76 Original article

Species identification of dermatophytes isolated from human

superficial fungal infections by conventional and molecular
methods
Mohamed Tahaa, Mona Elfangaryb, Sabry Essac and Ahmed Younesa
a
Microbiology Department, Faculty of Veterinary
Medicine, Zagazig University, Zagazig, bDermatology
Department, Faculty of Medicine, Misr University for
Science and Technology (MUST) and cDepartment of
Mycoplasma, Animal Health Research Institute, Giza,
Egypt
Correspondence to Mohamed Taha, PhD, Microbiology
Department, Faculty of Veterinary Medicine, Zagazig
University, Egypt
Tel: + 20 111 134 1333; fax: + 20 222 986 682;
e-mail: mtahalab@yahoo.com
Received 6 April 2016
Accepted 29 August 2016
Journal of the Egyptian Women’s Dermatologic
Society 2017, 14:76–84
Background
Dermatophyte identification is based on the detection of fungal elements by direct
microscopy of clinical specimens combined with culture-based full identification.
Phenotypic identification includes macromorphological, micromorphological, and
physiological characteristics of the colonies. In the last few years, molecular
approaches have been proven to be useful for identification of dermatophyte species.
Objective
To investigate the conventional and molecular methods used for identification of
dermatophytes.
Materials and methods
Fifty-five specimens collected from human dermatophytosis cases were subjected to
mycological examination. Isolated dermatophytes were identified by phenotypic
methods. Molecular identification was done using PCR amplification of internal
transcribed spacer (ITS1 and ITS4), followed by restriction fragment length
polymorphism using the restriction endonuclease enzyme MvaI, application of PCR
using single repetitive oligonucleotide [(GACA)4], and DNA sequencing.
Results
The obtained dermatophyte isolates (n = 48) identified by phenotypic methods were as
follows: 15 Microsporum canis, 12 Trichophyton violaceum, 12 Trichophyton rubrum,
5 Epidermophyton floccosum, and 4 Trichophyton mentagrophytes. However,
restriction fragment length polymorphism using MvaI and repetitive (GACA)4 molecular
methods identified 19 dermatophyte isolates identical to those classified by
phenotypic methods. DNA sequencing reidentified one isolate formerly determined to
be T. rubrum into Trichophyton raubitschekii.
Conclusion
Although the molecular method is rapid and represents technological advancement in
the identification of dermatophytes, it is too expensive to be used as a routine method
and needs more effort. We recommend its use in the absence of skilled mycologists or
in atypical and variants of dermatophytes.

Keywords:
dermatophytes, molecular identification, phenotypic identification

J Egypt Women Dermatol Soc 14:76–84

& 2017 Egyptian Women’s Dermatologic Society
1687-1537

length polymorphism (RFLP), have brought greater

Introduction
Dermatophyte identification usually depends on conven-
tional methods based on detection of phenotypic
characteristics, such as direct microscopy and in-vitro
culture [1]. Morphological and physiological character-
istics can frequently vary; in fact the phenotypic features
can be easily influenced by external factors such as
temperature and the medium used [2].
In the last few years, molecular approaches have been
proven to be useful for solving taxonomic problems
regarding dermatophytes. Genotypic differences are
considered more stable and more precise than phenotypic
characteristics [3]. Molecular methods, such as PCR,
random amplification of polymorphic DNA, arbitrarily
primed PCR, PCR fingerprinting, and restriction fragment
1687-1537 & 2017 Egyptian Women’s Dermatologic Society
efficiency in distinguishing between species and strains
of dermatophytes [1,4–7]. On the other hand, PCR
immunosorbant assay (PCR-ELISA), line block PCR
(PCR-RLP), and multiplex real-time PCR are propagated
for the detection of dermatophytes in clinical materi-
als [8–10]. The present work was done to study the
phenotypic and molecular methods for identification of
dermatophytes.
Materials and methods
Samples
Fifty-five skin scrapings and/or hair were collected from
patients suffering from dermatophytosis who were attend-
ing the Dermatology Outpatient Clinic of Misr University
DOI: 10.1097/01.EWX.0000499598.84966.cb

Copyright r 2017 Egyptian Women’s Dermatologic Society. Unauthorized reproduction of this article is prohibited.

