Antifungal Susceptibilities of Rare Yeast Isolates

Abstract

The recent increase in the number of rare yeasts isolated from clinical specimens is a cause for concern, requiring accurate identification of these yeasts and assessment of their antifungal susceptibility to guide treatment. In this regard, we identified 196 rare yeasts isolated from various clinical specimens, mostly urine and respiratory tract specimens of patients hospitalized in intensive care unit and wards, using MALDI-TOF MS, and assessed their susceptibility to amphotericin B, fluconazole, voriconazole, posaconazole, itraconazole, isavuconazole and anidulafungin using the EUCAST broth microdilution method. Among the rare yeast species we isolated, Candida lusitaniae (13.8%) was the most common, followed by Magnusiomyces capitatus (13.3%), Candida fabianii (12.2%), and Trichosporon asahii (11.7%). Antifungal susceptibility testing revealed high echinocandin MIC values against Magnusiomyces spp., Trichosporon spp., and Rhodotorula mucilaginosa isolates. Similarly, we found high MIC values for fluconazole against the isolates of Magnusiomyces spp., T. asahii, R. mucilaginosa, and several Candida spp., including Candida guilliermondii, Candida pararugosa, Candida rugosa, Candida pelliculosa, Candida norvegensis, and Candida fabianii. We found similar MIC values across phylogenetically closely related species. In conclusion, prompt identification of rare yeasts and assessment of their antifungal susceptibilities are essential for effective treatment of the infections they cause.

1. Introduction

Rare yeasts that cause superficial infections in hosts with normal immune systems but can cause invasive fungal infections associated with high mortality and morbidity rates in immunosuppressed hosts or the presence of underlying risk factors are being isolated more commonly [1,2].

All phenotypic identification systems, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and molecular approaches can contribute to the identification of rare yeasts [2]. However, traditional fungal identification methods based on examination of morphological and phenotypic characteristics are complicated by the diversity of organisms that can cause infections [3,4]. In MALDI-TOF MS systems, which are widely used in microbiology laboratories today, the difficulty of updating the nomenclature in the database prevents clinical microbiology laboratories from successfully adapting to new species names [4].

The widespread use of molecular approaches in fungal identification in recent years has led to radical changes in fungal nomenclature and taxonomy. Long-recognized species have been transposed to new genera based on genotypic comparisons, or new genera and species have been created that include new organisms identified by detailed phylogenetic analyses [3,4]. Recent changes to the Fungal Nomenclature Code have also led to dramatic changes in the nomenclature of medically important molds and yeasts [5]. The binomial nomenclature system, which stipulated giving separate names to anamorphic (asexual) and teleomorphic (sexual) forms of fungi, was abandoned as of 1 January 2013 [5,6,7].

To reduce the instability caused by unnecessary and temporary nomenclature in this process, working groups and committees established under the auspices of the International Commission on the Taxonomy of Fungi (ICTF) and the Nomenclature Committee for Fungi (NCF) have published lists of protected and rejected names of key species/genera for which definitive changes have been approved [3]. Information on the classical names most commonly used in clinical laboratories and the recommended names to be reported for clinical use can be accessed from the Atlas of Clinical Fungi, Index Fungorum, and MycoBank [8,9,10].

As with their nomenclature, antifungal susceptibility values for rare yeasts are also not well established. In fact, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) has not reported clinical breakpoints for antifungal drugs against rare yeasts due to insufficient evidence, except for commonly isolated Candida species and Cryptococcus neoformans [11]. On the other hand, based on the study by Astvad et al., EUCAST developed a classification for the criteria for assessing antifungal drugs to be used against different fungal species based on two key assumptions [12]. The first assumption is that genetically related isolates would have similar pathogenicities and intrinsic susceptibility patterns. The second assumption is that rare yeasts, even those not phylogenetically related to other species, will likely respond to treatment, provided their minimum inhibitory concentration (MIC) values are similar to those observed in wild-type isolates of other, more commonly isolated species. Current treatment recommendations based on in vivo efficacy data and clinical experience were also taken into account in the EUCAST classification [11,13]. Accordingly, provisional MIC values were determined to guide amphotericin B, anidulafungin, fluconazole, and voriconazole treatments against thirty different yeasts until official clinical breakpoints are set by EUCAST [12].

