Embed Size (px)
Citation preview
Fonsecaea pugnacius, a Novel Agent of DisseminatedChromoblastomycosis
Conceição M. P. S. de Azevedo,a Renata R. Gomes,b Vania A. Vicente,b Daniel W. C. L. Santos,c Sirlei G. Marques,d
Mariana M. F. do Nascimento,b Caroline E. W. Andrade,b Raimunda R. Silva,a Flávio Queiroz-Telles,b,e G. Sybren de Hoogb,f
Department of Medicine, Federal University of Maranhão, Brazil, São Luis, MA, Brazila; Microbiology, Parasitology and Pathology Post-graduation Program, Department ofBasic Pathology, Federal University of Paraná, Curitiba, PR, Brazilb; Institute of Infectious Diseases Emilio Ribas, São Paulo, SP, Brazilc; University Hospital of FederalUniversity of Maranhão and Cedro Laboratories Maranhão, São Luis, MA, Brazild; Clinical Hospital of the Federal University of Paraná, Curitiba, PR, Brazile; Centraalbureauvoor Schimmelcultures KNAW Fungal Biodiversity Centre, Utrecht, The Netherlandsf
We report a fatal case of a chromoblastomycosis-like infection caused by a novel species of Fonsecaea in a 52-year-old immuno-competent Caucasian male from an area of chromoblastomycosis endemicity in Brazil. The patient had a 30-year history ofslowly evolving, verrucous lesions on the right upper arm which gradually affected the entire arm, the left hemifacial area, andthe nose. Subsequent dissemination to the brain was observed, which led to death of the patient. The internal transcribed spacer(ITS) and partial large subunit (LSU), BT2, and CDC42 genes of the isolates recovered from skin and brain were sequenced, con-firming the novelty of the species. The species is clinically unique in causing brain abscesses secondary to chromoblastomycosislesions despite the apparent intact immunity of the patient. Histopathologic appearances were very different, showing muriformcells in skin and hyphae in brain.
Chromoblastomycosis is a chronic cutaneous and subcutane-ous disease characterized by verrucose skin lesions, even-
tually leading to emerging cauliflower-like eruptions, with mu-riform cells in tissue provoking a granulomatous immuneresponse (1–3). Although traumatic inoculation from thornsor wood splinters is a likely source of onset of the disease (4),the infection process and route of dispersal have been insuffi-ciently clarified (5, 6), particularly in disseminated cases. Sev-eral members of the black yeast order Chaetothyriales in thegenera Cladophialophora, Exophiala, Fonsecaea, Phialophora, andRhinocladiella are able to cause chromoblastomycosis or chromo-blastomycosis-like infections, of which infections with C. carrioniiand F. pedrosoi are the most frequent (1, 7, 8).
Fonsecaea species are among the prevalent etiologic agents ofchromoblastomycosis in humid (sub)tropical climates (9). Thegenus Fonsecaea comprises three cryptic entities (F. pedrosoi, F.monophora, and F. nubica) potentially causing the same disease(10, 11). A specialized tissue form is produced, the “muriformcell,” consisting of spherical cells which develop one or morecross-septa. The invasive potentials of closely related siblings dif-fer significantly among species (12, 13). Fonsecaea pedrosoi and F.nubica are strictly associated with chromoblastomycosis and mu-riform cell formation, while F. monophora may also be involved indisseminated phaeohyphomycosis of the brain and other organsand with hyphae in tissue.
Cerebral infections by black fungi mostly involve the centralnervous system only, and the fungi rarely invade other organs. Theneurotropic species Cladophialophora bantiana is by far the mostfrequent agent (14, 15). In brain tissue, hyphal growth leads tocerebral abscesses and typically to death of the patient. Five caseshave been confirmed to have been caused by Fonsecaea monophora(16–19).
Fonsecaea infections are relatively frequent in the humid cli-mate zones of Brazil and southern China (7, 20). The state ofMaranhão in northeast Brazil is a region of chromoblastomycosishyperendemicity. Fonsecaea pedrosoi is classically assumed to be
responsible for almost 99% of the clinical cases (21, 22). However,since Fonsecaea agents of the disease are morphologically indistin-guishable (2, 5, 23) and specimens from cases have mostly notbeen cultured, it is possible that other agents have a higher prev-alence than currently thought. The three current agents have beendistinguished by multilocus sequencing, and several moleculartests are currently available for their distinction in the routinelaboratory (5, 24, 25). In the present report, we present findings ona case of chromoblastomycosis from Maranhão state which in-volved the skin and disseminated to the brain, having a fatalcourse. The strains recovered from skin, a lesion, and the brainabscess proved to be a new species of the genus Fonsecaea, which isdescribed here with phenotypic and molecular data.
CASE REPORT
The patient, a 52-year-old Caucasian male government official,born in Ceará and living in Cidelândia, Maranhão state, Brazil, forseveral decades, presented with a history of over 30 years of earlyverrucous lesions in the right upper arm, with slowly evolving
Received 9 March 2015 Returned for modification 15 April 2015Accepted 3 June 2015
Accepted manuscript posted online 17 June 2015
Citation de Azevedo CMPS, Gomes RR, Vicente VA, Santos DWCL, Marques SG, doNascimento MMF, Andrade CEW, Silva RR, Queiroz-Telles F, de Hoog GS. 2015.Fonsecaea pugnacius, a novel agent of disseminated chromoblastomycosis. J ClinMicrobiol 53:2674 –2685. doi:10.1128/JCM.00637-15.
Editor: D. W. Warnock
Address correspondence to Vania A. Vicente, [email protected], orG. Sybren de Hoog, [email protected].
C.M.P.S.D.A. and R.R.G. contributed equally to this article.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.00637-15.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.00637-15
2674 jcm.asm.org August 2015 Volume 53 Number 8Journal of Clinical Microbiology
on October 4, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
plaques, gradually affecting the entire arm, with much itching.Lesions appeared subsequently in the left hemifacial area, spread-ing to the nose and reaching the chin region (Fig. 1a and b). In July2012, the patient presented with a right hemiplegia. A skin biopsywas performed, demonstrating muriform cells in tissue (Fig. 1c),and the infection was diagnosed as chromoblastomycosis withisolation of CBM49 strains.
