Rapid Assays for Specific Detection of Fungi of Scopulariopsis and Microascus Genera and Scopulariopsis brevicaulis Species

Purpose Fungi of Scopulariopsis and Microascus genera cause a wide range of infections, with S. brevicaulis being the most prevalent aetiological agent of mould onychomycosis. Proper identification of these pathogens requires sporulating culture, which considerably delays the diagnosis. So far, sequencing of rDNA regions of clinical isolates has produced ambiguous results due to the lack of reference sequences in publicly available databases. Thus, there is a clear need for the development of new molecular methods that would provide simple, rapid and highly specific identification of Scopulariopsis and Microascus species. The objective of this study was to develop simple and fast assays based on PCR and real-time PCR for specific detection of fungi from Scopulariopsis and Microascus genera, and separately, S. brevicaulis species. Methods On the basis of alignment of β-tubulin gene sequences, Microascus/Scopulariopsis-specific primers were designed and S. brevicaulis-specific primers were reevaluated. DNA from cultured fungal isolates, extracted in a two-step procedure, was used in Microascus/Scopulariopsis-specific and S. brevicaulis-specific PCR and real-time PCR followed by electrophoresis or melting temperature analysis, respectively. Results The specificity of the assays was confirmed, as positive results were obtained only for Scopulariopsis spp. and Microascus spp. isolates tested in Microascus/Scopulariopsis-specific assay, and only for S. brevicaulis and S. koningii (syn. S. brevicaulis) isolates in a S. brevicaulis-specific assay, respectively, and no positive results were obtained neither for other moulds, dermatophytes, yeast-like fungi, nor for human DNA. Conclusions The developed assays enable fast and unambiguous identification of Microascus spp. and Scopulariopsis spp. pathogens.

The data considering Scopulariopsis and Microascus antifungal susceptibility are scarce and often inconsistent. The very few reports available have recognised them as a multidrug-resistant fungi [41,42]. Noteworthy, the lack of correlation between in vitro drug susceptibility (MIC determination results) and clinical outcomes has been demonstrated [39,41].
The recovery of Scopulariopsis and Microascus species from clinical samples is relatively easy, as these fungi grow well on routine laboratory media. Yet, it is still difficult to perform species identification based on morphological criteria. Moreover, Microascus/Scopulariopsis infections, and disseminated infections in particular might be clinically and histologically indistinguishable from aspergillosis, fusariosis or zygomycosis [43,44]. Since, in the majority of clinical reports on Scopulariopsis spp. infections, morphological identification of the aetiological agent has not been confirmed at the molecular level, the actual prevalence of Scopulariopsis species, other than S. brevicaulis, is unknown [6].
In this paper, we present PCR and real-time PCRbased assays developed for the detection of cultured isolates of Scopulariopsis and Microascus genera, as well as S. brevicaulis species.

Strains and Isolates
In the present study, we used a total of 219 fungal strains, representing 103 fungal species (Table 1). The strains were obtained from international culture collections (CBS-KNAW Fungal Biodiversity Centre; BCCM/IHEM Biomedical Fungi and Yeasts Collection-Belgian Coordinated Collections of Microorganisms; Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures) and Molecular Biotechnology and Microbiology Department (MBMD) collection of fungi (Gdańsk University of Technology, Gdańsk, Poland). Identification of all MBMD isolates was performed by observation of macro-and micromorphology and then confirmed by sequencing of the ITS region, as described by White et al. [45]. Moreover, in case of Alternaria spp., Aspergillus spp. and Scopulariopsis spp. MBMD isolates b-tubulin gene sequencing was performed, as described by Glass and Donaldson [46].

DNA Extraction
Isolates were cultured on Sabouraud glucose agar (Biomerieux, Marcy l'Etoile, France) and incubated for up to 14 days at room temperature. DNA from fungal samples (pieces of mycelium of 3-5 mm S. brevicaulis-specific PCR assay was performed the same way as previously described [48]. Real  assay; SbFor, SbRev in S. brevicaulis-specific assay) at 100 lM, and 2 ll of DNA. PCR was performed in a LightCycler Ò Nano Instrument (Roche, Basel, Switzerland). The cycling conditions in Microascus/ Scopulariopsis-specific assay included an initial denaturation for 3 min at 95°C followed by 40 cycles of 15 s at 94°C, 15 s at 68°C and 30 s at 72°C. The time-temperature profile in S. brevicaulis-specific assay started with initial denaturation for 3 min at 94°C followed by 40 cycles of 10 s at 94°C, 10 s at 60°C and 15 s at 72°C. The presence of specific amplicons was examined upon melting temperature analysis (80°C to 95°C at 0.1°C/s ramp rate), which followed cycling.

