Antifungal Susceptibility of Emerging Dimorphic Pathogens in the Family Ajellomycetaceae

ABSTRACT

The in vitro susceptibilities of 24 molecularly identified dimorphic fungi belonging to the genera Adiaspiromyces, Blastomyces, and Emergomyces within the family Ajellomycetaceae were tested against 8 standard antifungal agents using CLSI document M38-A2. Amphotericin B and posaconazole had the lowest geometric mean MICs (<0.05 μg/ml) followed by itraconazole (<0.07 μg/ml), voriconazole (<0.15 μg/ml), and isavuconazole (<0.42 μg/ml) while fluconazole was not active. Micafungin demonstrated good in vitro antifungal activity against Emergomyces (geometric mean minimum effective concentration [GM MEC] 0.1 μg/ml) and Blastomyces (GM MEC <0.017 μg/ml).

TEXT

Fungi in the family Ajellomycetaceae are well known systemic dimorphic pathogens. Recent phylogenetic studies resulted in the establishment of two new genera (Adiaspiromyces and Emergomyces) in addition to the well-known genera Blastomyces, Histoplasma, and Paracoccidioides (1–3). The genus Adiaspiromyces now accommodates the former Emmonsia crescens and the environmental species Adiaspiromyces terricola (2). The genus Emergomyces contains Emergomyces pasteurianus as a type species (formerly Emmonsia pasteuriana) and four newly introduced species, Emergomyces africanus (1), Emergomyces orientalis (4), Emergomyces canadensis, and Emergomyces europaeus (2). Blastomyces was reorganized to include three new species, B. silverae, B. helicus, and B. percursus along with Blastomyces parvus (formerly Emmonsia parva), B. gilchristii, and the well-known human pathogen B. dermatitidis (1, 2).

These genera and their species differ in their pathogenic forms (adiaspores, small yeasts and large yeasts), host range, and clinical manifestation. Adiaspiromyces crescens is primarily a causative agent of pulmonary infections in terrestrial mammals (5) but has been reported occasionally as a causative agent of pulmonary infections in humans (6). The pathogenic form is called adiaspore, a large spore with dimensions ranging from 20 to 140 μm (7). Since human infections are rare, there are only few reports on antifungal susceptibility testing (5) or treatment (6).

In contrast, Emergomyces species are human pathogens and not yet isolated from the environment or from animals. Almost all infected human hosts were HIV infected. Emergomyces species differ from the other species within the same family by producing budding yeasts (less than 5 μm) in vivo rather than adiaspores and often disseminate with secondary cutaneous manifestation, referred to as emergomycosis (8). Two species have been tested for antifungal susceptibility: Emergomyces africanus, the causative agent of an outbreak in South Africa (8, 9), and Emergomyces pasteurianus (10). Emergomyces infections have been treated mostly with amphotericin B, itraconazole, or fluconazole (4, 11; S. Sanche, A. Wong, L. Sigler, S. Angel, and S. Peterson, presented at Focus on Fungal Infections, Miami, FL, 2005, abstract 87) but no clinical trial has been conducted to date.

The genus Blastomyces is more versatile than Adiaspiromyces and Emergomyces. Blastomyces dermatitidis, B. gilchristii, and B. percursus are human pathogens with broad-based large yeasts (more than 5 μm) as the pathogenic form (2). Blastomyces dermatitidis and B. gilchristii cause blastomycosis mostly in North America (12), with few cases reported from the Asia-Pacific region (13, 14). There are several antifungal susceptibility studies (15–17) and treatment suggestions (18, 19). Blastomyces percursus was recently described as a human pathogen in both immunocompetent and immunocompromised patients (20). The other two species, B. parvus (Emmonsia parva) and the newly described B. silverae cause infections in small animals but rarely in humans (2). No in vitro susceptibility data for these two species have been published.