for Science and Technology (MUST) Hospital during the
period from May 2010 to September 2011. The study
protocol was approved by the Research Ethics Committee
of MUST. The cases included were 25 tinea capitis, 15
tinea corporis, 10 tinea cruris, and 5 tinea pedis.
Media
The following media were used: dermasel agar (Oxoid;
OXOID Ltd., Basingstoke, Hampshire UK), dermatophyte
test medium (DTM; HiMedia, Mumbai, Maharashtra, India),
InTray DM (Biomed Diagnostic, White City, Oregan, USA),
Derm-Duet (Hardy Diagnostics, Santa Maria, California, USA)
(containing a biplate with one section of DTM and the other
section of rapid sporulation medium), bromocresol purple
(BCP) [11], rice lactritmel agar (RLA) [12], milk honey
bromothymol blue (MHB) medium [13], and rice grain (RG)
medium [14].
Isolation of dermatophytes
After cleaning the lesions with 70% ethyl alcohol, skin
scrapings and/or hair were collected from each case in a
sterile Petri dish (6 mm). Direct microscopic examination
was performed using KOH (20%). Specimens of skin
scrapings and hair were inoculated in dermasel agar,
DTM, Derm-Duet, and InTray DM. All inoculated media
were incubated at 301C for 2 weeks.
Identification of isolated dermatophytes
Phenotypic identification
Phenotypic identification was made as follows [15,16]:
(1) Through macromorphological examination, on the
basis of the reported rate of growth, consistency,
surface color, reverse color, and change in DTM color.
(2) Through micromorphological examination: using InTray
DM, the cartilage was examined through a clear viewing
window under a microscope for hyphae and conidia; in
the case of other media, first, subculturing on dermasel
agar was performed, and then specimens from the
growth were placed on a slide with drops of lactophenol
cotton blue (HiMedia), overlaid with a coverslip, and
examined under a microscope for modification of
hyphae, macroconidia, and microconidia.
(3) Through cultivation on differential media: subculture
of the isolated dermatophytes was performed on RG,
BCP, RLA, and MHB.
Molecular identification
Dermatophytes: Nineteen isolates formerly identified by
phenotypic methods were subjected to molecular identi-
fication.
DNA extraction: This was performed using a Qiagen
extraction kit (Qiagen, Hilden, North Rhine-Westphalia,
Germany) according to the manufacturer’s instructions.
Detection of DNA by PCR
Using general primers (internal transcribed spacer – ITS1 and
ITS4) [17]
Amplification reactions were carried out with 100 ml
total reaction volume containing 50 ml DreamTag Green
Master Mix 2  (Thermo Scientific Fermentas, Waltham,
Copyright r 2017 Egyptian Women’s Dermatologic Society. Unauthorized reproduction of this article is prohibited.
Species identification of dermatophytes Taha et al. 77
Massachusetts, USA) and 30 pmol of each of primers,
ITS1 (50 -TCCGTAGGTGAACCTGCGG-30 ) and ITS4
(50 -TCCTCCGCTTATTGATATGC-30 ) [17] prepared by
Sigma (Hamburg, Germany), and 10 ml DNA. The PCR
cycling conditions were 35 cycles of 951C for 1 min, 551C
for 1 min, and 721C for 2 min, followed by an extension
step of 721C for 10 min. PCR was carried out using a
thermal cycler (S1000; Bio-Rad, Hercules City, California,
USA). The resulting PCR products were digested with
the restriction endonuclease enzyme MvaI (Takara, Ostu,
Shiga, Japan), which recognizes the sequence 50 -CC(T/
A)GG-30 . The resulting products were separated in 2%
agarose gel stained with ethidium bromide, 1  tris–
borate–EDTA, and DNA ladder 100 bp plus DNA, Gene
Ruler (Thermo Scientific Fermentas). Images were
captured using ultraviolet transillumination and a digital
camera (Canon, Ota, Tokyo, Japan).
Using repetitive primer (GACA)4 [1] (prepared by Sigma)
Amplification reactions were carried out with total
reaction volume of 50 ml containing 25 ml reaction buffer
and 30 pmol of the primer. PCR was carried out for 39
cycles of denaturation at 931C for 1 min, annealing at
501C for 1 min, and extension at 721C for 1 min, followed
by a final extension step at 721C for 7 min. The resulting
PCR products were separated in 1% agarose gel stained
with ethidium bromide in 0.5  tris–borate–EDTA buffer
and stained with ethidium bromide; images were then
obtained as described above.
Sequencing
The purified PCR products were sent to Macrogen Lab
(Seoul, South Korea). The nucleic acid sequences
obtained from forward and reverse primer cycle sequen-
cing were analyzed by DNA baser software (http://
www.dnabaser.com/index.html ) to trim the sequences and
align them with previously published sequence data in
GenBank.
Results
From 55 samples of skin scrapings and hair, positive
microscopic results were obtained for 92.72% of samples
on KOH, whereas dermatophytes were isolated from 48
(87.27%) samples on dermasel agar and DTM, 38
(69.09%) on InTray DM, and 32 (58.18%) isolates on
Derm-Deut.
Phenotypic identification of isolated dermatophytes
Macromorphological and micromorphological identification
Forty-eight dermatophyte isolates were identified (as
shown in Table 1 and Figs 1–3) and classified into
Microsporum canis (n = 15), Trichophyton violaceum (n = 12),
Trichophyton rubrum (n = 12), Trichophyton mentagrophytes
(n = 4), and Epidermophyton floccosum (n = 5). M. canis and
T. violaceum were the causative agents of tinea capitis, and
T. rubrum, M. canis, and T. violaeum were the causative of
tinea corporis. Tinea cruris was caused by E. floccosum and
T. rubrum, and tinea pedis was caused by T. mentagrophytes.
78 Journal of the Egyptian Women’s Dermatologic Society

