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Rapid Assays for Specific Detection of Fungi
of Scopulariopsis and Microascus Genera and Scopulariopsis
brevicaulis Species
Milena Kordalewska .Tomasz Jagielski .
Anna Brillowska-Da˛browska
Received: 10 November 2015 / Accepted: 6 April 2016 / Published online: 2 June 2016
ÓThe Author(s) 2016. This article is published with open access at Springerlink.com
Abstract
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 Microas-
cus 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 Scopulari-
opsis and Microascus genera, and separately, S.
brevicaulis species.
Methods On the basis of alignment of b-tubulin gene
sequences, Microascus/Scopulariopsis-specific pri-
mers 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. brevi-
caulis-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 Scopulari-
opsis 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.
Keywords Detection Identification Microascus
PCR Real-time PCR Scopulariopsis
Introduction
The genus Scopulariopsis, erected by Bainier (1907),
contains both hyaline and dematiaceous moulds,
Milena Kordalewska and Tomasz Jagielski have contributed
equally to this work.
M. Kordalewska (&)A. Brillowska-Da˛browska
Department of Molecular Biotechnology and
Microbiology, Faculty of Chemistry, Gdan
´sk University
of Technology, Narutowicza 11/12, 80-233 Gdan
´sk,
Poland
e-mail: milena.kordalewska@pg.gda.pl
T. Jagielski
Department of Applied Microbiology, Faculty of Biology,
Institute of Microbiology, University of Warsaw,
Miecznikowa 1, 02-096 Warsaw, Poland
123
Mycopathologia (2016) 181:465–474
DOI 10.1007/s11046-016-0008-5
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
which propagate asexually by conidia. Most of their
teleomorphs are included in the genus Microascus
[1–5]. The anamorph–teleomorph connections have
already been established for many species. However,
the sexual states of some Scopulariopsis species are
still unknown [6].
Scopulariopsis spp. are saprobes with a worldwide
distribution. They are commonly isolated from soil,
air, plant debris, paper, dung and moist indoor
environments [7,8]. Traditionally, Scopulariopsis
and Microascus species have not been considered
common human pathogens. However, the number of
cases with these organisms as main perpetrators has
recently been on the rise. Some species are known to
be opportunistic pathogens, primarily causing super-
ficial tissue infections, and being one of the principal
causes of non-dermatophytic onychomycoses [9,10].
The prevalence of onychomycosis caused by S.
brevicaulis is estimated to make up 3–10 % of the
total number of mould onychomycosis cases globally.
Clinically, the condition is generally recognised as
distal and lateral subungual onychomycosis (DLSO)
[11,12]. Cases of cutaneous and subcutaneous infec-
tions have also been described as due to S. brevicaulis
[13,14]. Less commonly Scopulariopsis and Microas-
cus species have been reported as causes of other
infections including endocarditis [15–18], keratitis
[19,20], endophthalmitis [21], sinusitis [22,23],
pulmonary fungus ball [24,25], otomycosis [26,27],
pneumonia [28–30], peritonitis [31], cerebral phaeo-
hyphomycosis and brain abscess [32–34], dissemi-
nated infection with skin lesions including a patient
with acquired immune deficiency syndrome (AIDS)
[13], disseminated infection after bone marrow trans-
plantation [35,36], invasive infection after lung
[37,38] or heart and lung transplantation [39].
Among Scopulariopsis and Microascus species
most frequently isolated from all types of lesions, S.
brevicaulis ranks first, followed by S. acremonium,S.
brumptii,S. flava,M. niger,M. cinereus,M. cirrosus,
M. manginii, and M. trigonosporus [6,40].
The data considering Scopulariopsis and Microas-
cus 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, Microas-
cus/Scopulariopsis infections, and disseminated infec-
tions 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 aetio-
logical 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 PCR-
based assays developed for the detection of cultured
isolates of Scopulariopsis and Microascus genera, as
well as S. brevicaulis species.