We report the in vitro antifungal susceptibility of eight antifungals of the newly described species Emergomyces africanus, E. orientalis, E. canadensis, Blastomyces percursus, B. helicus, and B. silverae. Although in vitro antifungal susceptibility testing of these dimorphic fungi is not standardized, the obtained MIC data might help with treatment decisions for infections caused by them. Of 24 isolates, 14 (58.3%) originated from human patients, 4 (16.6%) from rodents, 2 (8.3%) from soil, and 1 (4.16%) each from a weasel, coyote dung, and a bird's nest; 1 was of unknown origin. It should be noted that these species are still rare, and thus only small numbers of strains per species are available: Emergomyces pasteurianus (n = 3), E. africanus (n = 5), E. orientalis (n = 1), E. canadensis (n = 2), Adiaspiromyces crescens (n = 4), B. gilchristii (n = 1), B. parvus (n = 2), B. helicus (n = 1), B. silverae (n = 3), and B. percursus (n = 1), with one strain of Blastomyces dermatitidis as reference. All strains were initially subcultured on malt extract agar (MEA) (Oxoid, UK) and molecularly identified using multilocus sequencing as previously described (1).

Susceptibility studies were conducted in a class 2 biosafety cabinet in biosafety level 2 (BSL 2) laboratories, except for B. dermatitidis and B. gilchristii, which were tested using a BSL 3 facility. The isolates were subcultured on potato dextrose agar (PDA) (Oxoid, UK), malt extract agar (MEA) (Oxoid, UK), and Sabouraud's glucose agar (SGA). Sporulation on PDA was most abundant, and up to 10 plates with this medium were used to collect sufficient amounts of conidia. Antifungal susceptibility testing (AFST) was determined by broth microdilution using CLSI document M38-A2 (21). The mold phase on PDA at 24°C was used for inoculum preparation, which was adjusted to an optical density at 530 nm, to present transmission in a range of 76 to 77%, corresponding to 2.5 to 5 ×10⁵ CFU per ml and diluted 1:10 in RPMI 1640 medium. Plates were incubated at 24°C for 6 days with the following drug concentration ranges: amphotericin B (Bristol Myers Squib, Woerden, The Netherlands), itraconazole (Janssen Cilag, Beerse, Belgium), voriconazole (Pfizer Central Research, Sandwich, UK), posaconazole (Merck, Sharp & Dome, Haarlem, The Netherlands), and isavuconazole (Basilea Pharmaceutica, Basel), 0.016 to 16 μg/ml; fluconazole (Pfizer), 0.063 to 64 μg/ml; and anidulafungin (Pfizer) and micafungin (Astellas Pharma, Toyama, Japan), 0.008 to 8 μg/ml. The quality of every new batch of MIC plates was tested by using two quality control (QC) strains, Candida parapsilosis ATCC 22016 and Aspergillus flavus ATCC 204304.

Table 1 summarizes the MICs of 24 isolates from three clinically related genera, Emergomyces, Adiaspiromyces, and Blastomyces. All Emergomyces species had MIC values of ≥64 μg/ml for fluconazole, except one Emergomyces canadensis, which had a MIC of 8 μg/ml. Adiaspiromyces species showed high MICs of 16 μg/ml, with one isolate having the highest value of 64 μg/ml. Blastomyces silverae showed the highest MIC for fluconazole with ≥64 μg/ml, followed by B. parvus and B. helicus with 32 μg/ml, respectively. Blastomyces dermatitidis, B. gilchristii, and B. percursus had the lowest MICs (2 μg/ml) of all species. For the other azoles, a small degree of variation was found at the generic level. Overall, all MIC values of azole compounds except fluconazole were low. Amphotericin B was the most active antifungal agent and had the lowest MICs against species of Adiaspiromyces, Emergomyces, and Blastomyces. At the genus level, geometric mean (GM) MIC and MIC ranges of amphotericin B were, respectively, 0.027 and <0.016 to 0.125 μg/ml for Adiaspiromyces, 0.049 and <0.016 to 0.25 μg/ml for Emergomyces, and 0.023 and <0.016 to 0.063 μg/ml for Blastomyces. GM MIC values of itraconazole, voriconazole, posaconazole and isavuconazole were comparable to the values of amphotericin B (Table 2).

