.016–4	0.031	0.125	0.28
Caspofungin	0.25–1	0.5	1	0.64
Anidulafungin	0.016–0.125	0.031	0.063	0.04
V. calidifluminalis (n = 2)
Amphotericin B	0.063–0.125	NC	NC	0.094
Flucytosine	0.5–1	NC	NC	0.75
Fluconazole	1–4	NC	NC	2.5
Itraconazole	≤0.16	NC	NC	0.016
Voriconazole	0.063–0.125	NC	NC	0,.094
Posaconazole	≤0.16	NC	NC	0.016
Caspofungin	0.5–1	NC	NC	0.75
Anidulafungin	0.031–0.063	NC	NC	0.047
O. mirabilis (n = 8)
Amphotericin B	1->16	NC	NC	12.625
Flucytosine	8–64	NC	NC	21
Fluconazole	≥64	NC	NC	64
Itraconazole	0.25–>64	NC	NC	10.09375
Voriconazole	2–8	NC	NC	5.75
Posaconazole	0.063–0.25	NC	NC	0.211
Caspofungin	0.5–1	NC	NC	0.75
Anidulafungin	0.031–0.125	NC	NC	0.05475
O. tshawytschae (n = 2)
Amphotericin B	0.125–>64	NC	NC	2
Flucytosine	8–>64	NC	NC	32.0625
Fluconazole	0.016–0.25	NC	NC	36
Itraconazole	0.125–0.5	NC	NC	0.133
Voriconazole	<0.016–0.125	NC	NC	0.3125
Posaconazole	1–2	NC	NC	0.125
Caspofungin	0.125–0.25	NC	NC	1.5
Anidulafungin	0.031–0.125	NC	NC	0.1875
O. constricta (n = 3)
Amphotericin B	1–2	NC	NC	1.33
Flucytosine	>64	NC	NC	64.00
Fluconazole	64–> 64	NC	NC	64.00
Itraconazole	0.063	NC	NC	0.06
Voriconazole	0.5–1	NC	NC	0.67
Posaconazole	0.016	NC	NC	0.02
Caspofungin	0.063–0.5	NC	NC	0.35
Anidulafungin	0.031	NC	NC	0.03
Ochroconis sp. (n = 1)
Amphotericin B	4	NC	NC	NC
Flucytosine	64	NC	NC	NC
Fluconazole	64	NC	NC	NC
Itraconazole	0.25	NC	NC	NC
Voriconazole	0.25	NC	NC	NC
Posaconazole	0.031	NC	NC	NC
Caspofungin	0.5	NC	NC	NC
Anidulafungin	0.016	NC	NC	NC
O. cordanae (n = 1)
Amphotericin B	1	NC	NC	NC
Flucytosine	16	NC	NC	NC
Fluconazole	64	NC	NC	NC
Itraconazole	0.25	NC	NC	NC
Voriconazole	2	NC	NC	NC
Posaconazole	0.016	NC	NC	NC
Caspofungin	0.5	NC	NC	NC
Anidulafungin	0.031	NC	NC	NC
O. humicola (n = 1)
Amphotericin B	1	NC	NC	NC
Flucytosine	64	NC	NC	NC
Fluconazole	64	NC	NC	NC
Itraconazole	0.125	NC	NC	NC
Voriconazole	1	NC	NC	NC
Posaconazole	0.063	NC	NC	NC
Caspofungin	1	NC	NC	NC
Anidulafungin	0.125	NC	NC	NC
O. minima (n = 1)
Amphotericin B	1	NC	NC	NC
Flucytosine	8	NC	NC	NC
Fluconazole	64	NC	NC	NC
Itraconazole	0.063	NC	NC	NC
Voriconazole	0.5	NC	NC	NC
Posaconazole	0.016	NC	NC	NC
Caspofungin	0.5	NC	NC	NC
Anidulafungin	0.031	NC	NC	NC
O. gamsii (n = 1)
Amphotericin B	1	NC	NC	NC
Flucytosine	64	NC	NC	NC
Fluconazole	64	NC	NC	NC
Itraconazole	0.25	NC	NC	NC
Voriconazole	2	NC	NC	NC
Posaconazole	0.031	NC	NC	NC
Caspofungin	0.5	NC	NC	NC
Anidulafungin	0.031	NC	NC	NC
O. verrucosa (n = 1)
Amphotericin B	0.25	NC	NC	NC
Flucytosine	16	NC	NC	NC
Fluconazole	64	NC	NC	NC
Itraconazole	0.25	NC	NC	NC
Voriconazole	2	NC	NC	NC
Posaconazole	0.031	NC	NC	NC
Caspofungin	0.25	NC	NC	NC
Anidulafungin	0.016	NC	NC	NC
O. anellii (n = 1)
Amphotericin B	1	NC	NC	NC
Flucytosine	4	NC	NC	NC
Fluconazole	64	NC	NC	NC
Itraconazole	0.031	NC	NC	NC
Voriconazole	0.5	NC	NC	NC
Posaconazole	0.031	NC	NC	NC
Caspofungin	0.5	NC	NC	NC
Anidulafungin	0.031	NC	NC	NC
O. lascauxensis (n = 1)
Amphotericin B	1	NC	NC	NC
Flucytosine	64	NC	NC	NC
Fluconazole	64	NC	NC	NC
Itraconazole	0.25	NC	NC	NC
Voriconazole	1	NC	NC	NC
Posaconazole	0.063	NC	NC	NC
Caspofungin	0.25	NC	NC	NC
Anidulafungin	0.031	NC	NC	NC