Materials and Methods
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 col-
lections (CBS-KNAW Fungal Biodiversity Centre;
BCCM/IHEM Biomedical Fungi and Yeasts Collec-
tion—Belgian Coordinated Collections of Micro-
organisms; Leibniz Institute DSMZ—German
Collection of Microorganisms and Cell Cultures) and
Molecular Biotechnology and Microbiology Depart-
ment (MBMD) collection of fungi (Gdan
´sk University
of Technology, Gdan
´sk, 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
466 Mycopathologia (2016) 181:465–474
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Table 1 Organisms used in the study
Organism Collection
Moulds
Scopulariopsis acremonium DSM 1987
S. asperula CBS 298.67
IHEM 2546
S. brevicaulis CBS 112377; CBS 119550;
CBS 118474; CBS 340.39
MBMD (human-derived isolate): W1;
MBMD (dog-derived isolate): 19P;
MBMD (rabbit-derived isolates): F9; F10
S. brumptii (now Fuscoannellis carbonaria) CBS 121662
S. brumptii (now Microascus paisii) CBS 116060
S. canadensis CBS 204.61
S. carbonaria (now F. carbonaria) CBS 205.61
S. chartarum (now M. chartarus) CBS 294.52
S. chartarum (now M. paisii) CBS 670.74
S. coprophila CBS 433.83
S. flava CBS 207.61
S. fusca (now S. asperula) CBS 117767
IHEM 14552; IHEM 25912
S. gracilis (now M. gracilis) CBS 369.70
S. koningii (now S. brevicaulis) CBS 289.38
S. murina (now M. murinus) CBS 830.70; CBS 621.70; CBS 864.71
S. parva CBS 209.61; CBS 271.76
Microascus albonigrescens CBS 313.71; CBS 109.69
M. cinereus CBS 664.71; CBS 195.61
IHEM 25417
M. cinereus (now M. gracilis) CBS 116059;
M. cirrosus CBS 116405; CBS 277.34
M. cirrosus (now M. pseudolongirostris) CBS 462.97;
M. longirostris CBS 415.64; CBS 196.61
M. manginii (now S. macurae) CBS 506.66
M. manginii (now S. candida) CBS 132.78
M. senegalensis CBS 594.78
M. singularis CBS 505.66
M. stoveri (now Pithoascus stoveri) CBS 176.71
M. trigonosporus var. terreus (now M. terreus) CBS 601.67
M. trigonosporus var.macrosporus (now M. macrosporus) CBS 662.71
M. trigonosporus var. trigonosporus (now M. alveolaris) CBS 494.70
Acremonium charticola MBMD (environmental isolates)
Acremonium kiliense (now Sarocaldium kiliense)
Acremonium strictum (now Sarocaldium strictum)
Alternaria alternata
A. brassicae
A. tenuissima
Alternaria sp.
Mycopathologia (2016) 181:465–474 467
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Table 1 continued
Organism Collection
Aspergillus clavatus
A. flavus
A. fumigatus
A. nidulans
A. niger
A. versicolor
Cladosporium cladosporioides
C. herbarum
C. macrocarpum
Fusarium culmorum
F. discolor
F. oxysporum
F. proliferatum
Fusarium solani (now Neocosmospora solani)
Mucor racemosus
M. circinelloides
Ochrocladosporium elatum
Penicillium chrysogenum
P. carneum
P. chrysogenum
P. commune
P. crustosum
P. digitatum
P. glabrum
P. hirsutum
P. italicum
P. melinii
P. paneum
P. polonicum
P. verrucosum
Penicillium sp.
Phoma herbarum
Pleospora papaveracea
Rhizopus oligosporus
R. oryzae
Trichoderma viride
Ulocladium chartarum
U. tuberculatum
Dermatophytes
Epidermophyton floccosum MBMD (human-derived isolates)
Microsporum audouinii
M. canis
M. gypseum
M. nanum
M. persicolor
468 Mycopathologia (2016) 181:465–474
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diameter) was extracted by a 10-min incubation of the
sample in 100 ll of extraction buffer (60 mM sodium
bicarbonate [NaHCO
3
], 250 mM potassium chloride
[KCl] and 50 mM Tris, pH 9.5) in 95 °C and
subsequent addition of 100 ll anti-inhibition buffer
(2 % bovine serum albumin). After vortex mixing, this
DNA-containing solution was used for PCR [47]. All
reagents for DNA extraction were purchased from
Sigma-Aldrich (Seelze, Germany).
PCR and Real-Time PCR Assays
On the basis of alignment (VectorNTI; InforMax, Inc.)
of b-tubulin gene (TUBB) sequences deposited in the
NCBI nucleotide database, Microascus/Scopulariop-
sis-specific primers ScopFor (50CATCTCGGGCGA
GCACGGTC 30) and ScopRev (50CCAGGAC
AGCACGGGGAACAT 30) were designed. Primers
were then synthesised by Genomed (Warsaw, Poland).
PCR mixtures, of 20 ll each, consisted of 10 llof29
PCR Master Mix Plus High GC (A&A Biotechnology,
Gdynia, Poland), 0.1 ll of each primer (ScopFor,
ScopRev) at 100 lM, and 2 ll of DNA. PCR was
performed in a 5345 Mastercycler ep gradient S
(Eppendorf, Hamburg, Germany). The time–temper-
ature profile included initial denaturation for 3 min at
94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at
68 °C, and 30 s at 72 °C. The presence of specific
285-bp amplicons was examined electrophoretically
on a 2 % agarose gel, stained with ethidium bromide.
S. brevicaulis-specific PCR assay was performed
the same way as previously described [48].
