Introduction

Filamentous fungi are a heterogeneous group of pathogens causing both superficial and systemic fungal infections. Dermatophytes are known to be the commonest pathogenic fungi causing skin or, rarely, subcutaneous infections. Besides bacteria, filamentous fungal species responsible for invasive fungal infections are one of the main causes of morbidity and mortality in hospitalized individuals. They pose a particular threat to cancer or immunocompromised patients (Schuster et al. 2017). Some of them are known to be resistant to systemic antifungals.

The most accurate identification of microorganisms including fungi uses molecular genetic methods. These include various PCR-based techniques, sometimes coupled with restriction endonuclease analysis or hybridization with probes. The most reliable results are obtained by DNA sequencing of target genes (ITS regions of rRNA) and comparison with reference databases. This is currently considered the gold standard in microbial identification. Even though these procedures are becoming increasingly affordable for routine diagnostic practice, they are still not common in mycology laboratories and are used mainly for morphological confirmation or identification of non-sporulating isolates (Wickes and Wiederhold 2018; Ciardo et al. 2010). The reasons are the need for specific equipment and separate rooms, still high sequencing costs and contamination risks.

During the routine diagnostic process in a mycology laboratory, fungi are mainly identified using techniques based on phenotypic characteristics (Ciardo et al. 2007). However, unlike bacteria and yeasts identified according to their biochemical properties, identification of filamentous fungi relies heavily upon their macro- and micromorphology. The appearance of the isolate on a growth medium is assessed, including pigmentation and growth rate; slide culture may be used to characterize the mycelium as well as the shape, size, and arrangement of spores (Larone 2018). This approach, however, may only be used to reliably identify these fungi to the genus level. Moreover, it often requires considerable experience. In case of non-sporulating fungi (sterile mycelia), such identification is practically impossible.

Recently, modern methods for identification of microorganisms including fungi have included an approach that utilizes analysis of mass spectra of cellular proteins called matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Compared to earlier conventional methods, the advantages are rapidity, simplicity, accuracy, and reliability. It is successfully used to identify bacteria and, in mycology laboratories, yeast microorganisms (Croxatto et al. 2012; Demirev and Fenselau 2008). However, the use of this approach is limited by initial equipment costs and, in case of filamentous fungi, also by a relatively small database of these fungi and quite specific structure of the hyphal and spore cell wall. Even though a procedure commonly used to identify yeasts may be sufficient, a modified protocol, known as extraction method, is recommended when preparing culture samples for identification of filamentous fungi (Bader 2013; Ranque et al. 2014).

The study aimed to compare the reliability of identification of selected filamentous fungal species with MALDI-TOF MS using direct (i.e., common for bacteria and yeasts) and extraction methods. Identification of filamentous fungi with both methods was related to identification based on morphological features.

Material and methods

Cultures for identification

Between September 2017 and December 2018, 193 isolates of filamentous fungi from various clinical samples (sputum; tracheal secretions; bronchoalveolar lavage fluid; urine; swabs from the external ear canal, nose, and rectum; wound and skin swabs; skin and nail scrapings; biopsy specimens) were collected from patients in the University Hospital Olomouc, Czech Republic, and included in the study. Prior to testing, they were inoculated onto Sabouraud glucose agar with chloramphenicol (SGAC) slants (Trios, Czech Republic) and kept for at least three months to perform repeated identification.

Morphological identification

Cultures grown on SGAC with thiamine (Trios, Czech Republic) for 3–14 days were identified based on their macromorphological (color and texture of the surface, appearance of the culture base) and micromorphological (appearance of hyphae; presence, shape, size, and arrangement of spores) characteristics. Larone’s Medically Important Fungi: A Guide to Identification served as a reference publication for morphological identification (Larone 2018).

