ABSTRACT
Trichosporon species are relevant etiologic agents of hospital-acquired infections. High mortality rates are attributed to Trichosporon deep-seated infections in immunocompromised individuals, making fast and accurate species identification relevant for hastening the discovery of best-targeted therapy. Recently, Trichosporon taxonomy has been reassessed, and three genera have been proposed for the pathogenic species: Trichosporon, Cutaneotrichosporon, and Apiotrichum. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has replaced old phenotypic methods for microorganism identification in clinical laboratories, but spectral profile databases have to be evaluated and improved for optimal species identification performance. Vitek MS (bioMérieux) is one of the commercially available MALDI-TOF MS platforms for pathogen identification, and its spectral profile databases remain poorly evaluated for Trichosporon, Cutaneotrichosporon, and Apiotrichum species identification. We herein evaluated and improved Vitek MS for the identification of the main clinical relevant species of Trichosporon, Cutaneotrichosporon, and Apiotrichum using a large set of strains and isolates belonging to different yeast collections in Brazil and France.
INTRODUCTION
Opportunistic non-Candida yeasts, including Trichosporon spp., are emerging pathogens of deep-seated infections in the context of immunodepression and/or invasive procedures (1, 2). In addition, outbreaks of catheter-related fungemia by these pathogens have been described in neonatal intensive care units (3).
Based on molecular phylogenetic analysis, Trichosporon pathogenic species were initially subdivided into three clades: clade Porosum, which included the species Trichosporon asahii, Trichosporon inkin, Trichosporon coremiiforme, Trichosporon asteroides, Trichosporon faecale, Trichosporon ovoides, Trichosporon japonicum, and Trichosporon lactis; clade Cutaneum, which included the species Trichosporon mucoides, Trichosporon dermatis, Trichosporon debeurmannianum, Trichosporon jirovecii, and Trichosporon cutaneum; and clade Gracile/Brassicae, which included the species Trichosporon mycotoxinivorans, Trichosporon montevideense, Trichosporon domesticum, and Trichosporon loubieri (4). Among these pathogenic species, T. asahii, T. asteroides, T. faecale, T. inkin, T. coremiiforme, T. dermatis, and T. mycotoxinivorans are the main species associated with deep-seated infections (2, 4). Recently, based on multiple-gene sequence analysis, Trichosporon taxonomy has been reassessed, and new genera have been proposed for the monophyletic clades, which include Trichosporon, Cutaneotrichosporon, and Apiotrichum for pathogenic species (5). These genera are intrinsically resistant to echinocandins, and resistance to other antifungal classes has been described (2, 4). Moreover, a distinct interspecies antifungal susceptibility profile, virulence, and pathogenicity have been suggested (2, 6). Thus, correct identification of these opportunistic pathogens not only at the genus level, but also at the species level, is recommended for optimal clinical management and/or for epidemiological purposes.
Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) has emerged as a useful technique for rapid and precise pathogen identification in clinical laboratories (7). Recent studies analyzing the performance of MALDI-TOF MS for the identification of more than 1,000 yeast isolates (>95% Candida) describe successful species identification ranging from 95 to 98% compared to DNA-based gold-standard techniques (8, 9). However, the performance of MALDI-TOF MS for the identification of non-Candida species is suboptimal, and well-constructed in-house spectral profiles databases are required to achieve better performance (8, 10). Likewise, studies that evaluated MALDI-TOF MS for the identification of clinically relevant species of Trichosporon, Cutaneotrichosporon, and Apiotrichum using the Bruker instrument showed that it required an upgrade of the spectral profile library (e.g., Biotyper) to achieve optimal species identification performance (10, 11).
In this work, we evaluated the Vitek MS instrument (bioMérieux, Marcy-L'Etoile, France) and its associated databases for the identification of Trichosporon, Cutaneotrichosporon, and Apiotrichum clinically relevant species, and we also constructed and validated an in-house spectral profile database for better identification of these pathogens.
RESULTS
The in vitro diagnostic (IVD) database reported correct species identification (ID) for 73.6%, 5%, and 0% of Trichosporon, Cutaneotrichosporon, and Apiotrichum organisms, respectively. All organisms belonging to the species T. faecale, T. coremiiforme, T. japonicum, T. lactis, Cutaneotrichosporon jirovecii, Cutaneotrichosporon debeurmannianum, Apiotrichum montevideense, Apiotrichum mycotoxinivorans, Apiotrichum domesticum, and Apiotrichum loubieri lacked identification. All 12 Cutaneotrichosporon dermatis organisms were consistently misidentified as Cutaneotrichosporon mucoides. One isolate of C. jirovecii and one isolate of A. montevideense were misidentified as Candida valida (confidence level of 50%) and Candida albicans (confidence level of 95.4%), respectively, but all results of the subsequent repeat analyses came out as “no identification.” Two T. inkin isolates were initially misidentified as T. ovoides (confidence levels of 50 and 99.8%), but correct species assignment was achieved after repeat analyses.
