Coelomycetous Fungi in the Clinical Setting: Morphological Convergence and Cryptic Diversity

Abstract

Human infections by coelomycetous fungi are becoming more frequent and range from superficial to systemic dissemination. Traumatic implantation of contaminated plant material is the most common cause. The typical morphological feature of these fungi is the production of asexual spores (conidia) within fruiting bodies called conidiomata. This study aimed to determine the distribution of the coelomycetes in clinical samples by a phenotypic and molecular study of a large set of isolates received from a USA reference mycological institution and by obtaining the in vitro antifungal susceptibility pattern of a selected group of strains against nine antifungals. A total of 230 isolates were identified by sequencing the D1 and D2 domains of the LSU nrRNA gene and by morphological characterization. Eleven orders of the phylum Ascomycota were identified: Pleosporales (the largest group; 66.1%), Botryosphaeriales (19.57%), Glomerellales (4.35%), Diaporthales (3.48%), Xylariales (2.17%), Hysteriales and Valsariales (0.87%), and Capnodiales , Helotiales , Hypocreales and Magnaporthales (0.43% each one). The most prevalent species were Neoscytalidium dimidiatum , Paraconiothyrium spp., Phoma herbarum , Didymella heteroderae and Epicoccum sorghinum . The most common anatomical site of isolation was superficial tissue (66.5%), followed by the respiratory tract (17.4%). Most of the isolates tested were susceptible to the majority of antifungals and only flucytosine showed poor antifungal activity.

Full text

Coelomycetous Fungi in the Clinical
Setting: Morphological Convergence and
Cryptic Diversity
Nicomedes Valenzuela-Lopez,
a,b
Deanna A. Sutton,
c
José F. Cano-Lira,
a
Katihuska Paredes,
a
Nathan Wiederhold,
c
Josep Guarro,
a
Alberto M. Stchigel
a
Unitat de Micologia, Facultat de Medicina i Ciències de la Salut and IISPV, Universitat Rovira i Virgili, Reus,
Spain
a
; Microbiology Unit, Medical Technology Department, Faculty of Health Science, University of
Antofagasta, Antofagasta, Chile
b
; Fungus Testing Laboratory, University of Texas Health Science Center, San
Antonio, Texas, USA
c
ABSTRACT Human infections by coelomycetous fungi are becoming more frequent
and range from superficial to systemic dissemination. Traumatic implantation of con-
taminated plant material is the most common cause. The typical morphological fea-
ture of these fungi is the production of asexual spores (conidia) within fruiting bod-
ies called conidiomata. This study aimed to determine the distribution of the
coelomycetes in clinical samples by a phenotypic and molecular study of a large set
of isolates received from a U.S. reference mycological institution and by obtaining
the in vitro antifungal susceptibility pattern of nine antifungals against a selected
group of isolates. A total of 230 isolates were identified by sequencing the D1 and
D2 domains of the large subunit (LSU) nuclear ribosomal RNA (nrRNA) gene and by
morphological characterization. Eleven orders of the phylum Ascomycota were iden-
tified: Pleosporales (the largest group; 66.1%), Botryosphaeriales (19.57%), Glomerel-
lales (4.35%), Diaporthales (3.48%), Xylariales (2.17%), Hysteriales and Valsariales
(0.87%), and Capnodiales,Helotiales,Hypocreales and Magnaporthales (0.43% each).
The most prevalent species were Neoscytalidium dimidiatum,Paraconiothyrium spp.,
Phoma herbarum,Didymella heteroderae, and Epicoccum sorghinum. The most com-
mon anatomical site of isolation was superficial tissue (66.5%), followed by the respi-
ratory tract (17.4%). Most of the isolates tested were susceptible to the majority of
antifungals, and only flucytosine showed poor antifungal activity.
KEYWORDS Colletotrichum, coelomycetous fungi, coelomycetes, mycosis,
Neoscytalidium,Phoma,Pyrenochaeta, antifungal susceptibility
The coelomycetous fungi constitute a large number of taxa characterized by the
production of conidia (asexual propagules) within a cavity lined by fungal or host
tissue, called conidiomata (1), and although the majority of the human-opportunistic
infections are caused by fungi producing conidia on conidiophores (modified hyphae,
with one or more conidiogenous cells, which develop free on the substrate), a signif-
icant number of mycoses are produced by coelomycetous fungi (2–4). Coelomycetous
fungi are mostly saprobic and parasites of terrestrial vascular plants, but they can also
infect vertebrates and other fungi. They are ubiquitous in soil, in salty and freshwater
environments, and in sewage (4). Although the term Coelomycetes is still occasionally
used to refer to these fungi, this name is obsolete and is currently considered to refer
to an artificial fungal class. The class Coelomycetes is defined in terms of the morpho-
logical characterization of the asexual reproductive structures and considers the type
and the shape of their conidiomata and the ontogeny of their conidia as the most
useful characteristics (5, 6); the class has traditionally been divided into the orders
Melanconiales and Sphaeropsidales, depending upon the production of either acervular
Received 2 November 2016 Returned for
modification 21 November 2016 Accepted
29 November 2016
Accepted manuscript posted online 7
December 2016
Citation Valenzuela-Lopez N, Sutton DA,
Cano-Lira JF, Paredes K, Wiederhold N,
Guarro J, Stchigel AM. 2017. Coelomycetous
fungi in the clinical setting: morphological
convergence and cryptic diversity. J Clin
Microbiol 55:552–567. https://doi.org/
10.1128/JCM.02221-16.
Editor David W. Warnock, University of
Manchester
Copyright © 2017 American Society for
Microbiology. All Rights Reserved.
Address correspondence to José F. Cano-Lira,
jose.cano@urv.cat.
MYCOLOGY
crossm
February 2017 Volume 55 Issue 2 jcm.asm.org 552Journal of Clinical Microbiology

(cup-shaped) and pycnidial (globose to pyriform) conidiomata, respectively, and the
Pycnothyriales, characterized by the production of pycnothyrial (shield-shaped, flat-
tened, or hemispherical) conidiomata (5, 6). However, molecular studies have demon-
strated that the taxonomy of the Coelomycetes, represented by nearly 1,000 genera and
7,000 species (1), is artificial. Recent studies, have distributed the coelomycetes into at
least three classes of the phylum Ascomycota, i.e., Dothideomycetes,Leotiomycetes, and
Sordariomycetes (7–9).
Infections by coelomycetous fungi are mostly acquired by traumatic implantation of
plant/woody material or soil particles contaminated by their conidia rather than by
inhalation of air-dispersed propagules (2, 4). The coelomycetes are responsible for a
large variety of clinical entities, such as dermatitis, onychomycosis, keratitis, endoph-
thalmitis, subcutaneous phaeohyphomycosis, cysts, mycetoma, sinusitis, osteomyelitis,
bursitis, brain abscesses, and disseminated infections (4). The appropriate treatment of
the infections produced by these fungi is unknown, mainly due to the wide spectrum
of taxa involved and to the difficulties in their identification when the typical repro-
ductive structures are not produced. However, the European Society of Clinical Micro-
biology and Infectious Diseases (ESCMID) and the European Conference of Medical
Mycology (ECMM) have provided joint clinical guidelines for the management of
phaeohyphomycosis, with some recommendations for the treatment of infections due
to the most usual genera of coelomycetes, such as Neoscytalidium,Phoma, and Pyr-
enochaeta, mainly based on the use of amphotericin B and triazoles (10).
For the reasons mentioned above, the spectrum of species of these fungi in the
clinical setting is practically unknown (4, 11). Therefore, the objective of this study has
been to determine the distribution pattern of the coelomycetous fungi isolated from
clinical specimens from the United States using molecular identification of a large set
of isolates based on the sequencing of the D1 and D2 (D1-D2) domains of the large
subunit (LSU) of the nuclear ribosomal RNA (nrRNA) gene. In addition, we have
characterized those isolates morphologically and determined the antifungal suscepti-
bility of a representative number of them to nine antifungal drugs.
RESULTS
A total of 86 (38%) isolates of the 230 studied were able to produce pycnidial
conidiomata; 10 (4%) developed acervuli, and 35 (15%) produced the typical ana-
morphs of Neoscytalidium. The other 99 isolates (43%) remained sterile. The most
common species was Neoscytalidium dimidiatum, representing 15% (35/230) of the
isolates, followed by Paraconiothyrium cyclothyrioides with 7% (16/230), and both were
isolated mostly from superficial tissues. The third most common taxon recovered was
Total=230
19.57% Botryosphaeriales
0.43% Capnodiales
3.48% Diaporthales
4.35% Glomerellales
0.43% Helotiales
0.43% Hypocreales
0.87% Hysteriales
0.87% incertae sed is
0.43% Magnaporthales
66.10% Pleosporales
0.87% Valsariales
2.17% Xylariales
1
2
3
4
5
6
7
8
9
10
11 12
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
FIG 1 Distribution, by orders, of coelomycetous fungus isolates from clinical samples.
Coelomycetous Fungi of Clinical Origin Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 553