Table 1. The frequency of dermatophytes in types of human dermatophytosis

Types Microsporum canis Trichophyton violaceum Trichophyton rubrum Trichophyton mentagrophytes Epidermophyton floccosum

Tinea capitis 12 10 – – –
Tinea corporis 3 2 9 – –
Tinea cruris – – 3 – 5
Tinea pedis – – – 4 –

Figure 1.

(a–h) Dermatophytes on dermasel agar. (a) Microsporum canis: fluffy white colonies. (b) Trichophyton violaceum: waxy with deep violet colonies.
(c) Trichophyton rubrum (surface): fluffy white colonies. (d) T. rubrum (reverse): red brown. (e) Trichophyton mentagrophytes (surface): granular to
fluffy creamy colonies. (f) T. mentagrophytes (reverse): yellow to reddish brown. (g) Epidermophyton floccosum: folded or flat and white to yellow.
(h) E. floccosum (reverse): orange to light brown.

Figure 2.

(a, b) Dermatophytes on InTray DM. (a) Microsporum canis: white colonies. The medium turned red. (b) M. canis (microscopy): macroconidia.

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 Species identification of dermatophytes Taha et al. 79

Figure 3.

(a–e) Microscopic criteria of dermatophytes (lactophenol cotton blue). (a) Microsporum canis: macroconidia with hook. (b) Trichophyton violaceum
(without stain): bizarre hyphae and chlamydospores. (c) Trichophyton rubrum: microconidia along the hyphae. (d) Trichophyton mentagrophytes:
macroconidia and microconidia. (e) Epidermophyton floccosum: clavate macroconidia have 3–4 cells.

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80 Journal of the Egyptian Women’s Dermatologic Society

Figure 4.

(a–d) Cultivation on differential media. (a) Microsporum canis on RG medium: yellow pigment. (b) Trichophyton mentagrophytes on bromocresol
purple: the medium turned purple. (c) M. canis on rice lactritmel agar: increase of pigmentation. (d) Dermatophytes on milk honey bromothymol blue:
variation of the medium color.

Identification by cultivation on differential media Molecular identification of dermatophytes

Cultivation of dermatophyte isolates on RG, RLA, BCP,
and MHB media confirmed the macromorphological and
micromorphological identification of dermatophytes. In-
creased pigmentation, changed medium color, hydrolysis
of casein (Fig. 4), and increased conidiation were the
factors used for this type of identification.
In the present study three molecular methods were used
for identification of dermatophytes: PCR for amplifica-
tion of ITS1 and ITS4 by common primer, followed by
RFLP using MvaI; PCR using single repetitive oligonu-
cleotides [(GACA)4]; and DNA sequencing, which was
done only for four representative isolates.