The research use only (RUO) database reported correct species ID for 68.4%, 5%, and 0% of Trichosporon, Cutaneotrichosporon, and Apiotrichum organisms, respectively. All organisms belonging to the species T. ovoides, T. faecale, T. japonicum, C. jirovecii, C. debeurmannianum, A. montevideense, A. mycotoxinivorans, and A. domesticum lacked identification. Among three organisms of T. coremiiforme, two had correct genus identification, despite being misclassified as T. asahii with a confidence level of 80 to 90%, even after repeated analysis. C. dermatis organisms were consistently misclassified as C. cutaneum/C. mucoides or had identification restricted to the genus level.
The SuperSpectra “Trichosporon_asahii_1_” and “Trichosporon_cutaneum/mucoides_1_” from the original RUO database were inactivated, since they were related to misidentifications of T. coremiiforme and C. dermatis, respectively. The upgraded RUO database improved the identification of species already represented in the original databases, such as T. inkin and T. asteroides. In addition, mass spectra of Trichosporon faecale, T. coremiiforme, T. ovoides, T. lactis, T. japonicum, C. dermatis, C. jirovecii, C. debeurmannianum, A. mycotoxinivorans, A. montevideense, A. loubieri, and A. domesticum had correct species assignment (without misidentifications) by our in-house SuperSpectra library.
The performances of the IVD and the original and upgraded RUO databases for Trichosporon, Cutaneotrichosporon, and Apiotrichum species identification are summarized in Table 1. Details of the misidentifications observed for IVD and RUO databases are provided in Table 2.
Performance of Vitek MS according to the different databases for the identification of Trichosporon, Cutaneotrichosporon, and Apiotrichum clinically relevant species
Misidentifications produced by the IVD and RUO databases
DISCUSSION
Despite the good performance for the identification of some relevant species, such as T. asahii, T. asteroides, and T. inkin, we showed that the current IVD database from the Vitek MS needs improvement for identification of other relevant species of the genus Trichosporon, and also for the species of the genera Cutaneotrichosporon and Apiotrichum. Furthermore, despite being a rare event, we found genus misidentifications when Cutaneotrichosporon and Apiotrichum isolates were analyzed. Thus, while improvement of the current IVD database of Vitek MS by the manufacturer is pending, we advise Vitek MS users not to abandon traditional phenotypic methods (e.g., macromorphology, micromorphology, and hydrolysis of urea) to report an “unidentified basidiomycetous yeast,” a surrogate marker of echinocandin resistance, while final identification is carried out by a reference laboratory through sequence analysis of the intergenic spacer 1 (IGS1) or D1/D2 domain of the 26S region of the ribosomal DNA.
The RUO database (SARAMIS) has proven to be an auxiliary tool when the IVD database fails to provide correct Candida species identification (12). On the contrary, in the case of Trichosporon, Cutaneotrichosporon, and Apiotrichum species, the RUO database appeared ineffective for a most sensitive identification of those species. Only the addition of in-house SuperSpectra and exclusion of the manufacturer's SuperSpectra, which was related to misidentifications of T. coremiiforme and C. dermatis isolates, were necessary to optimize its performance. Indeed, the identification of C. dermatis has gained relevance, since this species was recently related to panazole resistance (13). However, like conventional phenotypic methods, such as Vitek2, Vitek MS, and its IVD and RUO databases misidentified all C. dermatis isolates as C. mucoides (14). Despite being genetically closely related species, we were able to construct species-specific SuperSpectra of both C. dermatis and C. mucoides, achieving 100% correct species ID for both species in the validation step of our in-house database. The SARAMIS Premium SuperSpectra tool (bioMérieux) has shown to be a useful tool to create species-specific spectral profiles, since it compares the biomarkers with the whole SARAMIS spectral archive during the SuperSpectra construction process (15).
A. mycotoxinivorans (formerly Trichosporon mycotoxinivorans) is now considered a relevant pathogen for patients with cystic fibrosis (16). Moreover, this microorganism has been related to deep-seated infections in immunocompromised patients with invasive disposals in India (17). The use of Bruker's MALDI-TOF MS as a reliable tool for A. mycotoxinivorans species identification has helped strengthen the epidemiologic link of T. mycotoxinivorans to cystic fibrosis (18, 19). Thus, it appears that inclusion of the species in the database used for clinical diagnosis is now mandatory.
In conclusion, Vitek MS databases show good performance for the identification of common relevant species of Trichosporon, such as T. asahii, T. inkin, and T. asteroides. However, Vitek MS consistently misidentifies C. dermatis as C. mucoides, and other Trichosporon, Cutaneotrichosporon, and Apiotrichum clinical relevant species remain neglected by the current IVD and RUO databases. The in-house database built with well-identified organisms of clinically relevant Trichosporon, Cutaneotrichosporon, and Apiotrichum species outperformed the current IVD and RUO spectral profile databases. SARAMIS Premium allowed the construction of species-specific SuperSpectra that can differentiate closely related species of Trichosporon, Cutaneotrichosporon, and Apiotrichum.
MATERIALS AND METHODS
Fungal organisms.A total of 15 CBS-KNAW reference strains and 93 nonreplicate clinical isolates (blood, urine, stool, skin, and respiratory tract) from collections in Brazil (University of São Paulo, Federal University of São Paulo) and France (Hôpital Saint-Antoine, Paris) were analyzed.