In view of the foregoing, this study was carried out to identify rare yeasts isolated from various clinical specimens and assess their antifungal susceptibilities to amphotericin B, fluconazole, voriconazole, posaconazole, itraconazole, isavuconazole, and anidulafungin.

2. Materials and Methods

2.1. Fungal Isolates

The study material consisted of 196 rare yeast collection isolates sent to the ISLAB-2 Central Mycology Laboratory for identification and antifungal susceptibility testing from hospitals affiliated with the 2nd Service Region of the Istanbul Provincial Health Directorate between 1 June 2023 and 30 October 2024, and identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (VITEK MS and VITEK MS PRIME, bioMérieux, Marcy l’Etoile, France). Identification of the isolates was supported by their macroscopic and microscopic appearances on Sabouroud Dextrose agar [(SDA), (RTA, İstanbul, Turkey)] and Candida chromogenic agar (RTA, İstanbul, Turkey). The isolates were stored at −80 °C until testing, revived by passage twice in SDA prior to testing, and incubated at 36 ± 1 °C for 18–24 h.

2.2. Antifungal Susceptibility Testing

Antifungal susceptibility tests were carried out using the broth microdilution method per EUCAST E.Def 7.4 guidelines [14]. The antifungal agents tested were obtained from Sigma–Aldrich Chemical Co., St. Louis, MO, USA. The MIC values of amphotericin B, fluconazole, posaconazole, itraconazole, voriconazole, isavuconazole, and anidulafungin against the isolates were determined. Growth was assessed visually after 24-h incubation at 37 °C and spectrophotometrically at 450 nm using a Multiskan Go (ThermoFisherScientific, Waltham, MA, USA) microplate reader. The MIC value was defined as the lowest concentration that caused a 90% reduction in growth for amphotericin B and a 50% reduction for other drugs compared to growth not involving an antifungal agent [15].

2.3. Statistical Analysis

SPSS 30.0 (Statistical Product and Service Solutions for Windows, Version 30.0, IBM Corp., Armonk, NY, USA, 2024) software package was used in the statistical analyses of the collected data. The results of the statistical analyses were expressed using descriptive statistics, i.e., mean ± standard deviation values and median with minimum and maximum values in the case of continuous variables, and frequencies (n) and percentage (%) values in the case of categorical variables. In comparing the differences in variables between the groups, the Mann–Whitney U test was used for numerical variables, and the chi-squared test was used for categorical variables. Probability (p) statistics of <0.05 were deemed to indicate statistical significance.

3. Results

We identified 196 rare yeasts and studied their antifungal drug susceptibility during the study period. Compared to other common Candida species, i.e., Candida albicans complex (1685), Candida glabrata complex (Nakaseomyces glabratus) (653), Candida tropicalis (441), Candida auris (Candidozyma auris) (417), Candida parapsilosis (307), Candida kefyr (Kluvyeromyces marxianus) (243), and Candida krusei (Issatchenkia orientalis) (76), rare yeasts accounted for 4.9% of all growth samples. The names and isolation rates of the 196 rare yeasts that make up the study material are shown in

Table 1. Rare yeasts isolated in the study.