UTHSC DI16-306 (LN907449)
UTHSC DI16-307 (LN907450)
UTHSC DI16-302 (LN907445)
UTHSC DI16-294 (LN907437)
UTHSC DI16-282 (LN907425)
UTHSC DI16-255 (LN907398)
UTHSC DI16-228 (LN907371)
UTHSC DI16-212 (LN907355)
UTHSC DI16-205 (LN907348)
UTHSC DI16-204 (LN907347)
UTHSC DI16-200 (LN907343)
UTHSC DI16-199 (LN907342)
UTHSC DI16-308 (LN907451)
UTHSC DI16-319 (LN907462)
UTHSC DI16-365 (LN907508)
Phoma herbarum CBS 615.75 (EU754186)
UTHSC DI16-276 (LN880537)
Leptosphaeruli na australi s CBS 317.83 (EU754166)
UTHSC DI16-249 (LN907392)
UTHSC DI16-233 (LN907376)
UTHSC DI16-322 (LN907465)
UTHSC DI16-285 (LN907428)
UTHSC DI16-230 (LN907373)
UTHSC DI16-244 (LN907387)
UTHSC DI16-258 (LN907401)
UTHSC DI16-271 (LN907414)
UTHSC DI16-272 (LN907415)
UTHSC DI16-278 (LN907421)
UTHSC DI16-299 (LN907442)
UTHSC DI16-345 (LN907488)
Epicoc cum s orghinum CBS 179.80 (GU237978)
UTHSC DI16-338 (LN907481)
UTHSC DI16-301 (LN907444)
UTHSC DI16-288 (LN907431)
UTHSC DI16-280 (LN907423)
UTHSC DI16-257 (LN907400)
UTHSC DI16-206 (LN907349)
UTHSC DI16-202 (LN907345)
UTHSC DI16-201 (LN907344)
UTHSC DI16-197 (LN907340)
Epicoc cum ni grum CBS 173. 73T (GU237975)
UTHSC DI16-190 (LN907333)
UTHSC DI16-211 (LN907354)
UTHSC DI16-224 (LN907367)
UTHSC DI16-226 (LN907369)
UTHSC DI16-227 (LN907370)
UTHSC DI16-231 (LN907374)
UTHSC DI16-232 (LN907375)
UTHSC DI16-234 (LN907377)
UTHSC DI16-235 (LN907378)
UTHSC DI16-274 (LN907417)
UTHSC DI16-295 (LN907438)
UTHSC DI16-305 (LN907448)
Didymella heteroderae CBS 109.92T (GU238002)
UTHSC DI16-270 (LN907413)
1/72
0.99/76
0.05
0.99/-
0.98/-
sela
r
op
s
o
el
P
0.96/-
UTHSC DI16-291 (LN907434)
Asc ochyt a hordei var. hordei CBS 544.74 (E U754134)
UTHSC DI16-207 (LN907350)
UTHSC DI16-320 (LN907463)
UTHSC DI16-332 (LN907475)
UTHSC DI16-341 (LN907484)
UTHSC DI16-352 (LN907495)
UTHSC DI16-359 (LN907502)
UTHSC DI16-209 (LN907352)
Paraphoma radic ina CBS 111. 79T (KF251676)
UTHSC DI16-210 (LN907353)
Trematophoma sp. CBS 157.86 ( EU754221)
UTHSC DI16-296 (LN907439)
Paraphoma fimet i CBS 170. 70T (KF251674)
UTHSC DI16-324 (LN907467)
UTHSC DI16-260 (LN907403)
UTHSC DI16-264 (LN907407)
Edenia gomez pompae CBS 124106T (FJ839654)
Pleos pora herbarum CBS 191.86T (GU238160)
-/86
0.99/94
1/95
1/88
0.98/92
-/86
0.97/-
Neoascochyta desmazieri CBS 297.69T (KT389726)
Phoma clade I
Neoascochyta clade
Paraphoma clade
Pleospora clade
Didymella clade
Phoma clade II
FIG 2 Maximum-likelihood tree obtained from the D1-D2 of LSU (555 bp) sequences of the 322 strains, where 92 strains are
type or reference strains. In the tree, the branch lengths are proportional to phylogenetic distance. Bayesian posterior
probability scores of ⱖ0.95 and bootstrap support values of ⱖ70 are indicated on the nodes. The GenBank accession numbers
are given in parentheses. Saccharomyces castellii and S. cerevisiae were used to root the tree. The type (indicated by a
superscript T) and reference strains are shown in bold type.
Valenzuela-Lopez et al. Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 554

Phoma herbarum (6.5%, 15/230) from superficial and respiratory tract specimens,
followed by Didymella heteroderae (5%, 12/230) and Epicoccum sorghinum (4%, 10/230),
which were isolated from superficial tissues.
In the D1-D2 phylogenetic analysis, the isolates were distributed into 11 orders (Fig.
1), most of which belonged to the Pleosporales (66.1%) and the Botryosphaeriales
UTHSC DI16-229 (LN907372)
Pyrenoc haeta unguis -hominis CBS 378 .92 (GQ387621)
UTHSC DI16-213 (LN907356)
UTHSC DI16-273 (LN907416)
Coniothyri um palmarum CB S 400. 71 (JX681084)
Pyrenoc haeta nobili s CBS 407. 76T (EU754206)
Leptosphaeria rubefac iens CBS 387.80 (JF740311)
UTHSC DI16-192 (LN907335)
UTHSC DI16-290 (LN907433)
Leptosphaeria et heridgei CBS 125980 (JF740291)
UTHSC DI16-238 (LN907381)
UTHSC DI16-203 (LN907346)
UTHSC DI16-189 (LN907332)
UTHSC DI16-236 (LN907379)
Coniothyri um telephii CBS 18 8.71 (GQ387599)
UTHSC DI16-313 (LN907456)
Diederichomy ces c ladoniic ola CBS 128025 (JQ238625)
UTHSC DI16-191 (LN907334)
UTHSC DI16-337 (LN907480)
UTHSC DI16-283 (LN907426)
Neosetophom a samarorum CBS 138.96T (GQ387578)
UTHSC DI16-240 (LN907383)
UTHSC DI16-325 (LN907468)
UTHSC DI16-330 (LN907473)
UTHSC DI16-336 (LN907479)
UTHSC DI16-339 (LN907482)
Parast agonospora nodorum CBS 259.49 (KF251688)
Phaeosphaeria ory zae CBS 110110T (KF251689)
Phaeosphaeria papay ae S528 (KF251690)
Phaeosphaeriops is m usae CBS 120026 (DQ885894)
UTHSC DI16-303 (LN907446)
UTHSC DI16-297 (LN907440)
UTHSC DI16-298 (LN907441)
UTHSC DI10-289 (LN907432)
UTHSC DI16-193 (LN907336)
UTHSC DI16-198 (LN907341)
UTHSC DI16-225 (LN907368)
UTHSC DI16-275 (LN907418)
UTHSC DI16-277 (LN907420)
Pyrenoc haetopsi s leptos pora CBS 101635T (GQ387627)
0.99/84
1/99
0.99/82
1/-
1/-
Medicopsis clade
Acrocalymma clade
Pyrenochaetopsis clade
Coniothyrium clade
al
l
e
o
s
s
u
oR e
dalc
Biatriospora
clade
Trematosphaeria
clade
Keissleriella clade
flavescens clade
Camarographium
clade
Phaeosphaeria clade
Neosetophoma italica MFLU 14 C0809 (KP711361)
Pyrenochaetopsis decipiens CBS 343.85T (GQ387624)
Pyrenochaetopsis indica CBS 124454T (GQ387626)
0.98/84
0.05
selaropsoelP
UTHSC DI16-195 (LN907338)
Acrocalymma walkeri CBS 257. 93T (FJ795454)
UTHSC DI16-315 (LN907458)
Medicops is rom eroi CBS 252.60T (EU754207)
UTHSC DI16-242 (LN907385)
UTHSC DI16-309 (LN907452)
UTHSC DI16-310 (LN907453) UTHSC DI16-220 (LN907363)
UTHSC DI16-356 (LN907499)
Arthopyrenia salicis CBS 368. 94 (AY538339)
UTHSC DI16-362 (LN907505)
UTHSC DI16-334 (LN907477)
Roussoel la nitidula MFLUCC 11 0182 (KJ474843)
Roussoella hysterioides CBS 546.94T (KF443381)
UTHSC DI16-269 (LN907412)
UTHSC DI16-292 (LN907435)
UTHSC DI16-300 (LN907443)
UTHSC DI16-360 (LN907503)
Roussoel la percutanea CB S 868.95 (KF 366449)
UTHSC DI16-342 (LN907485)
UTHSC DI16-241 (LN907384)
Biatri ospora mack innonii CB S 674.75T (GQ387613)
Biat riospora m arina CY 1 228 (GQ925848)
UTHSC DI16-335 (LN907478)
Trematosphaeria pert usa CBS 122368T (FJ201990)
UTHSC DI16-281 (LN907424)
UTHSC DI16-237 (LN907380)
UTHSC DI16-286 (LN907429)
UTHSC DI16-354 (LN907497)
Trematosphaeria gris ea CBS 120271 (K F015613)
UTHSC DI16-326 (LN907469)
Keissleriella cladophila CBS 104.55 ( JX681090)
UTHSC DI16-355 (LN907498)
Paraconiot hyrium flavescens CBS 178. 93 (GU238075)
UTHSC DI16-358 (LN907501)
Pseudoc haetosphaeronem a larense CBS 640.73T (KF015611)
UTHSC DI16-361 (LN907504)
Camarographium koreanum CBS 117159T (JQ044451)
1/99
1/94
1/99
-/73
1/99
1/99
1/99
1/99
1/99
1/91
0.95/96
1/-
0.95/-
1/-
FIG 2 (Continued)
Coelomycetous Fungi of Clinical Origin Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 555