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 Species identification of dermatophytes Taha et al. 81

Figure 5. Figure 8.

Agarose gel electrophoresis of Trichophyton rubrum, Trichophyton

mentagrophytes, and Epidermophyton floccosum DNA products. Lane Agarose gel electrophoresis of MvaI restriction products of Microspor-
1: molecular weight marker; lanes 2–5: T. rubrum; lanes 6 and 7: um canis. Lane 1: molecular weight marker; lanes 4–7: fragments
T. mentagrophytes; and lane 8: E. floccosum. ranged from 100 to 500 bp (three bands).

Figure 6. Figure 9.

Agarose gel electrophoresis for PCR product of the internal transcribed

spacer 1 and internal transcribed spacer 4 of the Trichophyton Agarose gel electrophoresis of MvaI restriction products of Trichophy-
violaceum. Lane 1: molecular weight marker; lanes 2–8; T. violaceum. ton violaceum and Trichophyton rubrum. Lane 1: molecular weight;
lanes 2–8: T. violaceum; lanes 9–12: T. rubrum.

Figure 7.
Figure 10.

Agarose gel electrophoresis Microsporum canis DNA products. Lane Agarose gel electrophoresis of DNA products using (GACA)4. Lane 1:
1: molecular weight marker; lanes 2–8: M. canis. DNA ladder; lanes 2–7: Microsporum canis.

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82 Journal of the Egyptian Women’s Dermatologic Society

Identification of dermatophyte isolates by common Identification of dermatophyte by (GACA)4-based PCR

primer and RFLP
PCR with the ITS1/ITS4 primer set for 19 dermatophyte
isolates resulted in amplified products of B690 bp
specific for T. rubrum, T. mentagrophytes, E. floccosum, and
T. violaceum (Figs 5 and 6) and of 740 bp for M. canis
(Fig. 7).
MvaI digestion of these amplified products from each of
the dermatophyte isolates revealed unique restriction
patterns, with no interspecies variation. M. canis isolates
showed three band patterns, ranging from 100 to 500 bp
in size, with a marked size difference between the largest
and middle bands (Fig. 8).
On the other hand, both T. violaceum and T. rubrum isolates
resulted in four bands, with sizes ranging between 50 and
400 bp (Fig. 9).
Nineteen isolates were amplified with (GACA)4. The
results showed that the numbers of PCR bands ranged
from 9 to 13 (size range: 200–1300 bp).
(GACA)4-Based PCR of M. canis strains revealed the most
complex profiles, with up to 11 bands, ranging from 170
to 1200 bp in size. There was no interspecies variation
among M. canis isolates, all of which had the same band
pattern (Fig. 10).
(GACA)4-Based PCR for T. rubrum showed 5–12 bands
ranging from 200 to 2000. The result for E. floccosum
showed bands ranging from 50 to 1500 bp. T. mentagro-
phytes showed different bands ranging from 50 to 800 bp
(Fig. 11). T. violaceum gave nearly the same pattern, with
PCR bands ranging from 400 to 2599 bp.

Figure 11. DNA sequencing

The query sequences were compared with those in
GenBank database by Blast analysis. Whereas three of the
representative isolates (M. canis, T. violaceum, and
T. rubrum) were found to be identical in 97, 99, and
99% of cases to the same species in GenBank, the fourth
isolate formerly identified as T. rubrum was found to be
identical (99%) to Trichophyton raubitschekii, differing in
only two bases, in nucleotides 150 and 160 (Table 2
and Fig. 12).

