In recent years, with the development of organ transplantation and the widespread use of immunosuppressants and antibiotics, the number of cases of invasive infections caused by filamentous fungi have increased (Brown et al., 2012). In addition to that of infection caused by the most common filamentous fungus, Aspergillus, the incidence of infections caused by non-aspergillus filamentous fungi such as Fusarium, Mucorales, Scedosporium, and other rare fungi is also increasing (Miceli and Lee, 2011). Scedosporium, Acremonium-like, Scopulariopsis, and Microascus spp. are saprobic fungi commonly found in the environment, some species have been reported as pathogens of humans, and most are opportunistic (Ramirez-Garcia et al., 2018; Perez-Cantero and Guarro, 2020a,b). Within the genus Scedosporium, the Scedosporium apiospermum species complex and Scedosporium aurantiacum are most related to human diseases; the former currently contains Scedosporium apiospermum, Scedosporium boydii, Scedosporium ellipsoideum, Scedosporium angustum, and Scedosporium fusarium (Ramirez-Garcia et al., 2018). Aremonium-like spp. comprise a high diversity of morphologically and genetically related fungi, among which Aremonium egyptiacum and Sarocladium kiliense are the most commonly involved in human diseases (Perez-Cantero and Guarro, 2020b). Scopulariopsis-like spp. include a group of hyaline and dematiaceous fungi, and most of the clinically relevant species belong to the genera Scopulariopsis and Microascus (Perez-Cantero and Guarro, 2020a). The above pathogenic fungi have raised concern in the field of medical mycology, as they show intrinsic resistance to multiple antifungal drugs (Ramirez-Garcia et al., 2018; Perez-Cantero and Guarro, 2020a,b). Additionally, differences in in vitro susceptibility have been reported among species within Scedosporium and Scopulariopsis-like spp. (Lackner et al., 2012; Yao et al., 2015). Therefore, rapid and accurate identification of these fungi is important for timely treatment, as well as for the supplementation of epidemiological data and drug susceptibility studies of a large range of species. However, due to the taxonomic complexity of these genera and the interspecies morphological similarities, morphological methods are often unable to identify these fungi at the species level (Perdomo et al., 2011; Ramirez-Garcia et al., 2018; Perez-Cantero and Guarro, 2020a). DNA sequencing allows accurate identification but is expensive, labor intensive, time consuming, and not suitable for routine laboratory testing.

Over the last few years, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been increasingly used for the laboratory identification of yeast and filamentous fungi because of its high accuracy, simple operation and low cost (Wilkendorf et al., 2020). The main obstacles to the application of this technique to the identification of filamentous fungi are the lack of a sufficient database and of a rapid and effective protein extraction method (Wilkendorf et al., 2020). Few studies have been conducted on the identification of Scedosporium (Del Chierico et al., 2012; Normand et al., 2013; Ranque et al., 2014; Sitterle et al., 2014; Sleiman et al., 2016; Zvezdanova et al., 2019), Acremonium-like (Becker et al., 2014; Rychert et al., 2018), Scopulariopsis and Microascus species (Lau et al., 2013; Becker et al., 2014; Schulthess et al., 2014; Levesque et al., 2015; Riat et al., 2015; Stein et al., 2018; Sacheli et al., 2020) by MALDI-TOF MS. In this study, we developed an in-house database containing 29 strains covering 21 species of Scedosporium, Acremonium-like, Scopulariopsis, and Microascus and challenged the system with 52 clinical isolates to evaluate the identification performance of the Bruker Filamentous Fungi Library v1.0 (FFL v1.0) (Bruker Daltonics, Bremen, Germany) alone and FFL v1.0 plus the in-house database.

The mass spectra of Scedosporium, Acremonium-like, Scopulariopsis, and Microascus species are shown in Figures 1–3, respectively. The results of MALDI-TOF MS identification of the 52 Scedosporium, Acremonium-like, Scopulariopsis, and Microascus isolates by FFL v1.0 alone and by the combined databases are shown in Table 2.

Using FFL v1.0 alone, 60% (12/20) of Scedosporium isolates were identified with scores ≥ 2.0, while all of them were identified as Scedosporium apiospermum [anamorph] Pseudallescheria boydii [teleomorph], which was the only Scedosporium species represented in FFL v1.0, so clear species identification was not available. Seven isolates (35%) were identified to the genus level and also identified as S. apiospermum [ana] P. boydii [teleo]. Using the combination of FFL v1.0 and the BMU database, the correct species-level identification rates increased significantly to 75% (15/20) (p < 0.001), and all 10 S. boydii isolates were correctly identified. The remaining five isolates were misidentified as species within the S. apiospermum species complex (Table 2).