UTHSC DI16-208 (LN907351)
Montagnula aloes CBS 132531 (NG042676)
UTHSC DI16-251 (LN907394)
Montagnula opul enta CBS 168. 34 (NG027581)
se
la
ro
psoelP
UTHSC DI16-187 (LN907330)
Lophiostoma heterosporum CBS 644. 86 (AY016369)
Sporormiella minima CBS 524.50 (DQ678056)
UTHSC DI16-253 (LN907396)
Preuss ia longis poropsis 16551-g (GQ203742)
UTHSC DI16-287 (LN907430)
Exos porium stylobat um CBS 160.3 0T (JQ044447)
UTHSC DI16-316 (LN907459)
Anteagloni um parvulum SMH5223 (GQ221909)
UTHSC DI16-343 (LN907486)
Phyllosticta flevolandica CBS 998. 72T (DQ377927)
UTHSC DI16-369 (LN907512)
Myrmaec ium rubric osum CB S 139068 (KP 687885)
UTHSC DI16-368 (LN907511)
UTHSC DI16-254 (LN907397)
Chaetophoma s p. CBS 119963 (E U754143)
UTHSC DI16-353 (LN907496)
UTHSC DI16-318 (LN907461)
Neofusicoc cum andi num CBS 117453 (DQ377914)
UTHSC DI16-221 (LN907364)
Lasiodipl odia parva CBS 456.78T (KF766362)
Lasiodipl odia theobromae CB S 287.47 (DQ377858)
UTHSC DI16-364 (LN907507)
UTHSC DI16-214 (LN907357)
UTHSC DI16-217 (LN907360)
Aplosporella sterculiae CBS 342. 78 (JX681073)
UTHSC DI16-250 (LN907393)
UTHSC DI16-248 (LN907391)
Phaeobotry osphaeria visci CBS 100163 (E U754215)
UTHSC DI16-333 (LN907476)
Botry osphaeria dot hidea CBS 115476 (NG027577)
UTHSC DI16-321 (LN907464)
UTHSC DI16-312 (LN907455)
-/72
1/99
1/74
0.99/91
-/92
1/84
1/99
1/99
1/99
1/99
1/99
1/99
1/96
1/91
1/89
-/96
1/99
1X
1X
1.5X
s
el
ai
reahp
so
y
r
t
oB
1/-
0.99/-
0.99/-
Gloniopsis subrugosa CBS 123346 (FJ161210)
Rhytidhysteron rufulum CBS 306.38 (FJ469672)
0.5X
0.5X
1X
UTHSC DI16-284 (LN907427)
Phaeodothis winteri CB S 182. 58 (GU301857)
1/99
Bimuria novae-zelandiae CBS 107.79 (AY016356)
UTHSC DI16-256 (LN907399)
Valsariales
Hysteriales
0.05
1/63
Letendraea eurotioi des CBS 212.31 (AY787935)
1/69
UTHSC DI16-351 (LN907494)
UTHSC DI16-370 (LN907513)
UTHSC DI16-267 (LN907410)
UTHSC DI16-239 (LN907382)
1/86
UTHSC DI16-266 (LN907409)
UTHSC DI16-348 (LN907491)
Paraconi othyrium brasiliens e CBS 254. 88 (JX496171)
UTHSC DI16-311 (LN907454)
UTHSC DI16-357 (LN907500)
Curreya pity ophila CB S 149. 32 (JX681087)
UTHSC DI16-219 (LN907362)
UTHSC DI16-261 (LN907404)
Paraphaeosphaeria neglecta CB S 124078T (JX496152)
UTHSC DI16-263 (LN907406)
UTHSC DI16-363 (LN907506)
Paraconiothyrium fuckeli i CBS 797.95 (JX496226)
Paraconiothyrium cyc lothy rioides CB S 972.95T (JX496232)
UTHSC DI16-265 (LN907408)
UTHSC DI16-215 (LN907358)
UTHSC DI16-216 (LN907359)
UTHSC DI16-218 (LN907361)
UTHSC DI16-222 (LN907365)
UTHSC DI16-243 (LN907386)
UTHSC DI16-246 (LN907389)
UTHSC DI16-252 (LN907395)
UTHSC DI16-268 (LN907411)
UTHSC DI16-279 (LN907422)
UTHSC DI16-314 (LN907457)
UTHSC DI16-327 (LN907470)
UTHSC DI16-328 (LN907471)
UTHSC DI16-346 (LN907489)
UTHSC DI16-347 (LN907490)
UTHSC DI16-349 (LN907492)
UTHSC DI16-367 (LN907510)
Paraconiothyrium cyc lothy rioides CB S 432.75 (JX496201)
Paraconiothyrium estuarinum CBS 109850T (JX496129)
0.98/97
Didymosphaeriaceae
clade
Lophiostoma
clade
Exosporium
clade
Anteaglonium clade
Phyllosticta clade
Neofusicoccum clade
Lasiodiplodia clade
Aplosporella clade
Phaeobotryosphaeria clade
Botryosphaeria clade
FIG 2 (Continued)
Valenzuela-Lopez et al. Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 556

UTHSC DI14-331 (LN907310)
UTHSC DI14-332 (LN907311)
UTHSC DI14-336 (LN907315)
UTHSC DI14-306 (LN907285)
UTHSC DI14-307 (LN907286)
UTHSC DI14-308 (LN907287)
UTHSC DI14-309 (LN907288)
UTHSC DI14-310 (LN907289)
UTHSC DI14-311 (LN907290)
UTHSC DI14-312 (LN907291)
UTHSC DI14-313 (LN907292)
UTHSC DI14-314 (LN907293)
UTHSC DI14-315 (LN907294)
UTHSC DI14-316 (LN907295)
UTHSC DI14-317 (LN907296)
UTHSC DI14-318 (LN907297)
UTHSC DI14-319 (LN907298)
UTHSC DI14-320 (LN907299)
UTHSC DI14-321 (LN907300)
UTHSC DI14-322 (LN907301)
UTHSC DI14-323 (LN907302)
UTHSC DI14-324 (LN907303)
UTHSC DI14-325 (LN907304)
UTHSC DI14-326 (LN907305)
UTHSC DI14-327 (LN907306)
UTHSC DI14-328 (LN907307)
UTHSC DI14-329 (LN907308)
UTHSC DI14-330 (LN907309)
UTHSC DI14-333 (LN907312)
UTHSC DI14-334 (LN907313)
UTHSC DI14-335 (LN907314)
UTHSC DI14-337 (LN907316)
UTHSC DI14-338 (LN907317)
UTHSC DI14-339 (LN907318)
UTHSC DI14-340 (LN907319)
Neoscytalidium dimidiatum CBS 145. 78T (DQ377922)
UTHSC DI16-304 (LN907447)
Myc oleptodis cus terrest ris CB S 231. 53 (JN711859)
UTHSC DI16-196 (LN907339)
Cadophora fastigiat a DAOM 225754 (JN938877)
UTHSC DI16-245 (LN907388)
Pseudoc ercos pora vitis CBS 132012 (KF902011)
UTHSC DI16-350 (LN907493)
Phomatospora bellami nuta AFTOL-ID 766 (FJ176857)
UTHSC DI16-194 (LN907337)
UTHSC DI16-323 (LN907466)
UTHSC DI16-188 (LN907331)
Diatry pe dis ci formis CBS 197.49 (DQ470964)
Cryptos phaeria eunomia CBS 216.87 (KT425296)
UTHSC DI16-366 (LN907509)
UTHSC DI16-371 (LN907514)
1/99
1/86
1/100
1/96
-/73
1/99
1/99
0.99/81
1/100
1/100
1/100
0.96/75
1/99
2X
1X
2X
3X
1X
1X
4X
se
l
a
ir
ea
hpsoyrt
oB
Magnaporthales
Helotiales
Capnodiales
Incertae sedis
Xylariales
Pseudoc ercos pora oenother ae CBS 131885 (JQ324961)
1/96
0.99/90
Mycoleptodisc us indicus CBS 127677 (GU980697)
1/100
Peroneutypa scoparia MFLUCC 11-0615 (KU863140)
1/100
Protoventuria alpina CBS 140.83 (EU035444)
UTHSC DI16-329 (LN907472)
UTHSC DI16-331 (LN907474)
UTHSC DI16-317 (LN907460)
UTHSC DI16-293 (LN907436)
Diaporthe sclerotioides CBS 477 (A F439631)
UTHSC DI16-247 (LN907390)
UTHSC DI16-262 (LN907405)
UTHSC DI16-259 (LN907402)
UTHSC DI16-340 (LN907483)
Valsa ambiens CBS 1 09491 (EU255208)
UTHSC DI16-223 (LN907366)
Phialem oniopsis curvata CBS 490. 82T (KJ573448)
Phialem oniopsis ocularis CBS 110031 (K J573449)
UTHSC DI16-344 (LN907487)
Thyronec tria aus troameric ana A.R. 2808 (GQ505988)
UTHSC DI14-254 (LN907329)
Colletot richum gl oeosporioides CBS 79672 (AY 705727)
UTHSC DI14-250 (LN907325)
UTHSC DI14-248 (LN907323)
UTHSC DI14-245 (LN907320)
UTHSC DI14-249 (LN907324)
UTHSC DI14-246 (LN907321)
UTHSC DI14-252 (LN907327)
Colletot richum t runcatum CBS 112998 (JN940817)
UTHSC DI14-251 (LN907326)
UTHSC DI14-247 (LN907322)
Colletotrichum torulosum CBS 102667 (DQ286173)
UTHSC DI14-253 (LN907328)
Colletotrichum spaethianum CBS 101631 (JN940810)
Saccharomyces castellii NRRL Y 12630 (A Y048167)
Saccharomyces cerevisiae NRRL Y 12632 (AY 048154)
1/99
1/99
-/96
1/99
1/99
1/99
-/94
1/99
0.99/98
1/98
-/75
1/99
1/99
1/99
0.05
3X
1X
1X
2X
2X
se
l
a
h
trop
a
i
D
Incertae sedis
Hypocreales
s
e
la
l
l
er
e
molG
OUT GROUP
Saccharomycetales
1/-
Diatrype
clade
Peroneutypa
clade
Diaporthe
clade
Valsa
clade
Neoscytalidium
clade
Phomatospora
clade
Phialemoniopsis
clade
FIG 2 (Continued)
Coelomycetous Fungi of Clinical Origin Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 557