^aThe MIC₅₀ and MIC₉₀ values were calculated for those species with 10 or more isolates. NC, not calculated, because <10 strains per species were available for testing.

Overall, visual and spectrophotometric readings gave similar results for the MIC and MEC endpoints. The GM MICs for AMB, 5FC, FLC, ITC, VRC, and POS and the MEC values for CAS and AFG across the genera in this study are shown below, in increasing order. For Verruconis, the values (μg/ml) were as follows: AFG, 0.04; POS, 0.25; ITC, 0.37; AMB, 0.50; CAS, 0.65; VRC, 0.96; 5FC, 10.45; and FLC, 47.25. For Ochroconis, the values (μg/ml) were as follows: AFG, 0.06; POS, 0.11; CAS, 0.67; VRC, 2.76; ITC, 3.94; AMB, 5.68; 5FC, 34.48; and FLC, 61.33. The widest ranges were seen for FLC (range, 1 to ≥64 μg/ml) and 5FC (range, 0.5 to 64 μg/ml). The highest GM MICs were 47.25 μg/ml, for FLC, followed by 10.45 μg/ml, for 5FC. AMB MICs ranged from 0.125 to >16 μg/ml, and ITC had a MIC range of <0.016 to >16 μg/ml. POS exhibited potent activity against all strains, with MICs ranging from <0.0016 to 4 μg/ml, while the GM MIC of VRC (0.96 μg/ml) was 2 log₂ dilution steps less potent than that of POS (0.25 μg/ml) against thermotolerant strains and 6 log₂ dilution steps less active than in the Ochroconis species (2.76 μg/ml VRC versus 0.11 μg/ml POS). Notably, Ochroconis isolates had higher MICs of AMB, 5FC, FLC, ITC, and VRC than those for Verruconis strains. The two echinocandins showed susceptible profiles in their MECs. In most cases, AFG had a higher activity than that of CAS (AFG MEC of 0.04 μg/ml versus 0.65 μg/ml CAS against Verruconis strains, and AFG MEC of 0.06 μg/ml versus 0.67 μg/ml CAS against Ochroconis species). In addition, various susceptibility profiles were demonstrated within the genera. For Ochroconis, the triazole derivatives ITC and VRC and AMB offered significantly (P ≤ 0.05) higher susceptible profiles for O. mirabilis than for the other species. However, 5FC and FLC were found to be less active against V. gallopava than against V. calidifluminalis (P ≤ 0.05).

DISCUSSION

The genus Ochroconis was recently revised and currently contains 13 species (1). Species accepted within the lineage, within the order Venturiales, were keyed out on the basis of molecular phylogeny and phenotypic and physiologic characteristics. A new genus, Verruconis, was proposed for the neurotropic opportunist Ochroconis gallopava and its close relatives.

Notably, thermotolerance has a significant impact on the virulence potential of Ochroconis and Verruconis species, as shown previously in other melanized fungi. Species able to grow at temperatures of 37°C or above (e.g., Cladophialophora bantiana, Exophiala dermatitidis, and Exophiala jeanselmei) (25) may cause systemic or disseminated infections in mammals. The black yeast Exophiala dermatitidis has a maximum growth temperature of 42 to 45°C and has a natural habitat in association with birds and bats, which have a body temperature well above that of humans (26, 27). Mesophilic species with maximum growth temperatures of 27 to 33°C are restricted to cold-blooded vertebrates (25) or, occasionally, invertebrates (28, 29).