Classical Name Most Commonly Used in Clinical Laboratories	Recommended Name to Be Reported for Clinical Use	n (%)
Candida dubliniensis	Candida dubliniensis a,b,c	18 (9.2)
Candida fermentati	Candida fermentati a,b,c	1 (0.5)
Candida guilliermondii	Meyerozyma guilliermondii a,b,c	5 (2.6)
Candida haemulonis	Candida haemuli a,b or Candidozyma haemuli c	1 (0.5)
Candida inconspicua	Candida inconspicua a or Pichia inconspicua b,c	21 (10.7)
Candida lambica	Pichia fermentans a,b,c	1 (0.5)
Candida lusitaniae	Clavispora lusitaniae a,b,c	27 (13.8)
Candida lypolitica	Yarrowia lypolitica a	2 (1)
Candida metapsilosis	Candida metapsilosis a,b,c	3 (1.5)
Candida norvegensis	Pichia norvegensis a,b,c	2 (1)
Candida orthopsilosis	Candida orthopsilosis a,b,c	8 (4.1)
Candida palmioleophila	Candida palmioleophila b,c	1 (0.5)
Candida pararugosa	Wickerhamiella pararugosa b,c	3 (1.5)
Candida pelliculosa	Hansenula anomala a or Wickerhamomyces anomalus b,c	2 (1)
Candida rugosa	Diutina rugosa a,b,c	3 (1.5)
Candida fabianii	Cyberlindnera fabianii a,b,c	24 (12.2)
Cryptococcus neoformans	Cryptococcus neoformans a,b,c	1 (0.5)
Candida guilliermondii var. membranifaciens	Kodemea ohmeri b,c	1 (0.5)
Lodderomyces elongisporus	Lodderomyces elongisporus a,b,c	1 (0.5)
Rhodotorula mucilaginosa	Rhodotorula mucilaginosa a,b,c	6 (3.1)
Saccharomyces cerevisiae	Saccharomyces cerevisiae a,b,c	4 (2)
Saprochaete capitata	Saprochaete capitata b or Magnusiomyces capitatus c	26 (13.3)
Saprochaete clavata	Magnusiomyces clavatus b,c	9 (4.6)
Trichosporon asahii	Trichosporon asahii a,b,c	23 (11.7)
Trichosporon domesticum	Apiotrichum domesticum b,c	2 (1)
Zygosaccharomyces rouxii	Zygosaccharomyces rouxii b,c	1 (0.5)

^a Atlas of Clinical Fungi; ^b Index Fungorum; ^c MycoBank.

. The appearance of some yeasts isolated in the study on SDA is shown in Figure 1, and their appearance on Candida chromogenic agar is shown in Figure 2.

We isolated the rare yeasts mostly from the urine and respiratory tract specimens of patients hospitalized in the intensive care unit (ICU) and wards. No significant difference was found between the urine samples of patients hospitalized in the ICU and wards in terms of the isolation rates of rare yeasts compared to other common Candida species (p = 0.403). On the other hand, a significant difference was found between the respiratory tract samples of patients hospitalized in the ICU and wards in terms of the isolation rates of rare yeasts compared to other common Candida species (p < 0.001). The isolation rates of other Candida species were significantly higher than those of rare yeasts in the blood samples of patients hospitalized in the ICU and wards (p = 0.004). Among the rare yeast species we isolated, Candida lusitaniae (Clavispora lusitaniae) (13.8%) was the most common, followed by Saprochaete capitata (Magnusiomyces capitatus) (13.3%), Candida fabianii (Cyberlindnera fabianii). (12.2%), and Trichosporon asahii (11.7%). Demographic characteristics of patients from whom rare yeasts were isolated and isolation rates by sample type are shown in

Table 2. Demographic Distribution of Rare Yeasts.

Parameters	Rare Yeasts (n = 196)
Gender
Male	109 (55.6)
Woman	87 (44.4)
Age
mean ± SS	63.1 ± 22.9
Median (Min–Max)	70.5 (0–95)
Department
Other	15 (7.7)
Service	96 (49)
ICU	85 (43.4)
Sample
BAL	14 (7.1)
Sputum	26 (13.3)
Skin scraping	2 (1)
Tissue	3 (1.5)
Urine	94 (48)
Blood	15 (7.7)
Catheter	7 (3.6)
Peritoneal fluid	1 (0.5)
Nail	10 (5.1)
Tracheal aspirate	20 (10.2)
Vaginal swab	3 (1.5)
Wound	1 (0.5)

ICU: Intensive Care Unit; BAL: Bronchoalveolar Lavage.

.

EUCAST has not provided clinical breakpoints for antifungal drugs against rare yeasts. Therefore, it is recommended that evaluations of clinical breakpoints for antifungal medicines against rare yeasts should be made according to clinical breakpoints specified for either species to which rare yeasts are phylogenetically similar or wild isolates of other common species. We found high MIC values against Magnusiomyces spp., Trichosporon spp., and Rhodotorula mucilaginosa, which reportedly have intrinsic echinocandin resistance. Similarly, we found high MIC values for fluconazole against the isolates of M. capitatus, Saprochaete clavata (Magnusiomyces clavatus), T. asahii, and R. mucilaginosa, as well as Candida guilliermondii (Meyerozyma guilliermondii), Candida pararugosa (Diutina pararugosa), Candida rugosa (Diutina rugosa), Candida pelliculosa (Wickerhamomyces anomalus), Candida norvegensis (Pichia norvegensis), and C. fabianii, which were formerly included in Candida species. Antifungal susceptibilities of yeasts isolated from more than one sample are shown in

Table 3. Distribution of Antifungal Susceptibilities of Rare Yeasts Growing in Multiple Samples.