Real-time PCR mixtures, of 20 ll each, consisted
of 10 llof29PCR Master Mix SYBR A (A&A
Biotechnology, Poland), 0.1 ll of each primer (Scop-
For, ScopRev in Microascus/Scopulariopsis-specific
Table 1 continued
Organism Collection
Trichophyton equinum
T. erinacei
T. interdigitale
T. mentagrophytes
T. rubrum
T. schoenleinii
T. soudanense
T. terrestre
T. tonsurans
T. verrucosum
T. violaceum
Yeast-like fungi
Candida albicans MBMD (human-derived isolates)
C. catenulata
C. glabrata
C. guillermondii
C. kefyr
C. krusei
C. magnoliae
C. parapsilosis
C. tropicalis
C. utilis
Geotrichum sp. MBMD (environmental isolates)
Rhodotorula mucilaginosa
Saccharomyces cerevisiae
Human MBMD
Mycopathologia (2016) 181:465–474 469
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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 °Cto95°C at 0.1 °C/s ramp rate), which
followed cycling.
Results
Microascus/Scopulariopsis-Specific PCR
and Real-Time PCR Assay Results
A 285-bp PCR product corresponding to Scopulari-
opsis/Microascus was observed for all 48 Scopulari-
opsis 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).
S. brevicaulis-Specific PCR and Real-Time PCR
Assay Results
A 223-bp PCR product corresponding to S. brevicaulis
was observed for 8/8 S. brevicaulis and 1/1 S. koningii
(syn. S. brevicaulis) DNA samples. No PCR products
were detected for 20 other Scopulariopsis spp. strains,
19 Microascus spp. strains, 76 other mould isolates, 65
dermatophyte isolates, 30 yeast-like isolates or one
human DNA (100 % sensitivity and 100 % specificity
for PCR) (Fig. 3).
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 micro-
morphology. 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 Scopular-
iopsis fungi are difficult to distinguish from other
moulds (e.g. Aspergillus,Fusarium) upon histopatho-
logical examination. Furthermore, the sensitivity of
confirmatory blood cultures is poor [44].
Fig. 1 Example of Scopulariopsis/Microascus-specific PCR
product analysis. Mmolecular size marker (fragment sizes 700,
500, 400, 300, 200 and 100 bp); results of Scopulariopsis/
Microascus-specific PCR performed for S. asperula CBS
298.67 (lane 1); S. brevicaulis CBS 112377 (lane 2); S. flava
CBS 207.61 (lane 3); S. fusca IHEM 14552 (lane 4); M. cinereus
CBS 195.61 (lane 5)
470 Mycopathologia (2016) 181:465–474
123
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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 fre-
quently used for species identification, exhibits a low
interspecific variation in Scopulariopsis and Microas-
cus 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 onychomy-
cosis. 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/
Fig. 2 Example of
Scopulariopsis/Microascus-
specific real-time PCR
product melting temperature
analysis performed for S.
asperula CBS 298.67 (1); S.
brumptii CBS 121662 (2); S.
brevicaulis CBS 119550 (3);
S. flava CBS 207.61 (4); S.
fusca CBS 117787 (5); M.
manginii CBS 195.61 (6);
negative control (7)
Fig. 3 Example of S. brevicaulis-specific PCR product analy-
sis. Mmolecular size marker (fragment sizes 700, 500, 400, 300,
200 and 100 bp); results of S. brevicaulis-specific PCR
performed for S. asperula CBS 298.67 (lane 1); S. fusca IHEM
14552 (lane 2); S. flava CBS 207.61 (lane 3); S. brevicaulis CBS
112377 (lane 4); S. brevicaulis human-derived isolate MBMD-
W1 (lane 5); S. brevicaulis rabbit-derived isolate MBMD-F9
(lane 6)
Mycopathologia (2016) 181:465–474 471
123
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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 Scopu-
lariopsis 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. Devel-
oped assays are rapid, easily performed and inter-
pretable, and can serve as useful adjunct tools for the
identification of the Scopulariopsis spp. and Microas-
cus spp. infections. However, further studies are
needed to confirm assay’s clinical applicability (sen-
sitivity, 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 high-
risk 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.
Acknowledgments The authors wish to express their thanks
to Prof. B. Dworecka-Kaszak and I. Da˛ browska, M.Sc. from the
Department of Preclinical Sciences of Warsaw University of
Life Sciences (Poland), A. Hryncewicz-Gwo
´z
´dz
´, MD, Ph.D.,
and K. Kalinowska, Ph.D. from the Department and Clinic of
Dermatology, Venereology and Allergology of Wroclaw
Medical University, for the identification and delivery of S.
brevicaulis isolates.
Funding The study was in part financed by the Polish Ministry
of Science and Higher Education [Iuventus Plus grant number
IP12013023672] to TJ.
Compliance with Ethical Standards
Conflict of interest The authors declare that they have no
conflict of interest.
Fig. 4 Example of S.
brevicaulis-specific real-
time PCR product melting
temperature analysis
performed for
Scopulariopsis brevicaulis
CBS 112377 (1); S.
brevicaulis animal-derived
isolate MBMD-19P (2); S.
asperula CBS 298.67 (3); S.
fusca IHEM 14552 (4); S.
flava CBS 207.61 (5); M.
longirostris CBS 415.64 (6);
negative control (7)
472 Mycopathologia (2016) 181:465–474
123
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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 unre-
stricted 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 Com-
mons license, and indicate if changes were made.
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