Using FFL v1.0 alone, none of the Acremonium-like isolates were reliably identified. When supplemented with the in-house database, all isolates were correctly identified to the species level. Even though FFL v1.0 contains one strain of Acremonium strictum (now named Sarocladium strictum), there was no consistent identification of the quadruplicate tests when challenged with one isolate of S. strictum used for BMU database construction.

When challenged with Scopulariopsis and Microascus isolates, FFL v1.0, which contains seven Scopulariopsis brevicaulis, one Scopulariopsis acremonium and one Scopulariopsis brumptii MSPs, identified 62.5% (10/16) and 6.3% (1/16) of the isolates at the species and genus levels, respectively. All of the identified isolates were S. brevicaulis. With the combined databases, the species-level identification rate increased significantly to 100% (16/16) (p = 0.031).

The species- and genus-levels identification rates of Scedosporium, Acremonium-like, Scopulariopsis, and Microascus isolates using the BMU database alone were the same as that using the combined databases.

The CCI matrix of test species indicated that mass spectra within the S. apiospermum complex were highly similar, while those within Acremonium-like, Scopulariopsis, and Microascus spp. were more diverse (Figure 4).

The MSP dendrogram of the Scedosporium isolates cannot differentiate species in the S. apiospermum complex (Figure 5). As for the Acremonium-like isolates, the MSP dendrogram showed clear separation of different species, and the topology of the dendrogram appeared to be similar to that of the multilocus phylogenetic tree (Figure 6). The MSP dendrogram of Scopulariopsis and Microascus spp. clustered 27 isolates to the Scopulariopsis clade and Microascus clade (Figure 7). Isolates of M. gracilis and M. croci clustered in one subclade, whereas isolates of M. onychoides and M. intricatus clustered in another subclade, and the above two pairs of species were also close in the phylogenetic tree. The other isolates belonging to the same species clustered together into respective subclades. However, the topology of the MSP dendrogram and that of the multilocus phylogenetic tree of Microascus isolates differed.

The identification of filamentous fungi by MALDI-TOF MS relies mainly on an available database and an efficient protein extraction method. Liquid culture can improve the accuracy of MALDI-TOF MS identification and is usually used for research purpose. In clinical practice, liquid culture can be added when the identification of solid culture is unsatisfactory. The main obstacle of MALDI-TOF MS identification of Scedosporium, Acremonium-like, and Scopulariopsis-like species is a lack of reference spectral data in commercial databases. Our study indicated that MALDI-TOF MS is a powerful technique for rapid and accurate identification of the above species when using the Bruker database complimented with an in-house database and liquid culture.

Due to the non-updated nomenclature and a lack of reference species of Scedosporium in FFL v1.0, clear species identification is unavailable. With a supplementary database, the species identification rate of Scedosporium spp. ranged between 76 and 100% (Del Chierico et al., 2012; Normand et al., 2013; Ranque et al., 2014; Sleiman et al., 2016; Zvezdanova et al., 2019), and our study also found a correct identification rate of 75% using the combined databases. However, we found that the mass spectra within the S. apiospermum complex were highly similar, indicated by the high CCI values between species, so the strains in the complex could not be accurately differentiated, which was also reported by Bernhard et al. (2016). Zvezdanova et al. (2019) also found that even though 9 strains of S. apiospermum were included in an in-house database, three strains of S. apiospermum were misidentified at the species level (misidentified as S. boydii [n = 1] or Lomentospora prolificans [n = 2]). However, Sleiman et al. (2016) found that 17 strains of Scedosporium spp. and L. prolificans were correctly identified with a supplementary database, but the strains in the S. apiospermum complex included only five S. apiospermum and one S. boydii isolates. Sitterle et al. (2014) found with an in-house database containing 47 reference strains that the Andromas system identified 64 strains of Scedosporium spp. (including S. boydii, S. apiospermum, S. aurantiacum, S. minutispora, and S. dehoogii) and L. prolificans to the species level using the direct smearing method (without protein extraction). This indicated that the ability of different MALDI-TOF MS systems to identify Scedosporium spp. may be different, and a larger number of strains for database construction may improve the ability of species differentiation. Due to the lack of species, we were unable to clarify whether MALDI-TOF MS can distinguish the S. apiospermum complex from other species in the genus. In addition, by comparing the MSP dendrogram with the drug sensitivity data (Wang et al., 2015), we didn’t find a relation between the spectra difference and the drug sensitivity in the test strains. In conclusion, our results showed that MALDI-TOF MS identification for Scedosporium spp. by the Bruker Biotyper was relatively reliable after protein extraction from liquid cultivation and database combination.