In magnetic resonance imaging (MRI) analysis, heterogeneouslesions with a necrotic and liquefied center were found in thefrontal and parietal right lobes of the brain, with an internal hem-orrhagic focus and areas of diffusion restriction on the periphery,presenting peripheral uptake by contrast. A large lesion measured4.5 cm in diameter, while two smaller infected areas were ob-served. The infection gradually expanded, leading to slight efface-ment of sulci and regional cisterns and compression of the rightlateral ventricle, without deviation from the midline lobe struc-tures. A second lesion with similar characteristics was located inthe left-capsular core region and measured about 2.0 cm in diam-eter, while a third cortical-subcortical lesion of smaller peripheraldimensions (about 1.3 cm in diameter) was observed in the leftfrontal lobe from which the CBM50 strain of Fonsecaea sp. wasisolated. Based on the preliminary diagnosis and the results of invitro susceptibility tests of a fungal cerebral abscess, treatmentusing 50 mg of liposomal amphotericin B (L-AMB) was started,followed by oral itraconazole at 400 mg/day for 6 months. Littleresponse of skin lesions or brain abscesses was observed, with thepatient presenting a worsening of symptoms. In January 2013, asurgery procedure for a brain abscess drainage was performed(Fig. 1d). The material collected showed the presence of fungal
cells in the walls of the abscess (Fig. 1f). After surgery, therapy withvoriconazole was started using two initial doses of 6 mg/kg of bodyweight spaced 12 h apart, followed by doses of 400 mg/day, as thepatient presented severe hypokalemia collateral effects due to theuse of amphotericin B. In May 2013, the patient died at 52 years ofage due to vascular leakage in the brainstem.
MATERIALS AND METHODSClinical samples. Two isolates were obtained on Sabouraud glucose agar(SGA) (Difco Laboratories, Paris, France) from skin and brain biopsyspecimens of a patient with disseminated chromoblastomycosis. Stockcultures were maintained on slants of 2% malt extract agar (MEA) andoatmeal agar (OA) at 24°C. For morphological studies, MEA slide cultureswere prepared and mounted in aniline blue. The cultures were depositedin the CBS collection (Table 1).
Physiology. Cardinal growth temperatures were determined on MEA.Plates were incubated in the dark for 3 weeks in a 21 to 36°C temperaturerange, with intervals of 3°C; growth was also recorded at 37 and 40°C.Experiments consisted of three simultaneous replicates for each testedstrain; averages of three measurements were calculated. Growth rates perspecies were obtained by calculation of the average growth of all isolatesproven to belong to that species and of the respective standard deviations.The optimum range (average � standard deviation) of growth tempera-tures and the maximum growth temperatures were determined using thetype strains of each species with three replicates.
Antifungal susceptibility testing was performed as described in CLSIdocument M38-A2, with some modifications according to a method de-scribed by Najafzadeh et al. (26). The antifungal agents were diluted infilter-sterilized RPMI 1640 medium (Sigma Chemical Co.) (using a 0.22-�m-pore-size filter) buffered to pH 7.0 with 0.165 M morpholinepro-panesulfonic acid (Sigma) with L-glutamine without bicarbonate to yield
FIG 1 Clinical case pictures. (a and b) Vegetating plaques, affecting the face (a) and right upper arm (b). (c) Histopathology of skin tissue biopsy. (d) Brainabscess drainage. (e) Fungal cells observed in the abscess walls. (f) Magnetic resonance image of brain lesion. (g) Histopathology of skin tissue biopsy specimen.
A Novel Agent of Disseminated Chromoblastomycosis
August 2015 Volume 53 Number 8 jcm.asm.org 2675Journal of Clinical Microbiology
on October 4, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
TA
BLE
1St
rain
san
alyz
eda
Nam
eSt
rain
no.
Cro
ss-r
efer
ence
no.
Sou
rce(
s)G
eogr
aph
ical
orig
in
Gen
Ban
kac
cess
ion
no.
ITS
BT
2C
DC
42
Fons
ecae
abr
asili
ensi
s*C
BS
1197
10dH
1681
8M
angr
ove
crab
,Scy
llase
rrat
aB
razi
lJN
1737
84Fo
nsec
aea
bras
ilien
sis
CB
S11
9718
dH16
816
Man
grov
ecr
ab,S
cylla
serr
ata
Bra
zil
JN17
3785
Fons
ecae
abr
asili
ensi
sC
BS
1270
12dH
2054
1T
ucu
mpa
lmtr
ee(l
ivin
gpl
ant)
Bra
zil,
Mar
anh
ãost
ate
JN17
3789
Fons
ecae
erec
ta*
CB
S12
5762
dH20
492
Shel
lofB
abas
suco
con
ut
(liv
ing
plan
t)B
razi
l,M
aran
hão
stat
eK
C88
6413
KF1
5521
9Fo
nsec
aeer
ecta
CB
S12
5763
dH20
513
Livi
ng
Jape
can
gapl
ant
Bra
zil,
Mar
anh
ãost
ate
KC
8864
14K
F155
221
Fons
ecae
min
ima
CB
S12
6865
dH20
511
Tu
cum
palm
tree
(liv
ing
plan
t)B
razi
l,M
aran
hão
stat
eK
C88
6420
KF1
5522
3Fo
nsec
aem
inim
aC
BS
1257
60dH
2046
3P
alm
leaf
Bra
zil,
Mar
anh
ãost
ate
KC
8864
16K
F155
222
Fons
ecae
min
ima
CB
S12
6024
dH21
195
Tu
cum
palm
tree
(liv
ing
plan
t)B
razi
l,M
aran
hão
stat
eK
C88
6419
Fons
ecae
am
onop
hora
CB
M20
;LM
ICR
O32
9B
iops
ysp
ecim
enfr
omch
rom
obla
stom
ycos
isle
sion
Bra
zil,
Mar
anh
ãost
ate
KR
7323
06K
R73
2312
KR
7323
18Fo
nsec
aea
mon
opho
ra*
CB
S26
9.37
dH12
659
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onSo
uth
Am
eric
aA
Y36
6906
EU
9385
47G
U19
7516
Fons
ecae
am
onop
hora
CB
S39