MALDI-TOF MS identification

With the direct method of protein extraction, the tested strain was inoculated onto SGAC with thiamine. After the culture grew sufficiently, part of the mycelium was removed with a needle, transferred onto a MALDI plate, and covered with 0.85 µL of formic acid. When dried, the mixture was layered with the same amount of matrix. The extraction method started with culturing in a test tube with Sabouraud broth placed in Multi Bio RS-24 rotator (Biosan, Lithuania) and incubated for 48 h at 20 rpm and room temperature. Then, the culture was left for sedimentation for 10 min. Subsequently, 1.5 mL of the sediment was transferred to a microtube and centrifuged at 10,000 rpm for 4 min. After the supernatant was discarded, the sediment was washed with 1 mL of sterile water and centrifugation with washing was repeated twice. After the last centrifugation, the sediment was suspended in 900 µL of ethanol and 300 µL of sterile water and again centrifuged at 10,000 rpm for 4 min. The supernatant was removed, and the formed pellet was dried at room temperature for 20 min. Depending on the pellet size, an adequate amount of formic acid (10–100 µL) and the same amount of acetonitrile were added, and the mixture was left for sedimentation at 2–3 min at room temperature. Then, 1 µL of the supernatant or sediment was transferred onto a MALDI target plate with a pipette and layered with microliters of matrix. Samples prepared by this method were analyzed using the Microflex LT system and IVD Maldi Biotyper 2.3 software (Bruker Daltonics).

Sequencing

The DNA of isolates that were not identified reliably or at all (n = 20) was sequenced as described in detail elsewhere (Glass and Donaldson 1995, Khot et al. 2009). Briefly, DNA was isolated with PrepMan Ultra Sample Preparation Reagent (Applied Biosystems). Then, it was subjected to PCR with Cepheid SmartCycler (Thermo Fischer Scientific) to amplify the benA gene, ITS1-5,8-ITS2 and D1-D2 LSU regions using primers listed in Table 1. The PCR conditions were as follows: initial denaturation 95 °C/30 s, cycles 45 × 95 °C/5 s, 55 °C/10 s, 72 °C/15 s, and melting analysis 50 to 95 °C at 0.1 °C/s using TB Green Premix Ex Taq (Takara, Japan). Melting temperatures were analyzed to confirm positive samples. These were purified using ExoSAP-IT (Affymetrix) and amplified using BigDye Terminator v1.1 Cycle Sequencing RR (Applied Biosystems) in the Biometra TRIO 48 thermocycler (Analytik Jena, Germany) according to the manufacturer’s instructions. Further purification followed using NucleoSEQ Columns (Macherey–Nagel, Germany). For sequencing, the product was loaded into sequencing plates and placed in an autosampler on ABI3130 platform (Applied Biosystems). Data obtained were analyzed with Sequencing Analysis Software (Thermo Fischer Scientific), exported, and compared with the BLAST and CBS databases.

Table 1 Primer sequences used in the study
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Results

Morphological identification

The results are summarized in Fig. 1. As seen from the graph, a total of 17 genera of filamentous fungi were identified. Some isolates were identified to the species level (n = 118; 61.1%), others are to the genus level only (n = 70; 36.3%), and the others could not be identified at all (sterile mycelium; n = 5; 2.6%). Within the genus Aspergillus (n = 97), a total of 7 species (92.8%) could be identified; another 7 isolates were identified to the genus level only (Aspergillus spp.; 7.2%). The most common species was Aspergillus fumigatus (n = 38; 39.2%), followed by Aspergillus niger (n = 21; 21.65%), Aspergillus flavus (n = 21; 21.65%), and Aspergillus terreus (n = 7; 7.2%). The second most frequent genus was Trichophyton (n = 25) which included 3 species, with another 4 isolates being identified to the genus level only. The most common species was Trichophyton rubrum (n = 10; 40%), followed by species complex Trichophyton interdigitale/Trichophyton mentagrophytes (n = 8; 32%). In case of the relatively frequent genera Penicillium (n = 19) and Fusarium (n = 12) and the family Mucoraceae (n = 14), none of the isolates could be reliably identified to the species level.