(19.57%), followed by the Glomerellales (4.35%), Diaporthales (3.48%), Xylariales (2.17%),
and Hysteriales and Valsariales (0.87% each). The orders Capnodiales,Helotiales,Hypo-
creales, and Magnaporthales were represented by only one isolate each (0.43%), and the
other isolates (0.87%) were incertae sedis (of uncertain taxonomic position).
Figure 2 shows the phylogenetic tree inferred from the analysis of 322 D1-D2
sequences corresponding to our set of isolates and numerous selected type or refer-
ence strains phylogenetically related to them. As mentioned above, the Pleosporales
contained the largest number of isolates (n⫽152), which were distributed into 22
clades and belonged, probably, to 61 species of 44 different genera. These clades have
been named according to the first taxon historically described.
Within the Pleosporales,Phoma clade I (phylogenetically not supported) included 27
isolates, distributed mainly in two genera: Leptosphaerulina, with four isolates charac-
terized by a phoma-like asexual morph, which clustered with a reference strain of
Leptosphaerulina australis, and Phoma, with 15 isolates placed close to a reference strain
of Phoma herbarum and morphologically characterized by producing pycnidia and
hyaline aseptate conidia. The taxonomic position of the other eight isolates of this clade
was unresolved; in fact, they formed a separate, unsupported sister clade and displayed
a phoma-like asexual morph. The University of Texas Health Science Center (UTHSC)
isolate DI16-270 also showed the typical morphology of Phoma (Phoma clade II) but has
been placed phylogenetically distant from the mentioned genera and probably be-
longs to a new genus.
The Didymella clade included 22 isolates, 10 of which clustered with a reference
strain of Epicoccum sorghinum; unfortunately, the morphological features of these
isolates could not be studied because they produced only sterile mycelia in all the
culture media tested. Twelve isolates grouped with the type strain of Didymella
heteroderae, producing a phoma-like asexual morph, but were particularly character-
ized by the production of chlamydospores in long chains.
The Neoascochyta clade included seven isolates, six clustering with the type strain of
Neascochyta desmazieri and another one placed together with a reference strain of
Ascochyta hordei var. hordei. Morphologically, the species of this clade are mainly
characterized by the production of one-septate conidia that vary in size.
The Paraphoma clade contained only one isolate, which showed an identical
sequence to the type strain of Paraphoma radicina and morphologically was charac-
terized by setose (covered with bristle-like structures) pycnidia and hyaline aseptate
conidia.
The Pleospora clade was made up of five isolates and, with the exception of one of
them, was distributed into three well-supported sister clades corresponding to the
genera Edenia,Paraphoma, and Trematophoma. The isolate that clustered with the type
strain of Paraphoma fimeti was separate from the type species of Paraphoma (Para-
phoma radicina) and showed glabrous pycnidia instead the setose pycnidia produced
by the rest of the species. Interestingly, instead of the ellipsoidal, subhyaline conidia
typical of Edenia spp., the isolate UTHSC DI16-324 produced fusiform, hyaline, two- to
three-septate conidia that are probably indicative of a new genus. The other isolates of
this clade remained sterile.
The Coniothyrium clade included nine isolates, and its topology shows that the
genera Coniothyrium,Leptosphaeria, and Pyrenochaeta are clearly polyphyletic using
this conserved marker. Three of these isolates formed a well-supported sister clade
together with a reference strain of Coniothyrium telephii, which is characterized by
setose pycnidia. The other six isolates were distributed into the genera Leptosphaeria
and Pyrenochaeta. These had a pyrenochaeta-like anamorph, producing conidiophores
within pycnidia and hyaline aseptate conidia.
The Phaeosphaeria clade grouped nine isolates, with four of them clustering with
Neosetophoma and producing confluent pycnidia and small hyaline conidia. The other
five isolates were associated with the genera Diederichomyces,Parastagonospora,Pha-
eosphaeria, and Phaeosphaeriopsis. Only one isolate (UTHSC DI16-325), morphologically
Valenzuela-Lopez et al. Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 558

resembling Phaeosphaeriopsis spp., was able to sporulate, displaying small conidio-
phores within pycnidia and one-septate, pigmented, variable-in-shape conidia.
The Pyrenochaetopsis clade included nine isolates, with four of them matching the
type strain of Pyrenochaetopsis leptospora, another four isolates forming a supported
sister clade separate from P. leptospora, and one not clustering to any of the type strains
included in the analysis. All of the isolates displayed the typical phoma-like morphol-
ogy, i.e., glabrous pycnidia and hyaline aseptate conidia, instead of setose pycnidia of
the genus Pyrenochaetopsis.
The five isolates assigned to the Acrocalymma and Medicopsis clades were grouped
with the type strains of Acrocalymma walkeri and Medicopsis romeroi, respectively, but
differed in 4.5% of the nucleotide sequences of the respective strains of reference.
These isolates remained sterile throughout.
The Roussoella clade was made up of eight isolates, two of which were associated
with a supposed reference strain of Arthopyrenia salicis (CBS 368.94), whose correct
identification was questioned by Liu et al. (12), and the remaining ones were associated
with Roussoella spp.; only three isolates were able to sporulate and had a morphology
similar to that of this genus, i.e., production of glabrous pycnidia and pigmented
aseptate conidia.
Two isolates nested in the Biatriospora clade but remained sterile. The Trematospha-
eria clade comprised five sterile isolates, two of which were phylogenetically related
with the type strain of Trematosphaeria pertusa and the rest of which were associated
with a reference strain of Trematosphaeria grisea.
The Keissleriella clade had only one isolate, which showed a phoma-like morphology
and clustered with a reference strain of Keissleriella cladophila. Another isolate was
associated with a reference strain of Paraconiothyrium flavescens and displayed a
morphology similar to that of Paraconiothyrium (pycnidia, phialidic conidiogenous cells,
and pigmented aseptate conidia); however, the taxonomic placement of that isolate
remains doubtful because it grouped phylogenetically distant from the type species of
the genus (Paraconiothyrium estuarinum). In the Camarographium clade, two sterile
isolates were located that were related to the genera Camarographium and
Pseudochaetosphaeronema.
The Didymosphaeriaceae clade comprised 33 isolates, of which 22 were phyloge-
netically related to Paraconiothyrium spp., 2 were related to Montagnula spp., and 2
were related to the type strain of Paraphaeosphaeria neglecta. Three isolates were
distributed into each of the genera Bimuria,Curreya, and Phaeodothis, and four isolates
formed a well-supported monophyletic sister clade separated from any known taxa of
the family. Only three isolates (UTHSC DI16-261, UTHSC DI16-266, and UTHSC DI16-363)
were able to sporulate, showing glabrous pycnidia and pale brown conidia displaying
morphological features similar to those of Paraconiothyrium spp. The Exosporium clade
comprised only two sterile isolates, one of which was related to the genus Preussia
while the other was related to Exosporium. The Anteaglonium,Lophiostoma, and
Phyllosticta clades comprised only one sterile isolate each one.
In the Valsariales clade, two isolates matched a reference strain of Myrmaecium
rubricosum. These isolates were characterized by producing free, well-differentiated
conidiophores instead of simply conidiogenous cells (phialides) inside the pycnidia.
The Hysteriales clade contained two sterile isolates, one related to an unidentified
strain of Chaetophoma and one of uncertain taxonomical placement but phylogeneti-
cally related to Chaetophoma,Gloniopsis, and Rhytidhysteron.
The second largest clade, corresponding to the order Botryosphaeriales, included 45
isolates distributed in six clades but mostly concentrated into the Neoscytalidium clade.
The fungi included in these clades were characterized by the production of stromatic
conidiomata (a hard, compact mass of cells or of vegetative hyphae), holoblastic
instead of phialidic conidiogenous cells, and aseptate, hyaline to brown, thick-walled
conidia. The Botryosphaeriales included the genera Botryosphaeria (three isolates),
Lasiodiplodia (two isolates), Neofusicoccum (one isolate), Aplosporella (two isolates), and
Phaeobotryosphaeria (two isolates). Additionally, 35 isolates of Neoscytalidium dimidia-
Coelomycetous Fungi of Clinical Origin Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 559

tum were also placed in this order. This fungus is characterized typically by the
production of holoarthric conidia (formed by disarticulation of the preexisting hyphae)
in chains.
The Capnodiales,Helotiales, and Magnaporthales clades each included only one
sterile isolate. Only the isolate of the Helotiales was not phylogenetically related to
any previously known described species. The isolate of the Capnodiales was closely
related to a reference strain of Pseudocercospora oenotherae. This genus is charac-
terized by producing stromata in the (plant) host, subhyaline to brown conidio-
phores, and small or large, subhyaline to brown conidia; unfortunately, our isolate
failed to sporulate. In the Magnaporthales, the isolate matched a reference strain of
Mycoleptodiscus indicus. This genus is characterized by producing sporodochia (a
cushion-like, densely aggregated group of conidiophores) and curved conidia; in
this case, the morphological study was not possible due to the absence of sporu-
lation of the isolate.
The Xylariales clade included five sterile isolates, three of which were related to the
genus Diatrype but phylogenetically distant from a reference strain of Diatrype disci-
formis. The remaining two isolates were associated with the Peroneutypa clade, with
one of them matching a reference strain of Peroneutypa scoparia and the other
uncertainly placed taxonomically.
The Diaporthales clade grouped eight isolates, six of which belonged to the Dia-
porthe clade and were characterized by the production of hyaline conidiophores within
pycnidial conidiomata, phialidic conidiogenous cells, and small conidia. The other two
sterile isolates were located in the Valsa clade.
The Hypocreales clade included only a single sterile isolate that matched a reference
strain of Thyronectria austroamericana.
The Glomerellales clade comprised 10 isolates, all of which belonged to the genus
Colletotrichum and were characterized by the production of acervular conidiomata,
phialidic conidiogenous cells, conidia variable in shape, and the presence of appres-
soria. Six of the isolates were identified as Colletotrichum gloeosporioides, two were
identified as Colletotrichum truncatum, and one was identified as Colletotrichum
spaethianum. One isolate (UTHSC DI14-247) was molecularly closely related to a refer-
ence strain of Colletotrichum torulosum.
Two isolates (UTHSC DI16-350 and UTHSC DI16-223) were not located in any of the
previously known orders and consequently were treated as incertae sedis. The first one
was assigned to the Phomatospora clade and the other, characterized by the produc-
tion of sporodochia and hyaline conidia, was identified as Phialemoniopsis curvata.
From a total of 224 clinical isolates, 153 were recovered mainly from superficial
tissues (epidermis and dermis) (66.5%), followed by 40 from the respiratory tract (17.4),
22 from miscellaneous deep tissues or fluids (9.6%), and 9 isolates from subcutaneous
tissues (3.9%) (Table 1).
Approximately half of all the fungi tested (44%; 101/230) were able to grow at 37°C
(Table 1); they were distributed within the orders at the following percentages: 100%
(10/10) of the Glomerellales, 100% (2/2) of Hysteriales, 100% (2/2) of the Valsariales, 98%
(44/45) of the Botryosphaeriales, 50% (1/2) of the isolates incertae sedis, and 28%
(42/152) of the Pleosporales.
Table 2 summarizes the results of the antifungal susceptibility testing. In general, all
the drugs tested, but especially terbinafine and amphotericin B, showed good activity
against the coelomycetous fungi, with terbinafine being the most active (geometric
mean [GM] of 0.04
␮
g/ml; MIC
90
of 0.03
␮
g/ml). Among the triazoles, itraconazole was
the least active, with an overall GM of 1
␮
g/ml and a MIC
90
of 16
␮
g/ml. Colletotrichum
gloeosporioides,Neoscytalidium dimidiatum, and Didymella heteroderae showed high
MICs for all the antifungals tested. Posaconazole and voriconazole demonstrated similar
in vitro potencies, with the only exceptions being activity against Colletotrichum
gloeosporioides and Neoascochyta desmazieri, for which the voriconazole GMs were 2.64
and 2
␮
g/ml, respectively, and against Neoscytalidium dimidiatum, for which the
posaconazole GM was 2.26
␮
g/ml. All the echinocandins showed good in vitro activity
Valenzuela-Lopez et al. Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 560