7.48
dH15
828
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onSo
uth
Am
eric
aE
U93
8585
EU
9385
55G
U19
7513
Fons
ecae
am
onop
hora
CB
S10
0430
dH15
137
Cer
ebra
lmyc
osis
Afr
ica
AY
3669
24Fo
nsec
aea
mon
opho
raC
BS
1022
29dH
1159
0D
ecay
ing
vege
tabl
eco
ver
Bra
zil,
Par
ana,
Ipor
aE
U93
8581
EU
9385
45G
U19
7518
Fons
ecae
am
onop
hora
CB
S10
2238
dH11
602
Soil
Bra
zil,
Par
ana,
Ipor
aA
Y36
6927
EU
9385
46G
U19
7517
Fons
ecae
am
onop
hora
CB
S10
2242
dH11
606
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onB
razi
l,Sa
nta
Cat
arin
ast
ate
EU
9385
83E
U93
8549
Fons
ecae
am
onop
hora
CB
S10
2243
dH11
607
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onB
razi
l,P
aran
ast
ate
EU
9385
79E
U93
8542
GU
1975
14Fo
nsec
aea
mon
opho
raC
BS
1022
46dH
1161
1B
iops
ysp
ecim
enfr
omch
rom
obla
stom
ycos
isle
sion
Bra
zil,
Par
ana
stat
eA
Y36
6928
EU
9385
43G
U19
7515
Fons
ecae
am
onop
hora
CB
S10
2248
dH11
613
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onB
razi
l,P
aran
ast
ate
AY
3669
26E
U93
8550
GU
1975
12Fo
nsec
aea
mon
opho
raC
BS
1158
30dH
1297
8C
ereb
ralm
ycos
isB
razi
l,M
inas
Ger
ais
stat
eE
U93
8582
EU
9385
48Fo
nsec
aea
mon
opho
raC
BS
1172
36dH
1533
0B
rain
biop
sysp
ecim
ens
Un
know
nA
B24
0948
EU
9385
51G
U19
7519
Fons
ecae
am
onop
hora
CB
S11
7238
dH13
130
Bra
inbi
opsy
spec
imen
sE
ngl
and
AB
2409
49E
U93
8553
Fons
ecae
am
onop
hora
CB
S11
7542
dH14
523
Bra
inbi
opsy
spec
imen
sU
SA,M
assa
chu
sett
sE
U93
8584
EU
9385
52Fo
nsec
aea
mon
opho
raC
BS
1217
21dH
1839
9;SU
MS0
246
Bio
psy
spec
imen
from
Ch
rom
obla
stom
ycos
isSo
uth
ern
Ch
ina
EF5
1376
8G
U19
7506
Fons
ecae
am
onop
hora
CB
S12
1724
dH18
402
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onSo
uth
ern
Ch
ina
GU
1975
08Fo
nsec
aea
mon
opho
raC
BS
1217
27dH
1840
5;SU
MS0
190
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onSo
uth
ern
Ch
ina
EF5
1376
4G
U19
7509
Fons
ecae
am
onop
hora
CB
S12
3849
dH20
215
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onA
fric
a,G
uin
eaFJ
7854
71FJ
7854
73Fo
nsec
aea
mul
tim
orph
osa*
CB
S98
0.96
NC
MH
1412
Cat
,su
bcu
tan
eou
ssp
ecim
enQ
uee
nsl
and
JF26
7657
HQ
6811
21Fo
nsec
aea
mul
tim
orph
osa
CB
S10
2224
dH11
584
Woo
d,G
revi
llea
Bra
zil,
Par
ana
stat
eE
U93
8595
HQ
6811
22Fo
nsec
aea
mul
tim
orph
osa
CB
S10
2226
dH11
587
Dec
ayin
gtr
eetr
un
kB
razi
lJF
2676
58H
Q68
1123
Fons
ecae
am
ulti
mor
phos
aC
BS
1022
35dH
1159
7W
ood
Bra
zil,
Par
ana
stat
eJF
2676
60H
Q68
1124
Fons
ecae
am
ulti
mor
phos
aC
BS
1022
40dH
1160
4So
ilco
ver
Bra
zil,
Par
ana
stat
eJF
2676
55Fo
nsec
aea
nubi
caC
BS
1217
20dH
1839
8;SU
MS0
251
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onSo
uth
ern
Ch
ina
KP
1321
98G
U19
7526
Fons
ecae
anu
bica
CB
S12
1733
dH18
411;
SUM
S001
1B
iops
ysp
ecim
enfr
omch
rom
obla
stom
ycos
isle
sion
Sou
ther
nC
hin
aK
P13
2199
GU
1975
26Fo
nsec
aea
nubi
caC
BS
1217
34dH
1841
2;SU
MS0
255
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onSo
uth
ern
Ch
ina
GU
1975
25Fo
nsec
aea
nubi
ca*
CB
S26
9.64
dH15
656
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onC
amer
oon
EU
9385
92E
U93
8574
GU
1975
24Fo
nsec
aea
nubi
ca*
CB
S27
0.37
dH15
657
Un
know
nFr
ance
GU
1975
22Fo
nsec
aea
nubi
caC
BS
277.
29dH
h15
668
Der
mat
itis
verr
uco
saB
razi
lE
U93
8594
EU
9385
77G
U19
7521
Fons
ecae
anu
bica
CB
S44
4.62
dH15
586
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onSu
rin
amA
Y36
6931
EU
9385
75G
U19
7523
Fons
ecae
anu
bica
CB
S55
7.76
AT
CC
2817
4U
nkn
own
Un
know
nE
U93
8593
EU
9385
76G
U19
7520
Fons
ecae
ape
dros
oiC
BM
01;L
MIC
RO
310
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onB
razi
l,M
aran
hão
stat
eK
R73
2301
KR
7323
07K
R73
2313
Fons
ecae
ape
dros
oiC
BM
02;L
MIC
RO
311
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onB
razi
l,M
aran
hão
stat
eK
R73
2302
KR
7323
08K
R73
2314
Fons
ecae
ape
dros
oiC
BM
03;L
MIC
RO
312
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onB
razi
l,M
aran
hão
stat
eK
R73
2303
KR
7323
09K
R73
2315
Fons
ecae
ape
dros
oiC
BM
04;L
MIC
RO
313
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onB
razi
l,M
aran
hão
stat
eK
R73
2304
KR
7323
10K
R73
2316
Fons
ecae
ape
dros
oiC
BM
05;L
MIC
RO
314
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onB
razi
l,M
aran
hão
stat
eK
R73
2305
KR
7323
11K
R73
2317
Fons
ecae
ape
dros
oiC
BS
1022
47dH
1161
2B
iops
ysp
ecim
enfr
omch
rom
obla
stom
ycos
isle
sion
Bra
zil
AY
3669
19E
U93
8566
GU
1974
90
de Azevedo et al.
2676 jcm.asm.org August 2015 Volume 53 Number 8Journal of Clinical Microbiology
on October 4, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
Fons
ecae
ape
dros
oiC
BS
1179
10dH
1447
7H
um
anm
ale,
chro
mob
last
omyc
osis
han
dle
sion
Ven
ezu
ela
GU
1975
00Fo
nsec
aea
pedr
osoi
CB
S12
2740
dH18
430
Ch
rom
obla
stom
ycos
is,f
oot
Mex
ico
EU
9385
90E
U93
8572
GU
1975
01Fo
nsec
aea
pedr
osoi
CB
S12
2741
dH18
431
Ch
rom
obla
stom
ycos
is,f
oot
Mex
ico
EU
9385
89E
U93
8570
GU
1974
98Fo
nsec
aea
pedr
osoi
CB
S12
2849
dH18
902
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onM
exic
o,M
exic
oC
ity
GU
1974
85Fo
nsec
aea
pedr
osoi
CB
S12
5749
dH20
488
Rot
tin
gw
ood
from
back
yard
ofpa
tien
t’s
hou
seB
razi
l,M
aran
hão
stat
eK
C88
6423
Fons
ecae
ape
dros
oiC
BS
212.