Fig. 1
figure 1

Results of morphological identification of filamentous fungi cultures. Note: different colors means different groups of fungi (blue = Aspergillus spp.; green = Mucoraceae; yellow = dermatophytes; orange = other filamentous fungi)

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MALDI direct method

Of the total of 193 isolates, nine (4.7%) were identified to the genus levels; the other were identified to the species level (n = 184). A total of 75 species were identified. The most common genus was Aspergillus (n = 73) which included 16 species. The most frequent species was A. fumigatus (n = 20; 27.4%), followed by A. flavus (n = 13; 17.8%), and A. terreus (n = 8; 10.95%). The second most common genus was Penicillium (n = 29) which included 16 species. The most frequent were Penicillium italicum (n = 13; 44.8%), Penicillium brevicompactum (n = 3), and Penicillium corylophilum (n = 3). Three isolates were identified to the genus level only. Within the genus Trichophyton (n = 19), six species were identified. The most frequent were Trichophyton tonsurans and T. interdigitale (both n = 5; 26.3%), followed by T. rubrum (n = 4; 21.05%). Seventeen isolates were found to belong to the genus Fusarium, of which 13 (76.5%) were identified to the species level. The most frequent were Fusarium proliferatum (n = 5), Fusarium solani (n = 3), and Fusarium oxysporum (n = 2). Six (85.71%) out of 7 Rhizopus isolates were identified to the species levels, namely, Rhizopus oryzae (n = 3), Rhizopus microsporus (n = 2), and Rhizopus stolonifer (n = 1).

MALDI extraction method

Identification of isolates from both supernatant and sediment yielded the same results. Moreover, there were only small differences in the identification score, both generally and for individual isolates (1.648 vs. 1.687). Nearly all isolates could be identified to the species level (n = 190, 98.45%) and only three to the genus levels. There were 43 species of filamentous fungi. The most common genera were Aspergillus (n = 102; 52.85%), Trichophyton (n = 21; 10.9%), and Penicillium (n = 19; 9.85%). Within the genus Aspergillus, the most frequent species were A. fumigatus (n = 40; 39.2%) and Aspergillus niger (n = 21; 20.6%). Of the 21 Trichophyton isolates, nine were identified as T. rubrum (42.85%), five as T. tonsurans, and three as Trichophyton benhamiae. There were six species of the genus Penicillium, with two isolates being identified to the genus level only. The most frequent species was Penicillium glabrum (n = 5), followed by P. corylophilum (n = 3), and Penicillium chrysogenum (n = 3).

Agreement in identification results

When comparing identification by morphological features to both MALDI-TOF MS methods, as many as 187 isolates could be identified to the genus level but only 118 to the species level. The results are shown in Fig. 2. The rates of agreement between isolate identification using morphological features and the MALDI-TOF MS direct method were 63% at the genus level and 35% at the species level. The agreement between isolate identification using morphology and the MALDI-TOF MS extraction method was 88% at the genus level and 75% at the species level.

Fig. 2
figure 2

Rates of agreement between culture identification using morphological methods and MALDI-TOF MS direct and extraction methods. Note: different colors means different groups of fungi (blue = Aspergillus spp.; green = Mucoraceae; yellow = dermatophytes; orange = other filamentous fungi)

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The rates of agreement between the two methods based on MALDI-TOF MS were 63% and 38.5% at the genus and species levels, respectively. In addition, the mean log-score value was significantly lower for identification by the direct method (1.127) than for the extraction method (1.668). Differences in log score values between the two methods are demonstrated in Table 2.

Table 2 Differences in log score values between MALDI-TOF MS direct and extraction methods
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Sequencing

Of the 20 tested isolates, two yeast microorganisms (Galactomyces geotrichum, Blastobotrys sp.) were identified by sequencing. The likely explanation is contamination of the original isolates of filamentous fungi. Another two isolates, identified as Cadophora sp. and Cryptendoxyla hypophloia by sequencing, are not included in the current version of the commercial MALDI-TOF MS database of filamentous fungi. Therefore, sixteen isolates were compared. In case of the MALDI-TOF MS extraction method, identical results were found for 14 cultures (87.5%). There was an agreement between DNA sequencing results and those obtained by the MALDI-TOF MS direct method in 7 cases (43.75%), with all these isolates being identified by the extraction method as well.