Yeast (n)	AB		ANI		POSA		VOR		FLZ		ISV		ITRA
	MIC Range	MIC 90	MIC Range	MIC 90	MIC Range	MIC 90	MIC Range	MIC 90	MIC Range	MIC 90	MIC Range	MIC 90	MIC Range	MIC 90
Candida lusitaniae (Clavispora lusitaniae) (27)	1–2	2	0.06–0.25	0.25	0.06–0.25	0.125	0.015–0.125	0.03	0.125–8	2	0.008–0.5	0.03	0.03–0.25	0.125
Saprochaete capitata (Magnusiomyces capitatus) (26)	1–2	2	2–>4	>4	0.5–2	1	0.5–2	2	2 –32	16	0.25–1	1	0.06–2	1
Candida fabianii (Cyberlindnera fabianii) (24)	1–2	2	0.015–0.25	0.125	0.5–1	0.5	0.125–1	0.25	4–32	16	0.06–0.25	0.125	0.125–1	0.25
Trichosporon asahii (23)	2–4	4	>4	>4	0.25–2	1	0.125–1	0.5	1– 32	8	0.125–0.5	0.5	0.5–1	1
Candida inconspicua (21)	1–4	2	0.015–0.125	0.03	0.25–0.5	0.5	0.25–1	0.5	16–>32	>32	0.015–0.5	0.25	0.06–0.5	0.25
Candida dubliniensis (18)	0.5–1	1	0.008–0.03	0.03	0.03–0.25	0.125	0.015–0.06	0.03	0.125–1	0.5	0.008–0.03	0.015	0.03–0.25	0.125
Saprochaete clavata (Magnusiomyces clavatus) (9)	1–4	2	2–>4	>4	0.5–2	2	0.5–1	1	8–16	16	0.5–1	0.5	0.25–1	1
Candida orthopsilosis (8)	1–2	1	0.5–2	2	0.06–0.25	0.25	0.125–0.25	0.125	1– 2	2	0.03–0.125	0.125	0.06–0.12	0.125
Rhodotorula mucilaginosa (6)	1–2	2	0.125–>4	>4	0.5–2	2	1– 8	8	2– 16	16	0.06–1	1	0.5–4	4
Candida guilliermondii (Meyerozyma guilliermondii) (5)	1	1	1–2	1	0.5–2	1	1– 4	2	8–32	16	0.5–>4	4	0.06–>16	16
Saccharomyces cerevisiae (4)	1	1	0.06–0.125	0.125	1	1	0.5–1	1	8–16	16	0.125–0.25	0.25	0.5–2	2
Candida metapsilosis (3)	1–2	1	0.25–0.5	0.5	0.125–0.25	0.125	0.06–0.125	0.125	0.5–2	2	0.03–0.125	0.03	0.06–0.125	0.06
Candida pararugosa (Diutina pararugosa) (3)	1	1	0.125–0.25	0.25	0.25–0.5	0.5	0.25–2	2	2– 8	8	0.125	0.125	0.25–0.5	0.5
Candida rugosa (Diutina rugosa) (3)	1–2	2	0.12–0.5	0.5	0.06–0.25	0.25	0.25–2	2	8	8	0.125	0.125	0.25–1	1
Trichosporon domesticum (2)	1–2	2	>4	>4	0.25–0.5	0.5	0.125–0.25	0.25	2	2	0.125	0.125	0.25–0.5	0.5
Candida pelliculosa (Wickerhamomyces anomalus) (2)	0.25–1	1	0.015	0.015	0.5–1	1	0.5	0.5	2–4	4	0.125–0.25	0.25	0.125–0.25	0.25
Candida norvegensis (Pichia norvegensis) (2)	2	2	0.12–4	4	0.25	0.25	0.06–4	4	8	8	0.25	0.25	0.25	0.25
Candida lypolitica (Yarrowia lypolitica) (2)	1	1	>4	>4	1–2	2	0.25	0.25	4–8	8	0.125–0.25	0.25	0.5	0.5

AB: Amfoterisin B; ANI: Anidulafungin; POSA: Posaconazole; VOR: Voriconazole; FLZ: Fluconazole; ISV: Isavuconazole; ITRA: Itraconazole; MIC: Minimal inhibitory concentration.