77dH
1554
9B
iops
ysp
ecim
enfr
omch
rom
obla
stom
ycos
isle
sion
Net
her
lan
dsA
Y36
6912
EU
9385
68G
U19
7495
Fons
ecae
ape
dros
oiC
BS
253.
49dH
1562
0B
iops
ysp
ecim
enfr
omch
rom
obla
stom
ycos
isle
sion
Uru
guay
AY
3669
21E
U93
8571
GU
1974
97Fo
nsec
aea
pedr
osoi
*C
BS
271.
37dH
1565
9B
iops
ysp
ecim
enfr
omch
rom
obla
stom
ycos
isle
sion
Sou
thA
mer
ica
AY
3669
14E
U93
8559
GU
1974
91Fo
nsec
aea
pedr
osoi
CB
S27
2.37
dH15
661
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onB
razi
lA
Y36
6917
GU
1974
89Fo
nsec
aea
pedr
osoi
CB
S27
3.66
dH15
663
Mou
sepa
ssag
e,so
ilV
enez
uel
aA
Y36
6916
EU
9385
65G
U19
7499
Fons
ecae
ape
dros
oiC
BS
274.
66dH
1566
5M
ouse
pass
age,
soil
Ven
ezu
ela
EU
9385
87E
U93
8561
GU
1974
96Fo
nsec
aea
pedr
osoi
CB
S28
5.47
dH15
680
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onP
uer
toR
ico
EU
9385
91E
U93
8573
GU
1975
02Fo
nsec
aea
pedr
osoi
CB
S34
2.34
MU
CL
9758
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
lesi
onP
uer
toR
ico
AY
3669
15E
U93
8569
GU
1975
03Fo
nsec
aea
pedr
osoi
CB
S67
0.66
dH16
157
Mou
sepa
ssag
e,so
ilV
enez
uel
aE
U93
8588
EU
9385
64G
U19
7492
Fons
ecae
ape
dros
oiC
BS
671.
66dH
1615
9M
ouse
pass
age,
soil
Ven
ezu
ela
EU
9385
86E
U93
8560
GU
1974
94Fo
nsec
aea
pugn
aciu
s*C
BS
1392
14C
BM
49;L
GM
F358
Bio
psy
spec
imen
from
chro
mob
last
omyc
osis
skin
lesi
onB
razi
l,M
aran
hão
stat
eK
R70
6553
KR
7065
47K
R70
6551
Fons
ecae
apu
gnac
ius
dH24
207
CB
M50
;LG
MF3
59B
iops
ysp
ecim
enfr
omch
rom
obla
stom
ycos
isbr
ain
lesi
onB
razi
l,M
aran
hão
stat
eK
R70
6554
KR
7065
48K
R70
6552
Cla
doph
ialo
phor
aar
xii*
CB
S30
6.94
IFM
5202
2H
um
an,T
rach
eala
bsce
ssG
erm
any
AB
1091
81G
U19
7529
Cla
doph
ialo
phor
aar
xii
CB
S40
9.96
dH15
849
Hu
man
,dis
sem
inat
edU
nkn
own
EU
1039
87E
U13
7192
Cla
doph
ialo
phor
aar
xii
CB
S10
2461
dH11
524
Bra
inU
SA,F
lori
da,M
iam
iA
Y85
7509
Cla
doph
ialo
phor
aba
ntia
naC
BS
444.
96D
isse
min
ated
infe
ctio
nin
dog
Sou
thA
fric
aE
U10
3994
Cla
doph
ialo
phor
aba
ntia
naC
BS
648.
96U
AM
H38
30D
og,l
iver
Bar
bado
sE
U10
3993
Cla
doph
ialo
phor
aba
ntia
naC
BS
678.
79U
AM
H49
92Sk
inle
sion
inca
tU
SAE
U10
3992
Cla
doph
ialo
phor
aba
ntia
naC
BS
1004
29A
TC
C24
928
Hu
man
,bra
inab
sces
sU
nkn
own
KF1
5521
2C
lado
phia
loph
ora
bant
iana
CB
S10
2586
dH11
331
Hu
man
,bra
inab
sces
sB
razi
lE
U10
3990
Cla
doph
ialo
phor
aba
ntia
naC
BS
1178
90dH
1447
6H
um
an,s
kin
infe
ctio
nV
enez
uel
aE
U13
7279
Cla
doph
ialo
phor
aba
ntia
naC
BS
1197
19dH
1451
5H
um
an,s
kin
infe
ctio
nT
hai
lan
dE
U10
3991
Cla
doph
ialo
phor
aca
rrio
nii
CB
S10
8.97
UN
EFM
D95
01Sk
in,p
atie
nt
wit
hch
rom
obla
stom
ycos
isV
enez
uel
aE
U13
7306
EU
1371
88C
lado
phia
loph
ora
carr
ioni
iC
BS
161.
54H
um
anA
ust
ralia
EU
1373
13E
U13
7198
Cla
doph
ialo
phor
aca
rrio
nii
CB
S16
5.54
Hu
man
Ven
ezu
ela
EU
1373
05E
U13
7187
Cla
doph
ialo
phor
aca
rrio
nii
CB
S40
6.96
UA
MH
4366
Hu
man
Au
stra
lia,Q
uee
nsl
and
EU
1373
17E
U13
7202
Cla
doph
ialo
phor
aca
rrio
nii
CB
S86
1.96
UN
EFM
9607
Dry
plan
tde
bris
Ven
ezu
ela
EU
1373
09E
U13
7194
Cla
doph
ialo
phor
ach
aeto
spir
aC
BS
491.