TABLE 1.

In vitro susceptibility of 24 Emergomyces, Adiaspiromyces, and Blastomyces strains

No.	Species name	CBS no.	MIC/MEC (μg/ml) fora:								Source	Origin
			AMBb	FLC	ITC	VOR	POS	ISA	ANID	MICA
1	Emergomyces pasteurianus	101426 (T)	0.031	>64	0.125	0.25	0.125	2	0.5	0.031	Human, HIV, disseminated	Italy
2		140361	0.063	64	0.063	0.25	0.063	0.5	0.5	0.016	Human, HIV, disseminated	S.Africa
3		139522	0.125	>64	0.25	0.25	0.063	1	0.031	0.063	Human, renal transplant, disseminated	China
4	Emergomyces africanus	140362	0.25	64	0.125	0.063	<0.016	0.125	1	1	Human, HIV, disseminated	S.Africa
5		136260 (T)	0.031	64	0.063	0.125	0.063	0.5	2	0.031	Human, HIV, disseminated	S.Africa
6		4164	0.063	64	0.063	0.063	0.031	0.125	2	1	Human, HIV, disseminated	S.Africa
7		140363	0.031	64	<0.016	0.063	0.031	0.25	1	0.063	Human, HIV, disseminated	S.Africa
8		139543	0.031	64	0.063	0.25	0.031	2	0.5	0.125	Human, HIV, disseminated	S.Africa
9	Emergomyces orientalis	124587	0.063	>64	0.031	0.125	0.031	0.25	1	0.125	Human, diabetes, disseminated	China
10	Emergomyces canadensis	139873	0.031	8	0.063	0.125	0.063	0.25	0.5	0.063	Human, transplant, disseminated	Canada
11		139872 (T)	<0.016	>64	0.031	0.125	0.031	0.25	2	0.25	Human, HIV, skin lesion	Canada
12	Adiaspiromyces crescens	139864	<0.016	16	0.031	0.063	<0.016	0.125	2	0.125	Rodent	S.Korea
13		191.55	<0.016	16	<0.016	<0.016	<0.016	<0.016	4	0.063	Rodent	Canada
14		177.60 (T)	<0.016	16	0.031	0.063	0.031	0.125	8	8	Rodent	Norway
15		139869	0.125	16	0.031	0.125	0.063	0.25	8	0.125	Environmental	Canada
16	Blastomyces dermatitidis	126.33	0.016	2	<0.016	0.063	0.016	0.063	0.031	0.008	No data	no data
17	Blastomyces gilchristii	134223	0.031	2	0.031	0.063	0.031	0.125	0.016	0.008	Human	Canada
18	Blastomyces parvus	139881 (T)	<0.016	32	0.016	0.125	0.031	0.25	0.5	0.031	Rodent	USA
19		139883	<0.016	32	<0.016	0.063	0.031	0.25	0.25	0.031	Soil	Italy
20	Blastomyces helicus	140056 (T)	<0.016	32	<0.016	<0.016	<0.016	<0.016	0.063	0.0031	Human, leukemia	Canada
21	Blastomyces silverae	139879 (T)	<0.016	64	0.125	0.125	0.125	0.25	1	0.063	Weasel	USA
22		139885	0.063	64	0.031	0.063	0.063	0.25	1	0.016	Coyote dung	Canada
23		139871	0.063	>64	0.125	0.125	0.063	0.5	0.25	0.031	Bird's nest	Canada
24	Blastomyces percursus	139878	<0.016	2	<0.016	0.031	0.031	0.031	0.25	0.031	Human, immunocompetent	Israel

^aMIC/MEC values determined according to CLSI document M38-A2.