The availability of in vitro susceptibility profiles according to the latest taxonomic studies of Ochroconis and Verruconis species (1) is scant. Our study provides the first antifungal susceptibility data on a large set of clinical and environmental strains from a wide range of sources and origins. Our results indicate that thermotolerance has a significant impact on the antifungal susceptibility of Ochroconis and Verruconis species. Both thermotolerant and mesophilic species had susceptibility profiles with a uniform pattern of low MICs for POS, AFG, and CAS. VRC, AMB, and ITC showed efficacy against Verruconis species, with 1-, 3-, and 4-log₂ less susceptibility, respectively, than Ochroconis species. The majority of strains demonstrated high MICs for 5FC and FLC, indicating poor activity of these drugs against the pathogens. Both echinocandins were found to have potent in vitro activity against Ochroconis species. This matches previously reported data for CAS, with a MEC of 0.25 μg/ml against Ochroconis tshawytschae (20) and 0.03 to 1 μg/ml against Verruconis gallopava (30, 31). This is in contrast with previously published data on most melanized fungi, which appear to be tolerant to echinocandins, probably due to the presence of melanin, which prevents penetration of antifungals into fungal cells (32). Nevertheless, O. mirabilis demonstrated less susceptibility to ITC, VRC, and AMB (P ≤ 0.05) than the other Ochroconis species, and V. calidifluminalis was more susceptible to 5FC and FLC than V. gallopava (P ≤ 0.05).

Given that the echinocandins and the triazole POS showed the highest in vitro activity against thermotolerant and mesophilic species, a possible treatment option for Ochroconis and Verruconis infections in both warm-blooded (human) and cold-blooded animals may be provided. The triazole POS is an expanded-spectrum triazole with fungicidal activity against a wide spectrum of molds, including Aspergillus species and members of the Mucorales, as well as enhanced activity against Candida and other yeasts (33). The echinocandins represent the newest class of antifungals that exhibit fungicidal activity against many Candida species, making this drug class a desirable alternative to the azole agents, which exhibit only static activity against yeasts. Because mammalian cells have no cell wall, the echinocandins have very few adverse effects in humans (33).

In general, the divergent antifungal profiles of the Verruconis and Ochroconis genera and the interspecies variability observed for O. mirabilis and V. calidifluminalis clearly suggest that routine in vitro susceptibility testing can be useful for obtaining reliable information on treatment options. Until now, there have been no guidelines for optimal antifungal regimens for Ochroconis and Verruconis species. Although various efficacies have been documented (34), several studies suggest that POS and ITC may provide optimal therapies for Ochroconis infection, followed by AMB and VRC, and that 5FC and FLC are the least effective drugs (6, 30, 31, 35, 36), which is in agreement with the in vitro results of the present study.

Treatment of Verruconis infections with VRC is supported by in vitro results, and it proved to be active in a chronic granulomatous disease (CGD) patient (34). VRC has an optimal oral bioavailability and penetration to the blood-brain barrier, indicating its use for cerebral infections. In some cases of V. gallopava infections, AMB was also used successfully in empirical antifungal therapy (37). However, further studies are required to establish the optimal treatment. In addition, as recommended for other fungal infections, supportive management strategies, such as surgical excision of lesions, are recommended whenever feasible (34). Early diagnosis and treatment are also mandatory in order to avoid dissemination to the brain, which carries a very poor prognosis (20).

In conclusion, although there are no clinically defined breakpoints for Verruconis and Ochroconis species and the lack of interpretative breakpoints makes MICs difficult to interpret, antifungal susceptibility testing can be helpful in guiding clinical management of patients with these infections. Based on the data presented in the current study, POS and echinocandins were the antimycotics with the best overall activity, having broad-spectrum activity against both thermotolerant and mesophilic species. The apparently good penetration of POS into the central nervous system (CNS), with the MIC falling well below the serum levels achievable with standard dosing regimens (38), combined with excellent in vitro data (39) and activity in animal models (40–43), supports the use of POS for difficult-to-treat disseminated brain infections. In the clinical setting, POS has been used successfully in cases of cerebral and disseminated phaeohyphomycosis (44, 45). In addition, POS and VRC are routinely recommended for treatment, prophylaxis, and salvage therapy of life-threatening fungal infections, such as Aspergillus diseases. POS also has a label indication for the treatment of less common infections, including chromoblastomycosis, mycetoma, and coccidioidomycosis. Therefore, standard dosing regimens and provisional target concentrations used for the prevention or treatment of invasive fungal infections (46) might be optimal tentative suggestions for Verruconis and Ochroconis infections.