To date, there have been no MALDI-TOF MS identification studies for a wide range of Acremonium-like species. Becker et al. (2014) correctly identified five out of six isolates of S. strictum to the species level with a supplemented database by the Bruker Biotyper system. Rychert et al. (2018) correctly identified 30 Acremonium sclerotigenum (now named A. egyptiacum) (Summerbell et al., 2018) isolates to the species level using the Vitek MS v3.0 database. Due to the limited species in these studies, the identification ability of MALDI-TOF MS for Acremonium-like species could not be defined. We conducted the first MALDI-TOF MS identification study on multiple species and genera of Acremonium-like spp. Due to a lack of reference MSPs, FFL v1.0 could not identify all of the Acremonium-like isolates. Although one S. strictum MSP was included in the commercial database, no identification results were found when tested with one S. strictum isolate. However, three of the four isolates of S. kiliense were identified as S. strictum, though with low scores (between 1.464 and 1.671, data not shown), suggesting that the reference strain of S. strictum in FFL v1.0 might actually be S. kiliense. Some isolates previously reported as S. strictum in the literature were also reconfirmed as S. kiliense (Perdomo et al., 2011; Summerbell et al., 2018), indicating the possibility of confusion. After supplementation of the BMU database, the identification accuracy by MALDI-TOF MS of Acremonium-like species was similar to that of multilocus sequencing, and the former method was faster, simpler and less expensive.

Only a few species and strains of Scopulariopsis-like fungi have yet been involved in MALDI-TOF MS identification studies. Using FFL v1.0, S. brevicaulis was correctly identified in some cases (Schulthess et al., 2014; Levesque et al., 2015; Riat et al., 2015; Stein et al., 2018; Sacheli et al., 2020), while S. candida, S. cinerea, S. brumptii, and M. cirrosus could not be identified (Schulthess et al., 2014; Levesque et al., 2015; Stein et al., 2018). The identification of Scopulariopsis-like fungi can be improved by constructing an in-house database (Lau et al., 2013; Becker et al., 2014). Lau et al. (2013) correctly identified three strains of S. brevicaulis to the species level using the Bruker Biotyper system plus the self-built NIH database. Becker et al. (2014) also correctly identified 12 strains of S. brevicaulis and one strain of S. candida with an in-house database. Our study found that FFL v1.0 performed well in the identification of S. brevicaulis but was unable to identify other species due to a lack of reference MSPs. After adding the reference MSPs of ten species, the Bruker Biotyper system was able to accurately identify Scopulariopsis and Microascus species.

The MSP dendrogram obtained by MALDI-TOF MS analysis indicates the relationship between the strains based on protein fingerprint differences and can be applied to the classification of microorganisms. This study found that the MSP dendrogram of Acremonium-like isolates clearly distinguished 7 different species, with similar topology to the multilocus phylogenetic tree. The MSP dendrogram of Scopulariopsis and Microascus spp. clustered isolates of the same species or close species to a branch, while the topology of Microascus spp. was inconsistent with that of the multilocus phylogenetic tree. These results indicated that the MSP dendrogram is efficient for species differentiation; however, there was a difference between molecular taxonomy at the nucleic acid and protein levels, which may be due to the varied protein expression related to the growth condition and life cycle of the fungi (Putignani et al., 2011). Shao et al. (2018) also found that the interspecific discrimination of MSP dendrograms and ITS-based trees of Rhizopus and Mucor species was consistent, while there were inconsistences at the intergeneric and intraspecific levels. Research on Lichtheimia species by Schrodl et al. (2012) showed that the MSP dendrogram clearly discriminated different species, but the topology was different from that of the phylogenetic tree. Therefore, MSP dendrogram is an excellent tool for interspecific differentiation but is limited in phylogenetic analysis (Shao et al., 2018). In addition, we found the MSP dendrogram cannot differentiate species in the S. apiospermum complex. Shao et al. (2020) also found that the MSP dendrogram was difficult to distinguish species in the Trichophyton mentagrophytes series. These results indicated discrimination of close species in some species complex by MSP dendrogram is difficult.

This study showed that with the supplementary database, MALDI-TOF MS represents a powerful tool to identify Scedosporium, Acremonium-like, Scopulariopsis, and Microascus species. As the test strains were all previously identified storage strains, and the species were rare and difficult to collect in a short time, blind test was not conducted. In the future, the identification effect will be clearer on the basis of continuous database expansion and amplification of test species and strains.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/genbank/, MW528357 to MW5283564.

JY designed the study. ZW collected the samples. LW, JS, YS, LY, and HW participated in the performance of the research. LW participated in the writing of the manuscript and data analysis. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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