Microascus/Scopulariopsis-Specific PCR and Real-Time PCR Assay Results
A 285-bp PCR product corresponding to Scopulariopsis/Microascus was observed for all 48 Scopulariopsis and Microascus spp. DNA samples. No PCR products were detected for 76 other mould isolates, 65 dermatophyte isolates, 30 yeast-like isolates or human DNA (100 % sensitivity and 100 % specificity) (Fig. 1). Similar results were obtained when real-time PCR was applied, as amplicon of T m range of 87.03-89.02°C (C t = 25.12 ± 4.28), corresponding to Scopulariopsis/Microascus spp., was observed only for 48 Scopulariopsis and Microascus spp. DNA samples and not for any other fungal or human DNA samples (Fig. 2).
Accordingly, as a result of real-time PCR, amplicon of T m = 87.76 ± 0.20°C (C t = 24.11 ± 4.38) corresponding to S. brevicaulis was observed only for 8/8 S. brevicaulis and 1/1 S. koningii (syn. S. brevicaulis) DNA samples and not for any other fungal or human DNA samples (Fig. 4).

Discussion
At present, identification of pathogenic fungi still largely relies on the evaluation of macro-and micromorphology. Distinction between Scopulariopsis and Microascus species by using morphological criteria remains useful since the features of conidia and sexual reproductive structures are quite characteristic at the genus level. Two well-recognised disadvantages of these methods, delaying the diagnostic outcome, are the amount of time elapsing from specimen delivery to the diagnostic result acquisition and the requirement of sporulating culture. Diagnosis of disseminated infections is particularly challenging since Scopulariopsis fungi are difficult to distinguish from other moulds (e.g. Aspergillus, Fusarium) upon histopathological examination. Furthermore, the sensitivity of confirmatory blood cultures is poor [44]. Molecular tools have increasingly been adopted in clinical laboratories for the identification of fungi. The sequence analysis of the ribosomal operon has been used for the identification of clinical strains of Scopulariopsis, yet the results may not have been fully reliable because of insufficient availability of reference sequences in the public databases [6,39,49]. Moreover, the D1/D2 region, the target most frequently used for species identification, exhibits a low interspecific variation in Scopulariopsis and Microascus genera [6]. Recently, Ropars et al. [50] performed a combined analysis of partial sequences of the large subunit (LSU) rRNA gene, b-tubulin (TUBB), and elongation factor 1-a (EF1-a) genes for the taxonomic circumscription of Scopulariopsis species, whereas Bontems et al. [51] developed a PCR-RFLP assay, based on 28S rDNA, for identification of fungi, including Scopulariopsis spp., involved in onychomycosis. However, all these methods are laborious and generate rather complicated patterns, thus making them unlikely to be implemented in routine laboratory diagnostics.
All this underlines a need for the development of new methods that would provide simple, rapid and highly specific identification of Scopulariopsis/ Microascus at both genus and species levels. In this study, we present PCR and real-time PCR-based assays that enable genus-specific detection of Scopulariopsis spp. and Microascus spp. DNA, as well as species-specific detection of S. brevicaulis in culture samples. b-Tubulin gene, formerly chosen as one of the targets in phylogenetic studies [50,52], was confirmed to be an adequate target for genus-specific (Microascus spp. and Scopulariopsis spp.) and species-specific (S. brevicaulis) identification. Developed assays are rapid, easily performed and interpretable, and can serve as useful adjunct tools for the identification of the Scopulariopsis spp. and Microascus spp. infections. However, further studies are needed to confirm assay's clinical applicability (sensitivity, direct amplification from various clinical specimens, etc.). As pointed out by Balajee et al. [53], an increasing number of clinical laboratories begins to assess the usefulness of DNA-based methods for identification of isolates recovered from culture of clinical samples in order to complement morphology-based methods (especially when an isolate displays atypical colour, features, or morphology) or to supplant them when culture results are delayed due to slow or absent sporulation [54]. Moreover, analysis of DNA-based methods results is almost entirely independent from diagnostician experience, and thus, it is easy to implement them in basic laboratories. Precise and timely identification of fungal isolates to species can be extremely important when recovered from highrisk patients, as fungal infections in these patients can be serious, difficult to treat and rapidly fatal [53]. Diagnostic procedures should always be guided by clinical history of the patient and clinician's suspicion of disease.

Compliance with Ethical Standards
Conflict of interest The authors declare that they have no conflict of interest.  (6); negative control (7) Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.