70P
icea
abie
s,ro
otD
enm
ark
EU
0354
05C
lado
phia
loph
ora
chae
tosp
ira
CB
S51
4.63
AT
CC
1627
4W
hea
tfi
eld
soil
Ger
man
yE
U03
5406
Cla
doph
ialo
phor
ade
vrie
sii*
CB
S14
7.84
AT
CC
5628
0D
isse
min
ated
infe
ctio
nin
hu
man
pati
ent
Gra
nd
Cay
man
Isla
nds
EU
1039
85C
lado
phia
loph
ora
devr
iesi
iC
BS
1270
19dH
2117
0W
ood
clot
hes
line
Bra
zil,
Mar
anh
ãost
ate
KC
8864
02C
lado
phia
loph
ora
devr
iesi
iC
BS
1278
21dH
2101
1C
ocon
ut
ofB
abas
supa
lmtr
eeB
razi
l,M
aran
hão
stat
eK
C88
6403
Cla
doph
ialo
phor
aem
mon
sii
CB
S64
0.96
dH17
418
Cat
,su
bcu
tan
eou
ssp
ecim
enU
nkn
own
EU
1039
95C
lado
phia
loph
ora
emm
onsi
i*C
BS
979.
96C
DC
B-3
875
Hu
man
,su
bcu
tan
eou
sle
sion
USA
,Vir
gin
iaE
U10
3996
Cla
doph
ialo
phor
aem
mon
sii
CB
S10
2594
CD
CB
-542
0H
um
an,u
lcer
ofle
fth
and
USA
,Vir
gin
iaK
F155
214
Cla
doph
ialo
phor
aem
mon
sii
dH13
029
Bra
inU
nkn
own
AY
8575
18C
lado
phia
loph
ora
imm
unda
*C
BS
834.
96dH
2128
7Fo
rear
m,h
um
anU
SAE
U13
7318
EU
1372
03C
lado
phia
loph
ora
imm
unda
CB
S10
9797
dH11
474
Bio
filt
erin
ocu
late
dw
ith
soil
Ger
man
y,K
aise
rsla
ute
rnFJ
3852
71E
U13
7260
Cla
doph
ialo
phor
aim
mun
daC
BS
1105
51dH
1525
0So
ilu
nde
rga
solin
est
atio
nT
he
Net
her
lan
dsA
Y85
7510
EU
1372
61C
lado
phia
loph
ora
imm
unda
CB
S12
3977
NC
PF
4725
Hu
man
,su
bcu
tan
eou
ssp
ecim
enU
nit
edK
ingd
omE
U13
7320
EU
1372
60C
lado
phia
loph
ora
min
oura
e*C
BS
556.
83A
TC
C52
853
Dec
ayin
gw
ood
Japa
nA
Y25
1087
Cla
doph
ialo
phor
am
inou
rae
dH18
460
Pol
lute
dso
il,pe
trol
refi
ner
yU
nkn
own
KF1
5521
5
(Con
tin
ued
onfo
llow
ing
page
)
A Novel Agent of Disseminated Chromoblastomycosis
August 2015 Volume 53 Number 8 jcm.asm.org 2677Journal of Clinical Microbiology
on October 4, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
two times their concentrations and dispensed into 96-well flat-bottommicrodilution trays at final concentrations of 0.016 to 16 �g/ml for am-photericin B (Bristol-Myers Squibb, Woerden, The Netherlands), itra-conazole (Janssen Research Foundation, Beerse, Belgium), and voricona-zole (Pfizer Central Research, Sandwich, United Kingdom) and of 0.063to 64 �g/ml for fluconazole (Pfizer). Paecilomyces variotii ATCC 22319.Candida parapsilosis ATCC 22019, and C. krusei ATCC 6258 served asquality control strains. MICs of amphotericin B, itraconazole, and vori-conazole were determined visually with an inverted mirror by comparisonof growth in the wells containing the drug with that of the drug-freecontrol.
DNA isolation, amplification, and sequencing. Colonies were culti-vated on Sabouraud’s glucose agar (SGA), and genomic DNA extractionwas undertaken using an UltraClean Microbial DNA kit (MO Bio, Carls-bad, CA, USA) according to the manufacturer’s instructions. The largesubunit (LSU) of the nuclear rRNA gene was amplified using primers NL1and LR5 (27). Three gene regions were chosen for species delimitation: theribosomal DNA (rDNA) internal transcribed spacer (ITS) region, the par-tial cell division cycle gene (CDC42), and �-tubulin (BT2). ITS ampliconswere generated with primers V9G and LS266 (28, 29) and were sequencedwith primers ITS1 and ITS4. CDC42 amplification and sequencing weredone with cdc42w and cdc42f (23) and BT2 amplification and sequencingwith Bt-2a and T2 (30). Amplification reaction mixtures had a total vol-ume of 12.5 �l which was composed of 1� PCR buffer, 2.0 mM MgCl2, 25�M deoxynucleoside triphosphates (dNTPs), 0.5 �M (each) forward andreverse primers, 1 U of BioTaq DNA polymerase, and 10 ng of genomicDNA. Amplification was performed in an ABI Prism 2720 thermocycler(Applied Biosystems, Foster City, USA), as follows: 95°C for 4 min, fol-lowed by 35 cycles consisting of 95°C for 45 s, 52°C for 30 s, and 72°C for2 min, and a delay at 72°C for 7 min. Annealing temperatures werechanged to 52°C, 55°C, and 58°C for the ITS region, CDC42, and BT2.Amplicons were cleaned with exonuclease I and shrimp alkaline phospha-tase (SAP) according to the manufacturer’s instructions. Amplicons weresequenced with BigDye Terminator cycle sequencing kit v. 3.1 (AppliedBiosystems, Foster City, CA, USA) according to the manufacturer’s in-structions, and reaction mixtures were purified with Sephadex G-50 Fine(GE Healthcare Bio-Sciences, Uppsala, Sweden). Sequences were ana-lyzed on an ABI Prism 3700 DNA sequencer (Applied Biosystems, FosterCity, CA, USA).
Phylogenetic analysis. Consensus sequences of the ITS region, BT2,CDC42, and the LSU were visually inspected using MEGA v.6 software(31). Alignments were made using the online MAFFT interface (32). TheITS region, BT2, and CDC42 were first analyzed separately, and then aphylogenetic multilocus analysis was used to investigate relationships be-tween 99 CBS reference strains of Cladophialophora (n � 37) and Fonse-caea (n � 62), with Cladophialophora yegresii and C. carrionii comprisingthe outgroup (Table 1). Data representing conflicts were estimated us-ing the partition homogeneity test available in PAUP* v. 4.Ob10 (33). Wedid the LSU analyses to assess the phylogenetic position of the speciesanalyzed in this study. The phylogenetic analyses of the small subunit(SSU) and LSU groups previously recognized in the Herpotrichiellaceae byde Hoog et al. in 2011 (34), by Feng et al. in 2012 (27), and by Vicente et al.in 2014 (5) were taken as a basis for clade delimitation. The trees wereconstructed with 1,000 bootstrap replicates using the maximum likeli-hood function implemented in Mega v.6 software (31), and the best evo-lutionary model corresponding to the data set was used. Bootstrap valuesequal to or greater than 80% were considered statistically significant (35).