against these fungi, with a GM of 0.06
␮
g/ml. Flucytosine was the least active antifungal
tested, with elevated MICs against all isolates.
DISCUSSION
This is, to our knowledge, the largest taxonomic study on coelomycetous fungi of
clinical origin. It has demonstrated, based on DNA sequencing, a wider diversity of taxa
than previously reported. Although two recent reviews have reported approximately 35
species of coelomycetes involved in human infections (3, 4), the present study identifies
88 species; unfortunately, the role of many of them as pathogens for human still
remains uncertain because the clinical data of the patients are not allowed to be
TABLE 1 Anatomical sites of coelomycetous fungus isolates from clinical specimens
Order Clade
No. of isolates obtained from:
37°C
growth
Total no. of
isolates
Superficial
tissue
Subcutaneous
tissue
Deep
tissue/fluids
Respiratory
tract
Environment
and animal
Botryosphaeriales Aplosporella 2⫹2
Botryosphaeria 3⫹3
Lasiodiplodia 2⫹2
Neofusicoccum 1⫺1
Neoscytalidium 27 3 5 ⫹35
Phaeobotryosphaeria 2⫹2
Capnodiales 1⫺1
Diaporthales Diaporthe 321⫹6
Valsa 11⫹2
Glomerellales 71 1 1 ⫹10
Helotiales 1⫺1
Hypocreales 1⫹1
Hysteriales 11⫹2
incertae sedis Phialemoniopsis 1⫹1
Phomatospora 1⫺1
Magnaporthales 1⫹1
Pleosporales Acrocalymma 1⫺1
Anteaglonium 1⫺1
Biatriospora 11⫺2
Camarographium 2⫺2
Coniothyrium 71 1 ⫺9
Didymella 17 1 4 ⫹22
Didymosphaeriaceae 26 1 2 3 1 ⫺33
Exosporium 11⫺2
flavescens 1⫺1
Keissleriella 1⫺1
Lophiostoma 1⫺1
Medicopsis 31 ⫹4
Neoascochyta 61⫺7
Paraphoma 1⫺1
Phaeosphaeria 21 1 4 1 ⫺9
Phoma I 13 3 10 1 ⫺27
Phoma II 1 ⫺1
Phyllosticta 1⫹1
Pleospora 113⫺5
Pyrenochaetopsis 612⫺9
Roussoella 521⫹8
Trematosphaeria 41 ⫹5
Valsariales 2⫹2
Xylariales Diatrype 111 ⫺3
Peroneutypa 11⫺2
Total no. of isolates (%) 153 (66.5) 9 (3.9) 22 (9.6) 40 (17.4) 6 (2.6) 230 (100)
Coelomycetous Fungi of Clinical Origin Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 561

published. In general, the coelomycetous fungi are involved in many kinds of mycoses,
with superficial to deep infections, onychomycosis, cutaneous infections, keratitis, and
endophthalmitis being relatively frequent. In general, the most commonly reported
species clinically are Colletotrichum spp. (13–20), Neoscytalidium dimidiatum (21–25),
and Phoma spp. (11, 26–35). Our study partly confirms the data from previous studies
in which Neoscytalidium dimidiatum (approximately 15%), Paraconiothyrium cyclothyri-
oides (approximately 7%), and Phoma herbarum (approximately 6.5%) were the most
common species, having been recovered mainly from superficial tissue and respiratory
tract specimens. However, of these fungi, the only species that is relatively easy to
identify by phenotypic criteria is N.dimidiatum, which is the best known coelomycetous
fungus found clinically (22, 23, 36). The identification of the other fungi mentioned
above generally requires the use of molecular tools due to the difficulty of achieving in
vitro sporulation. Although Paraconiothyrium cyclothyrioides was relatively common in
our studied samples, there are only two clinical reports that refer to this species. Both
TABLE 2 Results of in vitro antifungal susceptibility testing of coelomycetous fungi
Taxon (no. of isolates) Parameter
a
Value for the drug (
␮
g/ml)
b
AMB VRC ITC PSC AFG CFG MFG TRB 5FC
Neoascochyta desmazieri (5) GM 0.44 2 0.57 0.21 0.03 0.03 0.03 0.03 1.15
Range 0.25–1 1–4 0.25–1 0.06–0.5 0.03–0.06 ⱕ0.03 ⱕ0.03 ⱕ0.03 0.5–2
MIC
90
0.5 2 1 0.5 0.03 0.03 0.03 0.03 2
Colletotrichum gloeosporioides (5) GM 0.57 2.64 8 0.87 0.03 0.03 0.03 0.03 16
Range 0.03–2 0.5–4 1–16 0.5–1 ⱕ0.03 ⱕ0.03 ⱕ0.03 ⱕ0.03 ⱖ16
MIC
90
2 4 16 1 0.03 0.03 0.03 0.03 16
Epicoccum sorghinum (8) GM 0.25 0.92 0.59 0.30 0.03 0.04 0.03 0.03 2.97
Range 0.12–1 0.5–2 0.5–1 0.12–0.5 0.03–0.06 0.03–0.5 ⱕ0.03 ⱕ0.03 1–8
MIC
90
0.5 1 1 0.5 0.03 0.03 0.03 0.03 4
Neoscytalidium dimidiatum (16) GM 0.22 0.59 2.56 2.26 0.13 0.2 0.47 0.08 2.83
Range 0.06–1 0.03–16 0.06–16 0.03–16 0.03–0.5 0.03–1 0.06–8 0.03–2 0.25–16
MIC
90
0.5 4 16 16 0.25 0.5 4 0.03 8
Paraconiothyrium cyclothyrioides (15) GM 0.25 0.25 0.3 0.15 0.03 0.03 0.03 0.03 2.61
Range 0.03–8 0.06–0.5 0.06–0.5 0.03–0.5 ⱕ0.03 ⱕ0.03 ⱕ0.03 ⱕ0.03 1–16
MIC
90
0.5 0.5 0.5 0.25 0.03 0.03 0.03 0.03 4
Didymella heteroderae (11) GM 1.76 1.87 3.31 1.07 0.34 0.13 0.14 0.03 4
Range 0.5–8 0.06–16 0.5–16 0.5–2 0.03–8 0.03–4 0.03–2 ⱕ0.03 1–16
MIC
90
4 16 16 2 8 4 2 0.03 16
Phoma herbarum (10) GM 0.43 0.57 0.81 0.40 0.04 0.04 0.03 0.03 2
Range 0.12–2 0.06–4 0.25–4 0.12–1 0.03–0.12 0.03–0.12 0.03–0.06 ⱕ0.03 0.5–16
MIC
90
1 1 1 1 0.06 0.12 0.06 0.03 16
Phoma sp. (7) GM 0.1 0.17 0.17 0.14 0.03 0.03 0.03 0.03 1.78
Range 0.03–4 0.03–2 0.03–2 0.03–1 ⱕ0.03 ⱕ0.03 ⱕ0.03 ⱕ0.03 0.5–16
MIC
90
0.25 1 0.5 0.5 0.03 0.03 0.03 0.03 4
Diaporthe sclerotioides (4) GM 0.06 0.21 2 0.5 0.04 0.03 0.03 0.03 4
Range 0.03–0.12 0.12–0.25 1–4 0.5 0.03–0.06 ⱕ0.03 ⱕ0.03 ⱕ0.03 0.5–16
MIC
90
0.12 0.25 2 0.5 0.03 0.03 0.03 0.03 8
Pyrenochaetopsis leptospora (4) GM 0.7 0.59 0.7 0.21 0.03 0.03 0.03 0.03 4
Range 0.03–4 0.25–2 0.06–16 0.03–1 ⱕ0.03 ⱕ0.03 ⱕ0.03 ⱕ0.03 0.5–16
MIC
90
2 1 1 0.5 0.03 0.03 0.03 0.03 16
Overall (85) GM 0.33 0.61 1 0.46 0.06 0.06 0.06 0.04 2.9
Range 0.03–8 0.03–16 0.03–16 0.03–16 0.03–8 0.03–4 0.03–8 0.03–2 0.25–32
MIC
90
2 4 16 16 0.25 0.5 2 0.03 16
a
GM, geometric mean; MIC
90
, drug concentration that inhibited 90% of isolates.
b
AMB, amphotericin B; VRC, voriconazole; ITC, itraconazole; PSC, posaconazole; AFG, anidulafungin; CFG, caspofungin; MFG, micafungin; TRB, terbinafine; 5FC,
flucytosine.
Valenzuela-Lopez et al. Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 562