Trichosporon spp., Cutaneotrichosporon spp., and Apiotrichum spp. were represented by 76, 20, and 12 organisms, respectively. For all these organisms, species identification was carried out by sequence analysis of the IGS1 or D1/D2 domain of the 26S region of the ribosomal DNA, with primers and amplification parameters that were previously described (20, 21).
Sample preparation for MALDI-TOF MS analysis.Strains and isolates maintained as frozen stocks at −80°C in yeast extract-peptone-dextrose medium were subcultured on Sabouraud's dextrose agar (SDA) plates and incubated for 48 h at 30°C before MALDI-TOF MS analysis. Due to the dry and rough morphology of some Trichosporon isolates, homogenous smearing of colonies on the target plate was troublesome, and a standard protein extraction protocol with ethanol and formic acid was used for all analyses. In brief, one loop of yeast biomass was transferred into a 1.5-ml tube (Eppendorf) containing 300 μl of purified water and mixed thoroughly. Subsequently, 900 μl of absolute ethanol was added to each tube and mixed for 1 min. The samples were centrifuged for 2 min at 13,000 rpm, and the supernatant was removed. The pellet was dried at room temperature, and 50 μl of formic acid (70%) was added. In addition, an equivalent volume of acetonitrile was added, and the mixture was centrifuged for 2 min at 13,000 rpm. Finally, 1 μl of the clear supernatant was spotted in quadruplicate onto a disposable MALDI target slide composed of a polypropylene carrier with a stainless steel layer (bioMérieux). After air-drying, each spot was overlaid with 1 μl of α-cyano-4-hydroxycinnamic acid (HCCA) matrix (bioMérieux).
MALDI-TOF MS analysis by bioMérieux IVD and RUO databases.Measurements were performed on a Vitek MS instrument (bioMérieux) equipped with both in vitro diagnostic (IVD) and research use only (RUO; SARAMIS) databases (bioMérieux). For the IVD analysis, spectra were obtained using the Vitek MS automation control and Myla software (bioMérieux), using the manufacturer's suggested settings. For each acquisition group, a standard (Escherichia coli ATCC 8739) was included to calibrate the instrument and validate the run. The spectra were analyzed by the Vitek MS version 3.2 IVD database (bioMérieux). The software compares the spectrum obtained to the expected spectrum of each organism or organism group (e.g., bacteria or fungi), and high-confidence-level identification was considered when a single species showed a probability of ID of ≥60%. For the RUO analysis, spectra were generated using the Launchpad version 2.8 software (bioMérieux) and compared to the SARAMIS version 4.13 database (bioMérieux). Peak matches that yield identification results with confidence values exceeding 75% are reported.
For both IVD and RUO results, we considered accurate identification if the correct species was mentioned despite the report of “Trichosporon” for the new genera Cutaneotrichosporon and Apiotrichum. Organisms that were initially not identified or misidentified were reanalyzed another two times to check the repeatability and reproducibility of these results.
Construction of an in-house SuperSpectra library for upgrading the RUO database.For SuperSpectra construction, mass spectra of 32 organisms, representing all the species investigated in this study, were imported into the SARAMIS Premium software package (bioMérieux) (Table 3). Then, 10 high-quality mass spectrum replicates of a given species from one or two organisms (≥120 masses, ≥70 similarity) were selected to create a species-specific SuperSpectrum with the SARAMIS Premium SuperSpectra tool (bioMérieux), according to the manufacturer's instructions. The specificity of the potential biomarker masses was determined by comparison against the whole SARAMIS spectral archive (bioMérieux), and a SuperSpectrum with 40 masses had to have at least 20 species-specific biomarkers. The RUO spectral profile database was upgraded with the addition of 22 in-house SuperSpectra, including three for the species T. asahii, T. inkin, and T. asteroides, two for the species C. dermatis, and one for the species T. faecale, T. ovoides, T. coremiiforme, T. lactis, C. mucoides, C. jirovecii, C. debeurmannianum, A. domesticum, A. montevideense, A. mycotoxinivorans, and A. loubieri (Table 3).
Organisms used to construct the in-house SuperSpectra library
Finally, the original SuperSpectra from the SARAMIS version 4.13 database (bioMérieux) related to misidentifications were inactivated, and the upgraded database with the in-house SuperSpectra was challenged for identification of all strains and isolates. Organisms that were initially not identified or misidentified were reanalyzed another two times to check the repeatability and reproducibility of these results.
ACKNOWLEDGMENTS
We thank Adriana L. Motta and Maria Isabel Cunha for excellent technical assistance.
The Trichosporon, Cutaneotrichosporon, and Apiotrichum SuperSpectra produced in this work are freely available by contacting the corresponding author.
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sector.
The work of A.L.C. is supported by a grant from National Council of Technological and Scientific Development (CNPQ 307510/2015-8).
FOOTNOTES
- Received 19 March 2017.
- Returned for modification 4 April 2017.
- Accepted 16 May 2017.
- Accepted manuscript posted online 24 May 2017.
- Copyright © 2017 American Society for Microbiology.