Nucleotide sequence accession numbers. The DNA sequences (ofB-tubulin, the ITS region, and CDC42) of new species described in thisstudy have been deposited in GenBank, and the accession numbers ofclinical and reference strains are presented in Table 1. The GenBank ac-cession numbers of the large subunit of the nuclear rRNA gene of strainsCBS 139214 (CBM49) and dH 24207 (CBM50) of the new species F.pugnacius are KR706549 and KR706550, respectively.T
AB
LE1
(Con
tin
ued
)
Nam
eSt
rain
no.
Cro
ss-r
efer
ence
no.
Sou
rce(
s)G
eogr
aph
ical
orig
in
Gen
Ban
kac
cess
ion
no.
ITS
BT
2C
DC
42
Cla
doph
ialo
phor
am
inou
rae
CB
S12
2275
dH18
466
Pol
lute
dso
ilfr
ompe
trol
Bra
zil
KF1
5521
6C
lado
phia
loph
ora
satu
rnic
aC
BS
1022
30dH
1159
1V
eget
able
cove
ran
dso
ilB
razi
l,P
aran
ást
ate
AY
8575
08C
lado
phia
loph
ora
satu
rnic
aC
BS
1096
28dH
1233
3D
ead
tree
Uru
guay
EU
1039
83C
lado
phia
loph
ora
satu
rnic
a*C
BS
1187
24dH
1293
9In
terd
igit
alto
ele
sion
,ch
ildB
razi
l,P
aran
ast
ate
EU
1039
84C
lado
phia
loph
ora
yegr
esii
CB
S11
4405
UN
EFM
SgSr
3St
enoc
ereu
sgr
iseu
spl
ant
Ven
ezu
ela
EU
1373
22E
U13
7209
Cla
doph
ialo
phor
aye
gres
iiC
BS
1144
06U
NE
FMSg
SR1
Sten
ocer
eus
gris
eus
plan
tV
enez
uel
aE
U13
7323
EU
1372
08C
lado
phia
loph
ora
yegr
esii
CB
S11
4407
UN
EFM
SgSR
2St
enoc
ereu
sgr
iseu
spl
ant
Ven
ezu
ela
EU
1373
24a
AT
CC
,Am
eric
anT
ype
Cu
ltu
reC
olle
ctio
n;C
BM
,ch
rom
obla
stom
ycos
iscu
ltu
reco
llect
ion
ofFe
dera
lUn
iver
sity
ofM
aran
hão
Bra
zil;
CB
S,C
BS
Fun
galB
iodi
vers
ity
Cen
tre,
Utr
ech
t,T
he
Net
her
lan
ds;d
H,C
olle
ctio
nde
Hoo
gSy
bren
,h
ouse
dat
CB
S;C
DC
,Cen
ters
for
Dis
ease
Con
trol
and
Pre
ven
tion
,Atl
anta
,GA
,USA
(sam
eas
NC
DC
);IF
M,R
esea
rch
Cen
ter
for
Pat
hog
enic
Fun
gian
dM
icro
bial
Tox
icos
es,C
hib
aU
niv
ersi
ty,C
hib
a,Ja
pan
;LM
ICR
O,C
ult
ure
Col
lect
ion
ofL
abor
ator
yof
Mic
roor
gan
ism
s,Fe
dera
lUn
iver
sity
ofP
aran
a,C
uri
tiba
,Bra
zil;
MU
CL,
Un
iver
sité
Cat
hol
iqu
ede
Lou
vain
,Lou
vain
-la-
Neu
ve,B
elgi
um
;NC
MH
,Th
eN
orth
Car
olin
aM
emor
ialH
ospi
tal,
Un
iver
sity
ofN
orth
Car
olin
a,C
hap
elH
ill,N
C,U
SA;N
CP
F,T
he
Nat
ion
alC
olle
ctio
nof
Pat
hog
enic
Fun
gi,U
nit
edK
ingd
om;S
UM
S,Si
dney
Un
iver
sity
Mu
seu
ms,
Sidn
ey,A
ust
ralia
;UA
MH
,Un
iver
sity
ofA
lber
taM
old
Her
bari
um
and
Cu
ltu
reC
olle
ctio
n,
Edm
onto
n,C
anad
a;U
NE
FM,U
niv
ersi
dad
Nac
ion
alE
xper
imen
talF
ran
cisc
ode
Mir
anda
,Ven
ezu
ela;
BT
2,pa
rtia
lbet
a-tu
bulin
gen
e;IT
S,in
tern
altr
ansc
ribe
dsp
acer
regi
ons
ofth
erD
NA
and
inte
rven
ing
5.8S
nu
clea
rri
boso
mal
DN
A(n
rDN
A);
CD
C42
,par
tial
cell
divi
sion
cycl
ege
ne.
de Azevedo et al.
2678 jcm.asm.org August 2015 Volume 53 Number 8Journal of Clinical Microbiology
on October 4, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
RESULTSPhylogeny. As shown by comparisons to a selected set of partialLSU rDNA sequences of members of Chaetothyriales available atCBS, the strains under study from a patient with severe chromo-blastomycosis and systemic infection were identical and located ina “bantiana clade” (34) containing Fonsecaea species known asagents of chromoblastomycosis, as well as the pathogenic Clado-phialophora species C. bantiana, C. arxii, and C. devriesii and theenvironmental species C. minourae, F. erecta, and F. minima (seeFig. S1 in the supplemental material).
Multilocus sequence analyses using the ITS region, BT2, andCDC42 were performed for elucidation of the positions of strainswith respect to agents of skin and brain disease within the Chae-tothyriales. The tree was constructed using the maximum likeli-hood method and the Kimura 2-parameter substitution model. Atotal of 1,607 sites were evaluated for the ITS region, BT2, andCDC42, corresponding to 602, 394, and 610 sites, respectively. Ofthese, 1,042 were conserved, 510 were variable, 419 were parsimo-niously informative (pi), and 36 were unique. The empirical basefrequencies were 0.02270457 for pi(A), 0.027624 for pi(C),0.023773281 for pi(G), and 0.02589833 for pi(T), with 1,000 boot-strap inferences and with Cladophialophora yegresii and C. carrio-nii selected as the outgroup. Five subclades were observed corre-sponding to F. monophora subdivided in two clusters (A and B)which grouped strains from different geographical regions indi-cating populations of a single species. Fonsecaea pedrosoi, F. nu-bica, and an unnamed group all showed high bootstrap supportvalues (Fig. 2). The unnamed group was a sister group of theknown Fonsecaea agents of chromoblastomycosis and unambigu-ously different from described environmental Fonsecaea species.