cases are from immunocompromised patients; in one case P.cyclothyrioides caused skin
lesions of the lower extremities, and in the second case it produced a systemic
coinfection together with Phaeoacremonium parasiticum (37, 38). Even though Phoma
sporulates easily, it is commonly misidentified as other related genera, such as Asco-
chyta, because the genera have similar morphologies, physiologies, and nucleotide
sequences (39, 40). Boerema et al. carried out one of the most comprehensive revisions
of the taxonomy of the genus Phoma. Using systematic criteria that predominated then,
approximately 220 species were accepted, distributed into nine sections (41). In a
recent multilocus study based on the sequence data of the 18S nrRNA (SSU) and LSU
genes, other authors demonstrated that such classification was totally artificial (42).
Currently, Phoma sensu stricto is included in the family Didymellaceae, and the other
Phoma-like fungi belong to other phylogenetic families, i.e., Cucurbitariaceae,Lepto-
sphaeriaceae,Phaeosphaeriaceae, etc. (39, 40, 42, 43).
It is of note that one of the frequently isolated species in our study, Didymella
heteroderae (5.2% of isolation frequency), has never been mentioned as an etiologic
agent of human infections even though our results reveal its ability to grow and to
sporulate at 37°C, which is uncommon in that genus and suggests its potential
pathogenicity.
An important clinical presentation of the coelomycetous fungi is eumycetoma,
which is restricted to a specific group of pleosporalean species of fungi, namely,
Medicopsis romeroi (formerly, Pyrenochaeta romeroi)(
44–46), Biatriospora mackinnonii
(formerly Pyrenochaeta mackinnonii)(
46), and Trematosphaeria grisea (formerly, Ma-
durella grisea)(
47–49), among others. However, in the present study only nine of the
isolates that were isolated from superficial and, less frequently, from deep tissues
belonged to these genera. This might be explained by the fact that the habitat of these
fungi is usually restricted to arid zones of East Africa and India and, occasionally, South
America (46, 50, 51).
Despite several studies in recent years devoted to infections by coelomycetous
fungi, little clinical data exist. The first well-documented review of human infections
caused by these fungi was carried out by Punithalingham (11), who referenced a total
of 12 species belonging primarily to the genera Botryodiplodia,Dothiorella,Hender-
sonula,Phoma,Phyllosticta,Pseudochaetosphaeronema, and Pyrenochaeta. In that work,
a morphological description of these taxa and their clinical origin was provided,
together with a dichotomous key for their identification. However, in our study, just
under 12% of the total isolates identified belonged to such genera. In a recent study,
Stchigel and Sutton (4) provided detailed information about the species of these fungi
isolated from clinical samples, described useful tools for their isolation and identifica-
tion, and gave general guidelines for infection management and treatment. These
authors concluded that these organisms are easy to isolate but that it was difficult to
induce in vitro fructification and sporulation. Our results are in agreement with theirs
because 43% of our isolates failed to sporulate, and it was only possible to identify
them and to determine their phylogenetic relationships by DNA sequencing.
The prevalence of coelomycetous fungi found in these clinical specimens—more
than 200 isolates recovered in a 9-year period— goes against the fact that so few
studies have described infections by them. This highlights the difficulty in conducting
a comprehensive study of these fungi and in establishing their real occurrence in
clinical settings. The taxonomy of these fungi is very complex because numerous
isolates are usually unable to sporulate in vitro or to produce different synanamorphs,
which sometimes predominate over the traditional coelomycete structures, making
their phenotypic recognition difficult; reliable identification can be done, therefore,
only by gene sequencing (9, 46, 52). However, even in this case, there are a very high
number of genera and species of coelomycetous fungi, and the phylogenetic bound-
aries of numerous taxa are still unresolved. Therefore, we carried out a phylogenetic
analysis of a large set of coelomycetous fungi using LSU sequences. This marker proved
useful for solving the phylogeny of most of the isolates included in the study, identi-
Coelomycetous Fungi of Clinical Origin Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 563

fying them, at least at genus level, and showing, in front of the internal transcribed
spacer (ITS), the advantage of an easy alignment of sequences.
The increasing use of molecular tools in fungal taxonomy has allowed the recog-
nition of numerous new taxa that are impossible to detect by traditional methods.
Recently, several new species of coelomycetous fungi, namely, Roussoella percutanea,
Truncatella angustata,Hongkongmyces pedis,Rhytidhysteron spp., Pseudochaetospha-
eronema martinelli, and Emarellia spp., have been involved in cases of subcutaneous
infections and eumycetoma (53–58), and some of our Pleosporales isolates, having
failed to sporulate, could represent new taxa.
Although clinical breakpoints for coelomycetous fungi have not been defined and
although in vitro antifungal susceptibility studies on these fungi are scarce, most of the
species seem to be inhibited by amphotericin B (4). Our results show that posaconazole
is the most active of the triazoles tested, and results for amphotericin B are similar in
vitro to those reported by Chowdhary et al. (10). Currently, only disseminated infections
due to N.dimidiatum have been conducted in animal models, and amphotericin B,
voriconazole, and posaconazole have been shown to be effective in the treatment of
this experimental mycosis (36). Guidelines for the management of infections due to
coelomycetous fungi include only a small group of taxa (Neoscytalidium,Phoma, and
Pyrenochaeta spp.) (10) although our study supports those protocols. A recent study by
Guégan et al. (59) analyzed several coelomycetous fungi that were implicated in human
mycosis and concluded that the surgical resection of infected tissues is advisable for
treating well-delimited lesions and that surgery together with new triazoles could be
used if lesions are extensive.
In conclusion, this study demonstrates that a wide variety of fungal taxa, identified
through their morphology as coelomycetous fungi, are involved in human infections in
the United States. However, more studies are necessary to understand the real preva-
lence of coelomycete infections throughout the world. The most active antifungal
drugs to treat them seem to be terbinafine, echinocandins, and amphotericin B, while
results for the azoles varied. Although the LSU gene sequence is useful for preliminary
identification and for establishing phylogenetic relationships between the majority of
coelomycetous fungi, future molecular studies testing a higher number genes are
essential to properly identify doubtful isolates at the species level.
MATERIALS AND METHODS
Fungal isolates and sequences. A total of 230 isolates of coelomycetous fungi were included in this
study, consisting of 224 from human clinical specimens, 3 from animal sources, and 3 from environ-
mental samples. All of the isolates were provided by the Fungus Testing Laboratory of the University of
Texas Health Science Center at San Antonio (UTHSC; San Antonio, Texas, USA). In addition, 92 D1-D2
sequences corresponding to type or reference strains were retrieved from GenBank and CBS databases
and included in the phylogenetic analysis.
Morphological and physiological characterization. For cultural characterization, the isolates were
grown on oatmeal agar (OA; 30 g of filtered oat flakes, 15 g of agar-agar, 1 liter of tap water) and malt
extract agar (MEA; 40 g of malt extract, 15 g of agar-agar, 1 liter of distilled water) at 20 ⫾1°C for 14 days
in darkness. The ability of the isolates to grow at 37°C was determined on potato dextrose agar (PDA;
Pronadisa, Madrid, Spain) after 7 days of incubation in darkness. The morphological features of the
vegetative and reproductive structures were studied using an Olympus CH2 light-field microscope
(Olympus Corporation, Tokyo, Japan) in wet mounts (on water and lactic acid) and slide cultures (isolates
grown on OA and MEA). The isolates were characterized phenotypically according to traditional criteria
(4, 5, 41, 60). Color standards are from Kornerup and Wanscher (61). Photomicrographs were taken with
an Axio-Imager M1 light-field microscope (Zeiss, Oberkochen, Germany).
DNA extraction, amplification, and sequencing. The total genomic DNA was extracted from
colonies grown on PDA after 7 days of incubation at 20 ⫾1°C, using a FastDNA kit protocol (Bio101; Vista,
CA) with a FastPrep FP120 instrument (Thermo Savant, Holbrook, NY) according to the manufacturer’s
protocol. DNA was quantified using a NanoDrop 2000 instrument (Thermo Scientific, Madrid, Spain). The
D1-D2 domains were amplified with the primer pair LR0R and LR5 (62). The amplicons were sequenced
in both directions with the same primer pair used for amplification at Macrogen Europe (Macrogen, Inc.,
Amsterdam, The Netherlands). The consensus sequences were obtained using SeqMan software, version
7.0.0 (DNAStar Lasergene, Madison, WI, USA).
Molecular identification and phylogenetic analysis. Preliminary molecular identification of the
isolates was made using the D1-D2 nucleotide sequences in blastn searches (https://blast.ncbi.nlm.nih.
gov/Blast.cgi) and the CBS database (www.cbs.knaw.nl). Only the sequences of type or reference strains
Valenzuela-Lopez et al. Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 564