^bAMB, amphotericin B; FLC, fluconazole; ITC, itraconazole; VOR, voriconazole; POS, posaconazole; ISA, isavuconazole; ANID, anidulafungin; MICA, micafungin.

TABLE 2.

MIC/MEC range, GM MIC/MEC, MIC/MEC₅₀, and MIC/MEC₉₀ values for Adiaspiromyces, Emergomyces, and Blastomyces to eight antifungal drugs

Genus (no. of strains)	Susceptibility parameterb	Susceptibility values fora:
		AMBc	FLC	ITC	VOR	POS	ISA	ANID	MICA
Adiaspiromyces (4)	MIC/MEC range	<0.016–0.125	16–64	<0.016–0.031	<0.016–0.125	0.016–0.063	<0.016–0.125	2- 8	0.125–8
	GM MIC/MEC	0.027	22.6	0.026	0.05	0.026	0.088	4.75	0.29
Emergomyces (11)	MIC/MEC range	<0.016–0.25	8–>64	<0.016–0.25	0.063–0.25	<0.016–0.125	0.125–2	0.031–2	0.016–1
	GM MIC/MEC	0.049	46.7	0.063	0.133	0.043	0.414	0.68	0.1
	MIC/MEC50	0.031	64	0.063	0.125	0.031	0.25	1	0.063
	MIC/MEC90	0.125	64	0.125	0.25	0.063	2	2	1
Blastomyces (9)	MIC/MEC range	<0.016–0.063	2– >64	<0.016–0.125	<0.016–0.25	<0.016–0.063	0.125–0.5	0.016–1	0.016–0.063
	GM MIC/MEC	0.023	16	0.029	0.062	0.036	0.125	0.18	0.017

^aMIC/MEC values determined according to CLSI document M38-A2.

^bMIC values were determined for amphotericin B and azole drugs; MEC values were determined for anidulafungin and micafungin.

^cAbbreviations are the same as in Table 1.

Anidulafungin had a low GM minimum effective concentration (MEC) and a MEC range of 0.68 and 0.031 to 2 μg/ml, respectively, for the genus Emergomyces. The GM MEC and MEC ranges were 0.18 and 0.016 to 1 μg/ml, respectively, for Blastomyces. Micafungin showed consistent low values with GM MEC and MEC ranges of 0.1 and 0.016 to 1 μg/ml, respectively, for Emergomyces. GM MEC and MEC ranges for Blastomyces were 0.017 and 0.016 to 0.063 μg/ml, respectively.

For all 11 strains of the newly described genus Emergomyces, posaconazole had the lowest GM MIC values followed by amphotericin B, itraconazole, voriconazole, isavuconazole, anidulafungin, and micafungin (Table 2).

We reported the antifungal susceptibility of 24 isolates from three genera within the Ajellomycetaceae family, Adiaspiromyces, Blastomyces, and Emergomyces. All three genera analyzed in this study sporulated slowly. This feature was also reported previously for Paracoccidioides (22), Emergomyces africanus (7), and Emmonsia pasteuriana (23). In contrast to Blastomyces dermatitidis and B. gilchristii, Emergomyces spp. do not constitute biosafety level 3 (BSL3) organisms, and therefore work was done in a class 2 biosafety cabinet. Blastomyces was handled, according to existing recommendations, in a BSL3 facility. Antifungal susceptibility testing of thermally dimorphic fungi is often limited to the mold phase, in which results may be misleading because the yeast phase is responsible for human disease. We limited antifungal susceptibility testing to the mold phase for the following reasons: (i) the pathogenic forms of these fungi differ greatly in the size and (ii) conversion time for some was very long and prone to contamination, with culturing for enough material taking longer than 1 month. Although it is a limitation that only the mold phase was tested, to our assurance recent studies of different endemic fungi using the mold and yeast phase for susceptibility testing found no differences in MICs between the two phases (8, 24).