, whereas antifungal susceptibilities of rare yeasts grown in only one sample are shown in

Table 4. Distribution of Antifungal Susceptibilities of Rare Yeasts Growing in a Single Sample.

Yeast	AB MIC	ANI MIC	POSA MIC	VOR MIC	FLZ MIC	ISV MIC	ITRA MIC
Candida fermentati	1	1	1	1	4	1	1
Candida haemulonis (Candida haemuli)	4	0.25	1	0.25	32	0.125	16
Candida lambica (Pichia fermentans)	0.25	0.015	0.5	0.5	4	0.25	0.25
Candida palmophilia	1	0.06	0.5	4	>32	1	1
Cryptococcus neoformans	2	>4	0.5	0.125	2	0.03	0.5
Kodemea ohmeri	0.25	4	0.25	0.125	8	0.125	0.25
Lodderomyces elongisporus	0.5	0.03	0.5	0.125	0.5	0.03	0.06
Zygosaccharomyces rouxii	1	0.015	0.03	0.03	0.125	0.06	0.25

AB: Amfoterisin B; ANI: Anidulafungin; POSA: Posaconazole; VOR: Voriconazole; FLZ: Fluconazole; ISV: Isavuconazole; ITRA: Itraconazole; MIC: Minimal inhibitory concentration.

.

4. Discussion

The accurate identification of rare yeasts and determination of their antifungal susceptibilities are crucial for guiding treatment and vaccination. Some antifungals have limited spectra of activity in rare yeast species due to intrinsic resistance. As with fluconazole, the antifungal efficacies of other antifungal drugs also vary [16]. Among the 196 rare yeast species we isolated, C. lusitaniae (13.8%) was the most common, followed by M. capitatus (13.3%), C. fabianii (12.2%), and T. asahii (11.7%). We found high MIC values of anidulafungin against isolates of Magnusiomyces spp., Trichosporon spp., and R. mucilaginosa, and of fluconazole against isolates of M. capitatus, M. clavatus, T. asahii, and R. mucilaginosa, as well as isolates of other species such as M. guilliermondii, D. pararugosa, D. rugosa, W. anomalus, P. norvegensis, and C. fabianii.

EUCAST has not reported clinical breakpoints for antifungal drugs against rare yeasts due to insufficient evidence. Then again, it has been recommended that antifungal drugs used in the treatment of isolates to which rare yeasts are genetically related be also used in the treatment of rare yeast infections, based on the assumption that their pathogenicity and intrinsic susceptibility patterns would be similar, as well as antifungal drugs that exhibit MIC values against wild-type isolates of other frequently isolated species that are not phylogenetically related to rare yeasts similar to those against isolates genetically related to rare yeasts [12,16].

In a study on rare yeasts isolated from fungemia cases, Tepe et al. [17] found that Trichosporon spp., Saprochaete spp., Cryptococcus spp., and Rhodotorula spp. had intrinsic resistance to echinocandin. We also isolated yeasts with intrinsic echinocandin resistance, such as Trichosporon spp. and Saprochaete spp., although rare (7.7%), among the rare yeasts we isolated from fungemia cases. Therefore, in patients with predisposing risk factors for the development of fungal infections, rare yeasts as well as Candida species should be kept in mind when deciding on empirical treatment. In a study on antifungal susceptibilities of rare yeasts according to EUCAST standards, Stavru et al. categorized the MIC values of antifungal drugs against rare yeasts for which there are no epidemiological breakpoints as susceptible/reduced susceptible in comparison with the epidemiological breakpoints of C. albicans. Accordingly, they reported that species of Meyerozyma, Diutina/Kodamaea, and Pichia clades showed reduced susceptibility to azoles, and Candida haemulonii, Candida pseudohaemulonii, and Candida duobushaemulonii species, which are closely related to C. auris, also showed reduced susceptibility to some antifungal drugs, mainly azoles [16]. In parallel, high MIC values were reported for azoles against Candida inconspicua, C. pararugosa, and P. norvegensis isolates [18]. We also found high MIC values of fluconazole against M. guilliermondii, P. norvegensis, D. pararugosa, D. rugosa, Candida palmophilia, C. haemuli, M. capitatus, T. asahii, and R. mucilaginosa.