deposited in CBS/GenBank databases were considered for identification purposes. A level of identity of
ⱖ98% was considered for species-level identification.
For the phylogenetic study, the sequences were aligned using the ClustalW application (63)ofthe
MEGA, version 6.06 (64), computer program, refined with MUSCLE (65), and manually adjusted using the
same software platform. Phylogenetic reconstructions were made by maximum-likelihood (ML) and
Bayesian inference (BI) with MEGA, version 6.06, and MrBayes, version 3.2.4 (66), respectively. The best
substitution model for the gene matrix (general time-reversal model incorporating invariable sites and
a discrete gamma distribution [GTR⫹I⫹G]) was estimated using MrModelTest, version 2.3 (67). For ML
analyses, a nearest-neighbor interchange was used as the heuristic method for tree inference. Support
for internal branches was assessed by 1,000 ML bootstrapped pseudoreplicates. Bootstrap support (BS)
of ⱖ70 was considered significant. For BI analyses, Markov chain Monte Carlo (MCMC) sampling was
carried out with 23 million generations, with samples taken every 1,000 generations. The 50% majority
rule consensus trees and posterior probability values (PP) were calculated after the first 25% of the
resulting trees was removed for burn-in. A PP value of ⱖ0.95 was considered significant. Saccharomyces
castellii (NRRL Y-12630; GenBank accession number AY048167) and Saccharomyces cerevisiae (NRRL
Y-12632; GenBank accession number AY048154) were used as outgroups.
Antifungal susceptibility testing. Using a broth microdilution reference method (68), the in vitro
antifungal susceptibilities of 85 isolates were determined of selected species of the genera Colletotri-
chum,Diaporthe,Didymella,Epicoccum,Neoascochyta,Neoscytalidium,Paraconiothyrium,Phoma sp., and
Pyrenochaetopsis. The following antifungals were tested: amphotericin B, voriconazole, posaconazole,
itraconazole, caspofungin, anidulafungin, micafungin, terbinafine, and flucytosine. The minimal effective
concentration (MEC) was determined after 48 h for the echinocandins, and the MIC was determined after
48 h and 72 h for the other drugs. Candida parapsilosis ATCC 22019 and Paecilomyces variotii ATCC
MYA-3630 were used as controls. The inocula for the coelomycetous fungi that did not sporulate were
prepared according to the method of Chowdhary et al. (69).
Accession number(s). The DNA sequences determined in this study have been deposited in
GenBank under accession numbers LN907285 to LN907514.
ACKNOWLEDGMENTS
This work was supported by the Spanish Ministerio de Economía y Competitividad,
grant CGL2013-43789-P.
We have no conflicts of interest to declare.
REFERENCES
1. Kirk PM, Cannon PF, Stalpers JA, Minter DW. 2008. Ainsworth & Bisby’s
dictionary of the fungi, 10th ed. CAB International, Wallingford, United
Kingdom.
2. Sutton DA. 1999. Coelomycetous fungi in human disease. A review:
clinical entities, pathogenesis, identification and therapy. Rev Iberoam
Micol 16:171–179.
3. Revankar SG, Sutton DA. 2010. Melanized fungi in human disease. Clin
Microbiol Rev 23:884 –928. https://doi.org/10.1128/CMR.00019-10.
4. Stchigel AM, Sutton DA. 2013. Coelomycete fungi in the clinical lab.
Curr Fungal Infect Rep 7:171–191. https://doi.org/10.1007/s12281
-013-0139-9.
5. Sutton BC. 1980. The Coelomycetes. Fungi Imperfecti with pycnidia,
acervuli and stromata. Commonwealth Mycological, Kew, England.
6. Nag Raj TR. 1993. Coelomycetous anamorphs with appendage-bearing
conidia. Mycologue Publications, Waterloo, Ontario, Canada.
7. Schoch CL, Crous PW, Groenewald JZ, Boehm EWA, Burgess TI, de
Gruyter J, de Hoog GS, Dixon LJ, Grube M, Gueidan C, Harada Y,
Hatakeyama S, Hirayama K, Hosoya T, Huhndorf SM, Hyde KD, Jones EBG,
Kohlmeyer J, Kruys A, Li YM, Lücking R, Lumbsch HT, Marvanová L,
Mbatchou JS, McVay AH, Miller AN, Mugambi GK, Muggia L, Nelsen MP,
Nelson P, Owensby CA, Phillips AJL, Phongpaichit S, Pointing SB, Pujade-
Renaud V, Raja HA, Plata ER, Robbertse B, Ruibal C, Sakayaroj J, Sano T,
Selbmann L, Shearer CA, Shirouzu T, Slippers B, Suetrong S, Tanaka K,
Volkmann-Kohlmeyer B, Wingfield MJ, Wood AR, Woudenberg JHC,
Yonezawa H, Zhang Y, Spatafora JW. 2009. A class-wide phylogenetic
assessment of Dothideomycetes. Stud Mycol 64:1–15. https://doi.org/
10.3114/sim.2009.64.01.
8. Maharachchikumbura SSN, Hyde KD, Groenewald JZ, Xu J, Crous PW.
2014. Pestalotiopsis revisited. Stud Mycol 79:121–186. https://doi.org/
10.1016/j.simyco.2014.09.005.
9. Wijayawardene NN, Hyde KD, Wanasinghe DN, Papizadeh M, Goo-
nasekara ID, Camporesi E, Bhat DJ, McKenzie EHC, Phillips AJL, Diederich
P, Tanaka K, Li WJ, Tangthirasunun N, Phookamsak R, Dai D-Q, Dissanay-
ake AJ, Weerakoon G, Maharachchikumbura SSN, Hashimoto A, Mat-
sumura M, Bahkali AH, Wang Y. 2016. Taxonomy and phylogeny of
dematiaceous coelomycetes. Fungal Divers 77:1–316. https://doi.org/
10.1007/s13225-016-0360-2.
10. Chowdhary A, Meis JF, Guarro J, de Hoog GS, Kathuria S, Arendrup MC,
Arikan-Akdagli S, Akova M, Boekhout T, Caira M, Guinea J, Chakrabarti A,
Dannaoui E, van Diepeningen A, Freiberger T, Groll AH, Hope WW,
Johnson E, Lackner M, Lagrou K, Lanternier F, Lass-Flörl C, Lortholary O,
Meletiadis J, Muñoz P, Pagano L, Petrikkos G, Richardson MD, Roilides E,
Skiada A, Tortorano AM, Ullmann AJ, Verweij PE, Cornely OA, Cuenca-
Estrella M. 2014. ESCMID and ECMM joint clinical guidelines for the
diagnosis and management of systemic phaeohyphomycosis: diseases
caused by black fungi. Clin Microbiol Infect 20:47–75. https://doi.org/
10.1111/1469-0691.12515.
11. Punithalingam E. 1979. Sphaeropsidales in culture from humans. Nova
Hedwigia 31:119–158.
12. Liu JK, Phookamsak R, Dai DQ, Tanaka K, Jones EG, Xu JC, Chukeatirote
E, Hyde K. 2014. Roussoellaceae, a new pleosporalean family to accom-
modate the genera Neoroussoella gen. nov., Roussoella and Roussoellop-
sis. Phytotaxa 181:1–33. https://doi.org/10.11646/phytotaxa.181.1.1.
13. Guarro J, Svidzinski TE, Zaror L, Forjaz MH, Gené J, Fischman O. 1998.
Subcutaneous hyalohyphomycosis caused by Colletotrichum gloeospori-
oides. J Clin Microbiol 36:3060 –3065.
14. Cano J, Guarro J, Gené J. 2004. Molecular and morphological identifica-
tion of Colletotrichum species of clinical interest. J Clin Microbiol 42:
2450 –2454. https://doi.org/10.1128/JCM.42.6.2450-2454.2004.
15. Castro LG, Da Silva Lacaz C, Guarro J, Gené J, Heins-Vaccari EM, de Freitas
Leite RS, Arriagada GL, Reguera MM, Ito EM, Valente NY, Nunes RS. 2001.
Phaeohyphomycotic cyst caused by Colletotrichum crassipes. J Clin Mi-
crobiol 39:2321–2324. https://doi.org/10.1128/JCM.39.6.2321-2324.2001.
16. Fernandez V, Dursun D, Miller D, Alfonso EC. 2002. Colletotrichum kera-
titis. Am J Ophthalmol 134:435– 438. https://doi.org/10.1016/S0002
-9394(02)01576-3.
17. Chakrabarti A, Shivaprakash MR, Singh R, Tarai B, George VK, Fomda BA,
Gupta A. 2008. Fungal endophthalmitis: fourteen years’ experience from
a center in India. Retina 28:1400 –1407. https://doi.org/10.1097/
IAE.0b013e318185e943.
Coelomycetous Fungi of Clinical Origin Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 565