Discussion

Diseases caused by fungi continue to be underestimated, particularly compared to the threats posed by bacterial infections resistant to antibiotic therapy or viral epidemics (Almeida et al. 2019). According to the literature, severe fungal infections are estimated to affect approximately 832 million people in 14 countries with the highest incidence rates in Asia, the Americas, Europe, and North Africa. In each of those countries, between 1.8 and 3% of the population suffer from some of the most severe types of fungal infections potentially causing chronic illness or even death (Life leading international fungal education 2017). Moreover, resistance to the limited armamentarium of antifungal drugs has gradually developed and become a serious problem, especially in the case of the most common systemic fungal infections caused by species of the genera Aspergillus and Candida (Rivero-Menendez et al. 2016, van Paassen 2016). It is therefore essential that new rapid diagnostic strategies and algorithms for antifungal therapy including novel drugs are developed.

MALDI-TOF MS is currently considered the most reliable method for identification of microorganisms in routine microbiology laboratories including those specializing in clinical mycology. The increasing role of MALDI-TOF MS in mycological diagnosis is mentioned in current recommendations for diagnosing rare fungal diseases (Hoenigl et al. 2020). Even though identification using MALDI-TOF MS is beneficial, affordable, and rapid, its result still needs to be morphologically verified. Primarily, this may be carried out at the genus level if the isolate sporulates. Agreement, particularly with the extraction method, is usually good, as confirmed by our results. For example, for 51 isolates obtained from sputum in the present study, the rate of agreement between morphological identification and the extraction method was 81.9%, as compared with only 44% agreement with the direct method. When comparing the direct and extraction methods, log score values obtained by the extraction method were significantly higher, as shown in Fig. 2. Another argument for considerably higher reliability of identification with the extraction method was the result of sequencing. There was agreement for 14 isolates whereas only two isolates were misidentified.

Sometimes, MALDI-TOF MS identification results may correct morphological identifica`tion of an isolate at the species level; an example may be the atypical morphology of the isolate due to ongoing antifungal therapy. In the course of the study, two isolates from the same patient were originally identified as belonging to the genus Monocilium, based on morphological features. After culture in a liquid medium with extraction, however, they were identified as A. fumigatus with scores of 2.204 and 1.912. Even when repeated, culture morphology failed to confirm this result. Sequencing, on the other hand, showed the same result as mass spectrometry identification.

Another advantage of the extraction method is correct species identification. In the vast majority of cases, Penicillium isolates are only identified to the genus level when morphological features are used. The extraction method, however, was able to identify all but two cultures to the species level. Also, dermatophyte species are difficult to identify. Out of 25 dermatophytes belonging to the genus Trichophyton in the present study, there was agreement between species identification with the extraction method and morphological characterization for 14 isolates. For the remaining 11 isolates, mainly zoophilic species, the original morphological identification results were corrected using this approach. An example may be a specimen morphologically characterized as T. interdigitale. The extraction method, however, identified it as T. erinacei, with scores of 2.161 and 1.890.

In their study, Becker et al. (2014) used MALDI-TOF MS, and their own in-house library containing 760 strains that was evaluated on 390 strains of filamentous fungi by comparing MALDI-TOF MS and classical morphological identification results. The use of MALDI-TOF MS resulted in the correct identification of 85.6% of isolates to the species level when taking into account the manufacturer’s cutoff value for reliability (i.e., log score ˃ 1.7). Without considering the value, as many as 95.4% of isolates were correctly identified to the species level. Theel et al. (2011) were able to correctly identify only 20.5% of dermatophyte isolates using MALDI-TOF MS and a commercial library. But, in combination with a supplemented library of another 20 dermatophyte spectra, the rate increased to 60%. The above studies suggest that identification of filamentous fungi by MALDI-TOF MS often depends on the size and quality of the reference library used.

Conclusion

Identification of filamentous fungi based on morphological features is the most available approach commonly used in diagnostic medical mycology laboratories. In many cases, however, it requires considerable experience and ability of strains to sporulate. For its rapidity, simplicity, and accuracy, MALDI-TOF MS is invaluable for identification of microorganisms. But given the cell wall characteristic of filamentous fungi, it is advisable, unlike in bacteria and yeasts, to use a slightly more complex procedure, the so-called extraction method. Moreover, identification of filamentous fungi is limited by a narrow spectrum of species in the commercial database. The optimal approach to identification of filamentous fungi is a combination of morphological features and the MALDI-TOF MS extraction method with a prospect of developing an in-house database. If identification with the above combination is ambiguous or impossible to perform, molecular genetic methods need to be used, in particular sequencing.