This is the first comparative in vitro susceptibility study of five newly described taxa of dimorphic pathogens, Emergomyces orientalis, Emergomyces canadensis, B. percursus, B. helicus, and B. silverae, and of B. parvus and B. gilchristii. In vitro susceptibility studies of Adiaspiromyces crescens, Emergomyces africanus, Emergomyces pasteurianus, and Blastomyces dermatitidis have been performed previously (5, 8–10). For Adiaspiromyces crescens, our results confirm the previous findings of low MICs when the mold phase was tested, with MIC values of 0.06, 0.25, 0.06, and 64 μg/ml for amphotericin B, itraconazole, voriconazole, and fluconazole, respectively (5). Furthermore, the low MIC ranges of amphotericin B and azoles and high MIC range of fluconazole for Emergomyces pasteurianus and Emergomyces africanus were in agreement with previous findings (9, 10). In addition, the GM MIC, MIC₅₀, and MIC₉₀ of Emergomyces africanus are in agreement with results described for 50 E. africanus strains, except for fluconazole and with slightly higher values for echinocandins (8).

Treatment of patients infected by members of the genus Emergomyces (formerly Emmonsia) has been done with amphotericin B deoxycholate (25), liposomal amphotericin B (26), combinations of amphotericin B and itraconazole (9, 10) or caspofungin (27), and voriconazole (28). In one case report, a patient infected with Emergomyces canadensis strain CBS 139873 (UAMH 10370) was treated with liposomal amphotericin B (S. Sanche et al., presented at Focus on Fungal Infections, Miami, FL, 2005, abstract 87). A patient suffering from rheumatoid arthritis who was infected with Emergomyces europaeus (Emmonsia sp.) was treated with oral itraconazole (11). A case of Emergomyces orientalis was treated with amphotericin B and itraconazole after shifting due to clinical failure with fluconazole (4). These successful results were in agreement with our in vitro findings for this genus.

Members of the genus Blastomyces had consistently low MIC/MEC values for amphotericin B, itraconazole, voriconazole, posaconazole, isavuconazole, and micafungin. Our results matched previously published data with low MIC ranges for itraconazole and high ranges for fluconazole (15). In a comparative study of 304 strains belonging to Blastomyces, Histoplasma, and Coccidioides, amphotericin B, itraconazole, and voriconazole were effective in vitro, with the MIC₉₀ being the lowest for itraconazole, followed by voriconazole and amphotericin B (16). We also report low anidulafungin GM MEC and MEC ranges for Emergomyces and Blastomyces. Micafungin exhibited potent activity against the mold phases of Emergomyces and Blastomyces but is weakly active against the yeast forms of Histoplasma capsulatum, B. dermatitidis, and Coccidioides immitis (17). In contrast there was no difference between mold and yeast phase testing of Histoplasma capsulatum for caspofungin (24). At present, based on in vitro susceptibility testing, it cannot be judged whether echinocandins (anidulafungin and micafungin) have clinical usefulness for dimorphic fungal infections, since for most fungi it remains uncertain which growth form correlates better with therapeutic outcome.

Our results with Blastomyces testing agree with recommended treatment of blastomycosis (18). Currently, there are no published treatment guidelines for patients with Adiaspiromyces and Emergomyces. Conidia of Adiaspiromyces do not multiply in host tissues, and it is generally a self-limiting disease. In few immunocompromised patients, antifungal treatment has been attempted apparently with successful outcome (29). According to published cases and international guidelines for the management of patients with dimorphic fungal infections (30, 31), the recommended treatment is amphotericin B, followed by an azole drug (either itraconazole or fluconazole). Our in vitro data are in agreement with these guidelines for amphotericin B and itraconazole but not for fluconazole when testing the mold phase. In conclusion, amphotericin B appears highly active against the mold phases of all tested strains within Ajellomycetaceae followed by posaconazole, itraconazole, voriconazole, and isavuconazole. The results of this study warrant further investigations of micafungin as a therapeutic agent for infections caused by dimorphic fungi.