18. Shivaprakash MR, Appannanavar SB, Dhaliwal M, Gupta A, Gupta S,
Gupta A, Chakrabarti A. 2011. Colletotrichum truncatum: An unusual
pathogen causing mycotic keratitis and endophthalmitis. J Clin Micro-
biol 49:2894–2898. https://doi.org/10.1128/JCM.00151-11.
19. Shiraishi A, Araki-Sasaki K, Mitani A, Miyamoto H, Sunada A, Ueda A,
Asari S, Zheng X, Yamamoto Y, Hara Y, Ohashi Y. 2011. Clinical charac-
teristics of keratitis due to Colletotrichum gloeosporioides. J Ocul Phar-
macol Ther 27:487– 491. https://doi.org/10.1089/jop.2011.0011.
20. Figtree M, Weeks K, Chan L, Leyton A, Bowes A, Giuffre B, Sullivan M,
Hudson BJ. 2013. Colletotrichum gloeosporioides sensu lato causing deep
soft tissue mycosis following a penetrating injury. Med Mycol Case Rep
2:40 – 43. https://doi.org/10.1016/j.mmcr.2013.01.003.
21. al-Rajhi AA, Awad AH, Al-Hedaithy SS, Forster RK, Caldwell KC. 1993.
Scytalidium dimidiatum fungal endophthalmitis. Br J Ophthalmol 77:
388 –390. https://doi.org/10.1136/bjo.77.6.388.
22. Elewski BE. 1996. Onychomycosis caused by Scytalidium dimidiatum.J
Am Acad Dermatol 35:336 –338. https://doi.org/10.1016/S0190
-9622(96)90664-7.
23. Madrid H, Ruíz-Cendoya M, Cano J, Stchigel A, Orofino R, Guarro J. 2009.
Genotyping and in vitro antifungal susceptibility of Neoscytalidium
dimidiatum isolates from different origins. Int J Antimicrob Agents 34:
351–354. https://doi.org/10.1016/j.ijantimicag.2009.05.006.
24. Machouart M, Menir P, Helenon R, Quist D, Desbois N. 2013. Scytalidium
and scytalidiosis: what’s new in 2012? J Mycol Med 23:40 – 46. https://
doi.org/10.1016/j.mycmed.2013.01.002.
25. Bakhshizadeh M, Hashemian HR, Najafzadeh MJ, Dolatabadi S, Zarrinfar
H. 2014. First report of rhinosinusitis caused by Neoscytalidium dimidia-
tum in Iran. J Med Microbiol 63:1017–1019. https://doi.org/10.1099/
jmm.0.065292-0.
26. Bakerspigel A. 1970. The isolation of Phoma hibernica from lesions on a
leg. Sabouraudia 7:261–264.
27. Punithalingam E. 1976. Phoma oculo hominis sp. nov. from corneal ulcer.
Trans Br Mycol Soc 67:142–143. https://doi.org/10.1016/S0007-1536
(76)80022-8.
28. Bakerspigel A, Lowe D, Rostas A. 1981. The isolation of Phoma eupyrena
from a human lesion. Arch Dermatol 117:362–363. https://doi.org/
10.1001/archderm.1981.01650060052024.
29. Shukla NP, Rajak RK, Agarwasl GP, Gupta D. 1984. Phoma minutispora as
a human pathogen. Mykosen 27:255–258.
30. Baker JG, Salkin IF, Forgacs P, Haines JH, Kemna ME. 1987. First report of
subcutaneous phaeohyphomycosis of the foot caused by Phoma minu-
tella. J Clin Microbiol 25:2395–2397.
31. Rai MK. 1989. Phoma sorghina infection in human being. Mycopatholo-
gia 105:167–170. https://doi.org/10.1007/BF00437250.
32. Rosen T, Rinaldi MJ, Tschen JA, Stern JK, Cernoch P. 1996. Cutaneous
lesions due to Pleurophoma (Phoma) complex. South Med J 89:431– 434.
https://doi.org/10.1097/00007611-199604000-00018.
33. Hirsh AH, Schiff TA. 1996. Subcutaneous phaeohyphomycosis caused by
an unusual pathogen: Phoma species. J Am Acad Dermatol 34:679 – 680.
https://doi.org/10.1016/S0190-9622(96)80083-1.
34. Tullio V, Banche G, Allizond V, Roana J, Mandras N, Scalas D, Panzone M,
Cervetti O, Valle S, Carlone N, Cuffini AM. 2010. Non-dermatophyte
moulds as skin and nail foot mycosis agents: Phoma herbarum,Chaeto-
mium globosum and Microascus cinereus. Fungal Biol 114:345–349.
https://doi.org/10.1016/j.funbio.2010.02.003.
35. Roehm CE, Salazar JC, Hagstrom N, Valdez TA. 2012. Phoma and Acre-
monium invasive fungal rhinosinusitis in congenital acute lymphocytic
leukemia and literature review. Int J Pediatr Otorhinolaryngol 76:
1387–1391. https://doi.org/10.1016/j.ijporl.2012.06.026.
36. Ruíz-Cendoya M, Madrid H, Pastor J, Guarro J. 2010. Evaluation of
antifungal therapy in a neutropenic murine model of Neoscytalidium
dimidiatum infection. Int J Antimicrob Agents 35:152–155. https://
doi.org/10.1016/j.ijantimicag.2009.09.028.
37. Gordon RA, Sutton DA, Thompson EH, Shrikanth V, Verkley GJ, Stielow
JB, Mays R, Oleske D, Morrison LK, Lapolla WJ, Galfione S, Tyring S,
Samathanam CA, Fu J, Wickes BL, Mulanovich V, Wanger A, Arias CA.
2012. Cutaneous phaeohyphomycosis caused by Paraconiothyrium cy-
clothyrioides. J Clin Microbiol 50:3795–3798. https://doi.org/10.1128/
JCM.01943-12.
38. Colombier MA, Alanio A, Denis B, Melica G, Garcia-Hermoso D, Levy B,
Peraldi MN, Glotz D, Bretagne S, Gallien S. 2015. Dual invasive infection
with Phaeoacremonium parasiticum and Paraconiothyrium cyclothyrioides
in a renal transplant recipient: Case report and comprehensive review of
the literature of Phaeoacremonium phaeohyphomycosis. J Clin Microbiol
53:2084 –2094. https://doi.org/10.1128/JCM.00295-15.
39. Aveskamp MM, de Gruyter J, Woudenberg JH, Verkley GJ, Crous PW.
2010. Highlights of the Didymellaceae: a polyphasic approach to char-
acterise Phoma and related pleosporalean genera. Stud Mycol 65:1– 60.
https://doi.org/10.3114/sim.2010.65.01.
40. Chen Q, Jiang JR, Zhang GZ, Cai L, Crous PW. 2015. Resolving the Phoma
enigma. Stud Mycol 82:137–217. https://doi.org/10.1016/j.simyco
.2015.10.003.
41. Boerema GH, de Gruyter J, Noordeloos ME, Hamers M. 2004. Phoma
identification manual. Differentiation of specific and infraspecific taxa in
culture. CABI Publishing, Cambridge, United Kingdom.
42. de Gruyter J, Aveskamp MM, Woudenberg JH, Verkley GJ, Groenewald
JZ, Crous PW. 2009. Molecular phylogeny of Phoma and allied anamorph
genera: towards a reclassification of the Phoma complex. Mycol Res
113:508 –519. https://doi.org/10.1016/j.mycres.2009.01.002.
43. de Gruyter J, Woudenberg JH, Aveskamp MM, Verkley GJ, Groenewald
JZ, Crous PW. 2013. Redisposition of Phoma-like anamorphs in Pleospo-
rales. Stud Mycol 75:1–36. https://doi.org/10.3114/sim0004.
44. Borelli D. 1979. Opportunistic fungi as producers of gray colonies and
mycetomata. Dermatologica 159:168 –174. https://doi.org/10.1159/
000250685.
45. Mathuram Thiyagarajan U, Bagul A, Nicholson ML. 2011. A nodulo-cystic
eumycetoma caused by Pyrenochaeta romeroi in a renal transplant
recipient: a case report. J Med Case Rep 5:460. https://doi.org/10.1186/
1752-1947-5-460.
46. Ahmed SA, Van De Sande WW, Stevens DA, Fahal A, Van Diepeningen
AD, Menken SB, de Hoog GS. 2014. Revision of agents of black-grain
eumycetoma in the order Pleosporales. Persoonia 33:141–154. https://
doi.org/10.3767/003158514X684744.
47. Butz WC, Ajello L. 1971. Black grain mycetoma: a case due to Madurella
grisea. Arch Dermatol 104:197–201. https://doi.org/10.1001/archderm
.1971.04000200085015.
48. Gulati V, Bakare S, Tibrewal S, Ismail N, Sayani J, Baghla DPS. 2012. A rare
presentation of concurrent Scedosporium apiospermum and Madurella
grisea eumycetoma in an immunocompetent host. Case Rep Pathol
2012:154201. https://doi.org/10.1155/2012/154201.
49. de Hoog GS, Van Diepeningen AD, Mahgoub ES, Van de Sande WWJ.
2012. New species of Madurella, causative agents of black-grain
mycetoma. J Clin Microbiol 50:988 –994. https://doi.org/10.1128/JCM
.05477-11.
50. Mackinnon JE, Ferrada-Urzúa LV, Montemayor L. 1943. Madurella grisea
n. sp. Mycopathologia 4:384 –393. https://doi.org/10.1007/BF01237166.
51. McGinnis MR. 1996. Mycetoma. Dermatol Clin 41:97–104.
52. Borman AM, Desnos-Ollivier M, Campbell CK, Bridge PD, Dannaoui E,
Johnson EM. 2016. Novel Taxa Associated with Human Fungal Black-
Grain Mycetomas: Emarellia grisea gen. nov., sp. nov., and Emarellia
paragrisea sp. nov. J Clin Microbiol 54:1738 –1745. https://doi.org/
10.1128/JCM.00477-16.
53. Ahmed SA, Stevens DA, Van de Sande WWJ, Meis JF, de Hoog GS. 2014.
Roussoella percutanea, a novel opportunistic pathogen causing subcu-
taneous mycoses. Med Mycol 52:689 – 698. https://doi.org/10.1093/
mmy/myu035.
54. Jagielski T, Zak I, Tyrak J, Bryk A. 2015. First probable case of subcuta-
neous infection due to Truncatella angustata: a new fungal pathogen of
humans? J Clin Microbiol 53:1961–1964. https://doi.org/10.1128/JCM
.00400-15.
55. Tsang CC, Chan JF, Trendell-Smith NJ, Ngan AH, Ling IW, Lau SK, Woo PC.
2014. Subcutaneous phaeohyphomycosis in a patient with IgG4-related
sclerosing disease caused by a novel ascomycete, Hongkongmyces pedis
gen. et sp. nov.: first report of human infection associated with the
family Lindgomycetaceae. Med Mycol 52:736 –747. https://doi.org/
10.1093/mmy/myu043.
56. Mahajan VK, Sharma V, Prabha N, Thakur K, Sharma NL, Rudramurthy SM,
Chauhan PS, Mehta KS, Abhinav C. 2014. A rare case of subcutaneous
phaeohyphomycosis caused by a Rhytidhysteron species: a clinico-
therapeutic experience. Int J Dermatol 53:1485–1489. https://doi.org/
10.1111/ijd.12529.
57. Mishra K, Das S, Goyal S, Gupta C, Rai G, Ansari MA, Saha R, Singal A.
2014. Subcutaneous mycoses caused by Rhytidhysteron species in an
immunocompetent patient. Med Mycol Case Rep 5:32–34. https://
doi.org/10.1016/j.mmcr.2014.07.002.
58. Ahmed SA, Desbois N, Quist D, Miossec C, Atoche C, Bonifaz A, de Hoog
GS. 2015. Phaeohyphomycosis caused by a novel species, Pseudochaeto-
Valenzuela-Lopez et al. Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 566

sphaeronema martinelli. J Clin Microbiol 53:2927–2934. https://doi.org/
10.1128/JCM.01456-15.
59. Guégan S, Garcia-Hermoso D, Sitbon K, Ahmed S, Moguelet P, Dromer F,
Lortholary O, French Mycosis Study Group. 2016. Ten-year experience of
cutaneous and/or subcutaneous infections due to coelomycetes in
France. Open Forum Infect Dis 3:ofw106. https://doi.org/10.1093/ofid/
ofw106.
60. de Hoog GS, Guarro J, Gené J, Figueras MJ. 2000. Atlas of clinical fungi,
2nd ed. Centraalbureau voor Schimmelcultures, Utrecht, The Nether-
lands.
61. Kornerup A, Wanscher JH. 1978. Methuen handbook of colour, 3rd ed.
Methuen, London, England.
62. Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of
enzymatically amplified ribosomal DNA from several Cryptococcus spe-
cies. J Bacteriol 172:4238 – 4246. https://doi.org/10.1128/jb.172.8.4238
-4246.1990.
63. Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W: improving the
sensitivity of progressive multiple sequence alignment through se-
quence weighting, position-specific gap penalties and weight matrix
choice. Nucleic Acids Res 22:4673– 4680. https://doi.org/10.1093/nar/
22.22.4673.
64. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6:
Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol
30:2725–2729. https://doi.org/10.1093/molbev/mst197.
65. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accu-
racy and high throughput. Nucleic Acids Res 32:1792–1797. https://
doi.org/10.1093/nar/gkh340.
66. Huelsenbeck JP, Ronquist F. 2001. MRBAYES: Bayesian inference of
phylogenetic trees. Bioinformatics 17:754 –755. https://doi.org/10.1093/
bioinformatics/17.8.754.
67. Nylander JA. 2004. MrModeltest v2. Uppsala University, Uppsala,
Sweden.
68. Clinical and Laboratory Standards Institute. 2008. Reference method for
broth dilution antifungal susceptibility testing of filamentous fungi;
approved standard, 2nd ed. Document M38-A2. Clinical and Laboratory
Standards Institute, Wayne, PA.
69. Chowdhary A, Kathuria S, Singh PK, Agarwal K, Gaur SN, Roy P, Rand-
hawa HS, Meis JF. 2013. Molecular characterization and in vitro antifun-
gal susceptibility profile of Schizophyllum commune, an emerging ba-
sidiomycete in bronchopulmonary mycoses. Antimicrob Agents
Chemother 57:2845–2848. https://doi.org/10.1128/AAC.02619-12.
Coelomycetous Fungi of Clinical Origin Journal of Clinical Microbiology
February 2017 Volume 55 Issue 2 jcm.asm.org 567