Discussion
Conventional methods for dermatophyte identification
are based on the detection of fungal elements by direct
microscopy of clinical specimens, combined with culture.
The aim of the present study was to compare between
conventional and molecular methods for identification of
dermatophyte isolates obtained from human superficial
Agarose gel electrophoresis of DNA products of dermatophyte isolates
using (GACA)4. Lane 1: molecular weight marker; lanes 2 and 3:
skin infection.
Epidermophyton floccosum; lanes 4 and 5: T. mentagrophytes.
Fifty-five human samples were subjected to mycological
examination. Whereas direct microscopic examination

Table 2. Sequencing of four dermatophyte isolates related to four species

Maximum Total Maximum
Serial nos Description score score identified (%)

1 Forward Trichophyton violaceum isolate UOA/HCPF13250 18S ribosomal RNA gene, partial sequence; 304 356 97
ITS1, 5.8S ribosomal RNA gene, and ITS2, complete sequence; and 28S ribosomal RNA gene,
partial sequence
Reverse T. violaceum isolate UOA/HCPF13250 18S ribosomal RNA gene, partial sequence; ITS1, 5.8S 304 401 94
ribosomal RNA gene, and ITS2, complete sequence; and 28S ribosomal RNA gene, partial
sequence
2 Forward Microsporum canis strain ATCC MYA4605 18S ribosomal RNA gene, partial sequence; ITS1, 5.8S 880 880 99
ribosomal RNA gene, and ITS2, complete sequence; and 28S ribosomal RNA gene, partial
sequence
Reverse M. canis strain ATCC MYA4605 18S ribosomal RNA gene, partial sequence; ITS1, 5.8S ribosomal 1227 1227 98
RNA gene, and ITS2, complete sequence; and 28S ribosomal RNA gene, partial sequence
3 Forward Trichophyton rubrum 5.8 ribosomal RNA gene and ITS1 and ITS2 DNA (strain CBS392.58) 1085 1488 96
Reverse T. rubrum 5.8 ribosomal RNA gene and ITS1 and ITS2 DNA (strain CBS392.58) 1184 1184 99
4 Forward Trichophyton raubitschekii strain BMU04349 18S ribosomal RNA gene, partial sequence; ITS1, 1134 1134 99
5.8S ribosomal RNA gene, and ITS2, complete sequence; and 28S ribosomal RNA gene, partial
sequence
Reverse T. raubitschekii strain BMU04349 18S ribosomal RNA gene, partial sequence; ITS1, 5.8S ribosomal 1134 1134 99
RNA gene, and ITS2, complete sequence; and 28S ribosomal RNA gene, partial sequence
ITS1, internal transcribed spacer 1; ITS2, internal transcribed spacer 2.