In the consensus tree of the ITS region and partial BT2 andCDC42 genes (Fig. 3), the unnamed strains designated CBM49(CBS 139214) from skin and CBM50 (dH 24207) from brain werefound to be identical for all partitions and grouped with remain-ing members of Fonsecaea causing chromoblastomycosis as a sis-ter clade with high statistical support in all trees (Fig. 3). Theunnamed group was located at a significant distance in all generegions analyzed (ITS region, 3.6%; CDC42, 5.7%; BT2, 7.0%)and was judged to represent a novel Fonsecaea species.
Morphology and physiology. The cardinal growth tempera-tures of the voucher strains of this new species indicated growthover the entire range between 21 and 37°C, with optimal develop-ment at 27 to 33°C. The maximum growth temperature of thestrains was 37°C, with no growth observed at 40°C (Fig. 4). Thestrains (CBM49 and CBM50) were susceptible to the tree antifun-gals tested, with MICs of 1 �g/ml for amphotericin B, 0.06 �g/mlfor itraconazole, and 0.5 �g/ml for voriconazole. The novel spe-cies is described as follows.
Taxonomy. Fonsecaea pugnacius R.R. Gomes, V.A. Vicente,C.M.P.S. Azevedo & G.S. de Hoog sp. nov. Figure 5. MycobankMB. Etymology: named after the aggressive infection caused in ahuman patient. Colonies on SGA at 30°C restricted, compact, cir-cular, velvety to hairy, olivaceous gray; reverse olivaceous black.Germinating cells and budding cells absent. Hyphae smoothwalled, pale olivaceous brown, 1.5 to 2.4 �m wide, septate every
FIG 2 Multilocus tree of Fonsecaea and Cladophialophora based on confi-dently aligned ITS and partial BT2 and CDC42 sequences constructed withmaximum likelihood implemented in MEGA 6.0. Bootstrap values of �80%from 1,000 resampled data sets are shown with branches. Cladophialophorayegresii and C. carrionii (Table 1) comprised the outgroup. Novel species andclassical Fonsecaea species causing chromoblastomycosis are indicated withbackgrounds shaded in different colors. *, type strain. Species names followedby boxes in green, environmental strains; ochre, agents of chromoblastomy-cosis; red, agents of cerebral infection. I, isolates from south China;
II, isolates from United Kingdom and Europe; III, isolates from Brazil; IV,isolates from South America; V, isolates from the United States; VI, isolatesfrom Africa.
A Novel Agent of Disseminated Chromoblastomycosis
August 2015 Volume 53 Number 8 jcm.asm.org 2679Journal of Clinical Microbiology
on October 4, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
7.3 to 18.4 �m, with the presence of torulose hyphae. Conidio-phores erect, olivaceous brown, apically densely branched; some-times slightly differentiated, loosely branched. Conidiogenouscells pale olivaceous brown, intercalary or terminal, with rather
flat denticles in the apical part bearing conidia. Conidia pale oli-vaceous, 1 celled, single or in short chains of 1 to 3 conidia, mostlyellipsoidal to tear shaped, sometimes subspherical, 2.5 to 3.9 by 2.3to 3.8 �m (subspherical) to 1.8 to 5.3 by 1.3 to 5.2 �m (tear
FIG 3 Phylogenetic trees of Fonsecaea and Cladophialophora based on confidently aligned ITS and partial BT2 and CDC42 sequences constructed with maximumlikelihood implemented in MEGA 6.0. Bootstrap values of �80% from 1,000 resampled data sets are shown with branches. Cladophialophora arxii was taken asthe outgroup. Novel species and classical Fonsecaea species causing chromoblastomycosis are differentiated with color shading. *, type strain of the species.
de Azevedo et al.
2680 jcm.asm.org August 2015 Volume 53 Number 8Journal of Clinical Microbiology
on October 4, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
shaped). Chlamydospore-like cells sometimes present. Teleo-morph unknown. Optimal growth at 27 to 33°C, scant growth at37°C, no growth at 40°C.
Holotype: herbarium specimen CBS-H 22056, type strain CBS139214, from skin lesion of human patient, Cidelândia, MaranhãoBrazil; additional isolate CBM50 (dH 24207) from brain tissue ofthe same patient.
DISCUSSION
Black yeast and related members of the fungal order Chaetothyria-les are implicated in a range of diseases with high morbidity andmortality. Fatal dissemination and neurotropism of several spe-cies have been reported in both immunocompromised and im-munocompetent individuals (36). Disseminated infections arevery refractory to treatment and commonly have a fatal outcome(1, 19, 37). Agents of cerebral infection mostly cause primary ce-rebral infection, where neurological symptoms are the first indi-cations of infection and spreading from other sites to the braintakes place unnoticed (38). The main neurotropic agents in theChaetothyriales are Cladophialophora bantiana (39, 40), Rhinocla-diella mackenziei (41–43), and Fonsecaea monophora (17). Exo-phiala dermatitidis is also neurotropic but tends to cause dissem-inated infections (44). The great majority of these cases werereported from humans, while reports of infections of other warm-blooded mammals are extremely rare (20). Another category isdisseminated infections with chromoblastomycosis-like lesionsbut without cerebral involvement, such as in Veronaea botryosa(45).
The genus Fonsecaea presently contains three species, which allare potential etiologic agents of human chromoblastomycosis (10,11). This disease is chronic, involves cutaneous and subcutaneoustissue, and is characterized by the presence of muriform cells andabsence of hyphae in tissue, provoking a granulomatous immuneresponse (46, 47). Sources of infection and dissemination remainenigmatic, however, and infections may be extremely recalcitrantto antifungal treatment.
Fonsecaea pedrosoi seems to be a pathogen strictly associatedwith chromoblastomycosis, while F. monophora is an opportunistwith a more variable clinical spectrum. Fonsecaea monophora isthe fourth species of Chaetothyriales and exhibits marked neurot-ropism. Mostly these were primary brain infections; i.e., the firstclinical symptoms were of a neurological nature and no portal ofentry could be ascertained (38), although an inhalation route waslikely, as was the case in several F. monophora cases (Table 2).Several of these cases were in immunocompetent hosts (Table 2).
Chromoblastomycosis usually occurs in the skin and subcuta-neous tissue, and the fungi rarely invade other organs (16, 49, 50).In the present study of a Fonsecaea pugnacius infection, we ob-served a very chronic case of chromoblastomycosis which finallycaused secondary cerebritis by dissemination to the brain, despiteapparent intact immunity of the patient. Remarkably, the histo-pathologies of skin and brain infections are very different: mu-riform cells are produced in skin, but hyphae are produced inbrain. This type of dissemination and the apparent conversionto another invasive morphology have not been observed in F.monophora, and the issue arises concerning whether this virulentability is characteristic for F. pugnacius. In this sense, the speciesseems to be unique; animal studies are necessary to address theseissues.