Echinocandins are recommended as first-line treatment for invasive candidiasis [19]. In a study investigating echinocandin resistance in yeasts using the broth microdilution method based on a MIC threshold value of ≥0.5 mg/L, Ollivier et al. reported that echinocandins had low MIC values against isolates of C. lusitaniae, Candida dubliniensis, M. guilliermondii, P. inconspicua, Saccharomyces cerevisiae, C. haemuli, and W. anomalus, and high MIC values against isolates of M.capitatus, M. clavatus, T. asahii, R. mucilaginosa, and Yarrowia lypolitica. We found MIC values generally similar to those reported in the above studies, except against isolates of some specific genera and species, which acquired resistance due to previous exposure to antifungal drugs [19].

In a study investigating the antifungal susceptibilities of rare yeasts isolated from various clinical samples, it was reported that amphotericin B, fluconazole and itraconazole showed high MIC values against C. haemuli isolates, fluconazole and caspofungin against M. capitatus isolates, fluconazole against K. ohmeri isolates, and fluconazole, posaconazole and caspofungin against R. mucilaginosa isolates [20]. We also found similar MIC values against these yeasts.

Onychomycosis is another fungal infection in which rare yeasts have been isolated as causative agents. A study reported Candida orthopsilosis (n = 9) as the most commonly isolated rare yeasts causing onychomycosis, followed by C. lusitaniae (n = 2), W. pararugosa (n = 2), Naganishia diffluens (n = 2), W. anomalus (n = 2), C.fabianii (n = 1), and Meyerozyma caribbica (n = 1) [21]. In the same study, fluconazole was found to have high MIC values against most of these species. Of the rare yeast isolates we identified, 5.1% were causative agents of onychomycosis. Of these isolates, R. mucilaginosa and T. asahii were isolated in three cases each, C. fermentati and M. guilliermondii in two cases each, and C. orthopsilosis and Zygosaccharomyces rouxii in one case each. Fluconazole exhibited high MIC values against all these species.

This study has some limitations. First, the epidemiology of rare yeasts varies across regions, countries, and even institutions. Although the participating centers had a large patient population and diverse populations, this study only presents data on strains identified in the ISLAB-2 service area. Therefore, our findings may not reflect species distribution or resistance patterns in other regions with different climates or patient demographics. Second is the lack of clinical data. The study included only in vitro antifungal susceptibility results. Clinical characteristics such as patient risk factors and thirty-day mortality rates could not be examined in detail due to limited access to clinical data. This situation caused insufficient confirmation of the clinical validity of the obtained MIC values. Another limitation is that molecular methods cannot be used in the identification of rare yeasts or in the search for antifungal resistance genes. In our study, yeasts isolated from axenic cultures, which were thought to have preserved their morphological, physiological and genetic characteristics, were identified by MALDI-TOF MS and supported by conventional methods. It has been concluded that identification should be supported by additional conventional methods to avoid possible misidentifications, especially in the identification of phylogenetically similar species. Finally, in this study, susceptibility results for seven traditional antifungal drugs were investigated. This presents a limited antifungal panel. In particular, in rare yeasts where antifungal resistance is observed, the inclusion of new antifungal drugs with different mechanisms of action, such as ibrexafungerp, rezafungin, fosmanogepix or oteseconazole, will allow the expansion of treatment alternatives and provide information for resistance monitoring programs.

5. Conclusions

EUCAST guidelines currently do not provide established clinical breakpoints for antifungal drugs against rare yeasts, yet recommend the use of antifungal drugs that exhibit MIC values against these rare yeasts similar to those against more common and possibly more pathogenic Candida species in the treatment of invasive infections in which rare yeasts have been isolated as causative agents. In this context, the identification and antifungal susceptibility results we obtained may contribute to epidemiological studies and guidelines on antifungal management of rare yeasts.