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Figure 12.
The two different bases in nucleotides 150 and 160 in the alignment of internal transcribed spacer sequences of Trichophyton raubitschekii
KX228395 with published sequence in GenBank by online Blast search. Dots indicate nucleotide positions identical to the corresponding T.
raubitschekii sequence. Nucleotide positions conserved in all sequences are designated by asterisks. Numbers refer to the nucleotide positions in
the T. raubitschekii sequence.
(KOH) was positive in 92.72% of cases, culture was
positive in 87.27%. These results are in agreement with
those of others [13,18] who attributed the higher
positivity of KOH to the fact that the cases were under
treatment, the culture was contaminated by rapidly
growing fungi giving no chance for slowly growing
dermatophytes to appear, and the use of unsuitable
media for certain dermatophytes, which need higher
nutritional requirements. In general, KOH and culture
complete each other, and, although direct microscopic
examination showed higher sensitivity, culture showed
higher specificity, as reported by Levitt et al. [19].
Comparison between the four media – dermasel agar,
DTM, InTray DM, and Derm-Deut – showed that
dermasel and DTM succeeded in the isolation of 48
dermatophyte isolates from a total of 55 specimens,
whereas InTray DM and Derm-Deut helped isolate 38
and 32 dermatophytes, respectively. The reason why the
two readily prepared media failed to identify all dermato-
phyte isolates may be that the medium was subjected to
dryness before the growth of slow-growing dermatophytes
as T. violaceum and the plates of Derm-Deut were rapidly
subjected to contamination by nondermatophyte molds.
These coincide with the observations of Robert and
Pihet [20], who found that the usefulness in office culture
systems is still a matter of debate.
Phenotypic identification of dermatophyte species relies
on the macromorphology of colonies (rate of growth,
texture, and color of the surface and the reverse side), and
micromorphology (presence and characters of macroconi-
dia and microconidia as well as modification of hyphae –
e.g. spirals, nodular organs, favic chandeliers, and pecti-
nate bodies). If identification is not reached, biochemical
tests such as urease, nutritional requirements, and in-vitro
hair penetration will help in its identification [21].
Besides RG medium, which is used for differentiation of
M. canis from Microsporum audouinii [14], and BCP
medium [15] for differentiation of T. mentagrophytes from
T. rubrum, other differential media were also propa-
gated [12,13,22,23] for differentiation between dermato-
phytes with ambiguous morphological and physiological
characters.
In the present work the identification of 48 isolates
obtained from human cases by macromorphology, micro-
morphology, and culture on four differential media (RG,
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Species identification of dermatophytes Taha et al. 83
LA, BCP, and MHB) revealed 15 isolates of M. canis, 12 of
T. violaceum, 12 of T. rubrum, 5 of E. floccosum, and 4 of T.
mentagrophytes. These results, although in accordance with
other studies [13,24] in which M. canis was the
predominant dermatophyte isolated in the last few years
in Egypt followed by T. violaceum and T. rubrum, differs
from others [25,26], which isolated T. violaceum as the
predominant dermatophyte followed by M. canis in tinea
capitis and tinea corporis. This variation in results may be
due to the differences in study location.
Nucleic acid-based techniques rely on genotypic differ-
ences, and are more precise than those based on
phenotypic features [27]. Recently, a number of methods
have been reported for molecular identification of
dermatophytes. In the present study three methods were
used: first, PCR for amplification of ITS1 and ITS4,
followed by RFLP using MvaI for 19 isolates of
dermatophyte formerly identified by phenotypic meth-
ods; second, application of PCR using single repetitive
oligonucleotides [(GACA)4] for the same 19 dermato-
phyte isolates identified by phenotypic and RFLP
methods; and lastly DNA sequencing, which was done
only for four representative isolates.
The amplified products using universal primers ITS1 and
ITS4 from T. violaceum, T. rubrum, T. mentagrophytes, and E.
floccosum were found at 690 bp, whereas M. canis was at
740 bp. These results are identical to those of other
studies [17].
MvaI digestion of amplified products in the first step
revealed a unique restriction pattern. Analysis of the
number and size of patterns identified the nineteen
dermatophyte isolates examined as species typically
detected by the phenotypic method. The results of the
current work by RFLP coincide with those of Jackson
et al. [28], who found that PCR-RFLP of the ITS region is
an easy method for identifying dermatophyte isolates.
In the current study all isolates identified by (GACA)4-
based PCR was identical to those recognized by the
phenotypic and PCR-RFLP methods. The present study
is in agreement with that by Roque et al. [29] and Liu
et al. [30], who found that repetitive primer (GACA)4 was
able to differentiate all dermatophytes into species. A
comparison of the two methods revealed that RFLP is
complex, needing much effort and time, whereas the
(GACA)4 method is simple and rapid.
84 Journal of the Egyptian Women’s Dermatologic Society
On the other hand, four dermatophyte isolates [M. canis
(n = 1), T. violaceum (n = 1), and T. rubrum (n = 2)]
formerly identified by RFLP and repetitive primer
(GACA)4 were sent for sequencing. The data were
analyzed by DNA software and compared with those in
GenBank. Whereas M. canis, T. violaceum, and one isolate of
T. rubrum were found to be identical, the other isolate of
T. rubrum obtained from a case of tinea corporis was found
to be identical (99%) for the sequence of T. raubitschekii,
a dermatophyte considered atypical and confused with
T. rubrum and rarely isolated from people who live or
travel to the Mediterranean area [31].
Although sequencing provides a very accurate and useful
method for the identification of dermatophytes, it is
highly expensive to be used in routine identification.
Therefore, it is recommended for identifying atypical
isolates of dermatophytes [32,33].
In conclusion, although the current study revealed the
capacity of phenotypic (morphological and differential
media) and molecular (RFLP and repetitive primer)
methods to classify dermatophytes into species, molecu-
lar methods are rapid and represent technological
advancements in laboratory diagnosis. DNA sequencing
proved to be more accurate and detected variants and
subtypes, although it is expensive and face some
problems in application in our country. Therefore, we
recommended its use only for identification of dermato-
phytes in atypical isolates.
Conflicts of interest
There are no conflicts of interest.
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