The invasive potentials of black yeast-like fungi differ signifi-cantly between species (13, 51). Fonsecaea pedrosoi and F. nubicaare narrowly associated with chromoblastomycosis, while F.monophora is also involved in phaeohyphomycosis of brain andother organs (11). Fonsecaea pugnacius behaves differently again.
Although there is no standard antifungal for the treatment ofcerebral phaeohyphomycosis, it is generally agreed that a combi-nation of medical and surgical treatment is required (17). Ourpatient was initially treated with liposomal amphotericin B, fol-lowed by oral itraconazole. The L-AMB treatment had to bechanged to voriconazole treatment because the patient developedsevere hypokalemia and presented worsening of symptoms. Sur-
FIG 4 Colony diameters at various temperatures ranging in 3°C increments from 21 to 40°C, measured after 3 weeks on 2% MEA, were calculated for Fonsecaeapugnacius and related species.
A Novel Agent of Disseminated Chromoblastomycosis
August 2015 Volume 53 Number 8 jcm.asm.org 2681Journal of Clinical Microbiology
on October 4, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
FIG 5 Fonsecaea pugnacius microscopic morphology. (a) Colonies on SGA. (b) Conidiogenous cells. (c) Conidia formed in coherent chains. (d to k) Conidio-phores and conidia. (l) Torulose hyphae. (m to r) Conidia and yeast cells. Bars, 10 �m.
2682 jcm.asm.org August 2015 Volume 53 Number 8Journal of Clinical Microbiology
on October 4, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
gery for brain abscess drainage was performed, but vascular leak-age led to death.
Fonsecaea represents a highly complex genus containing nu-merous cryptic species. Judging from LSU sequence analysis, F.pugnacius clusters with some known potential agents of systemicdisease, with the Fonsecaea agents of chromoblastomycosis as thenearest neighbors. Phylogenetically, Fonsecaea is intermingledwith Cladophialophora species, composing a bantiana clade (34)comprising mainly thermophilic species. The maximumgrowth temperature of F. pugnacius strains analyzed was found tobe 37°C, with no growth observed at 40°C (Fig. 4). These cardinaltemperatures are similar to those of Fonsecaea species causingchromoblastomycosis (5, 11, 52). Vicente et al. (22) noted thatthermotolerance is probably an important virulence factor inChaetothyriales. Cladophialophora species causing systemic infec-tion are able to grow at 40°C (50).
Although little is known about the pathogenic mechanism ofthese fungi, especially in immunocompetent hosts, neuroinvasiveinfection caused by Fonsecaea pugnacius seems to be differentfrom that caused by Cladophialophora species, such as has beenobserved in cases of infection by C. bantiana, which is exclusivelyan agent of primary human brain infection supposedly caused viainhalation and occurring mainly in immunocompromised hosts.The route of neuroinvasive infection by F. monophora is unclear(Table 2) and also occurs in individuals with apparently normalimmune systems. The new species F. pugnacius is a new agent ofchromoblastomycosis with an apparent ability to disseminate andcause fatality in immunocompetent individuals. Understandingthe route of dissemination and the factors involved in the switchfrom muriform cells to hyphae is important to develop novelstrategies for treatment and control of advanced cases of chromo-blastomycosis.
ACKNOWLEDGMENTS
We thank Bert Gerrits van de Ende from the CBS-KNAW Fungal Biodi-versity Centre for technical assistance.
The work of R.R.G., C.E.W.A., and M.M.F.D.N. was supported by aBrazilian government fellowship and by financial support (PVE-Project)from the Brazilian Federal Agency for Support and Evaluation of Gradu-ate Education (CAPES) and National Counsel of Technological and Sci-entific Development (CNPq). R.R.G., V.A.V., M.M.F.D.N., and C.E.W.A.received fellowships from Coordination for the Improvement of HigherEducation Personnel (CAPES), Brasilia, Brazil. R.R.G. and V.A.V. re-ceived fellowships from National Council for Scientific and TechnologicalDevelopment (CNPq), Brasilia, Brazil.
REFERENCES1. Queiroz-Telles F, Esterre P, Perez-Blanco M, Vitale RG, Salgado CG,
Bonifaz A. 2009. Chromoblastomycosis: an overview of clinical manifes-tation, diagnosis and treatment. Med Mycol 47:1–13. http://dx.doi.org/10.1080/13693780802538001.
2. De Hoog GS, Attili-Angelis D, Vicente VA, Gerrits van den Ende AHG,Queiroz-Telles F. 2004. Molecular ecology and pathogenic potential ofFonsecaea species. Med Mycol 42:405– 416. http://dx.doi.org/10.1080/13693780410001661464.
3. Bonifaz A, Carrasco-Gerard E, Saúl A. 2001. Chromoblastomycosis:clinical and mycologic experience of 51 cases. Mycoses 44:1–7. http://dx.doi.org/10.1046/j.1439-0507.2001.00613.x.
4. Salgado CG, da Silva JP, Diniz JA, da Silva MB, da Costa PF, TeixeiraC, Salgado UI. 2004. Isolation of Fonsecaea pedrosoi from thorns of Mi-mosa pudica, a probable natural source of chromoblastomycosis. Rev InstMed Trop São Paulo 46:33–36. http://dx.doi.org/10.1590/S0036-46652004000100006.
5. Vicente VA, Najafzadeh MJ, Sun J, Gomes RR, Robl D, Marques SG,
TA
BLE
2D
eepin
fections
caused
byFonsecaea
agents
ofch
romoblastom
ycosisa
Speciesan
dstrain
Clin
icalfeature
Host
status
Neu
rotropismD
issemin
ationor
skinin
volvemen
tM
uriform
cellsin
skin
Hyph
aeor
other
forms
inbrain
oroth
erlocation
Age
(yrs)/sex/race/geograph
icallocation
Portalofen
trySpecim
enstu
diedR
eference
orsou
rceIm
mu
nologicalstatu
s
Fonsecaeam
onophoraC
BS
117238P
rimary
cerebralSkin
lesions
absent
Absen
tY
es53/M
/wh
ite/Un
itedK
ingdom
Un
know
nB
rainbiopsy
17Im
mu
nocom
petent
Fonsecaeam
onophoraC
BS
117236P
rimary
cerebral(?)Skin
lesions
absent
with
tibiallesions
Absen
tM
assofcells
inbrain
(brown
colored,fu
ngal)
62/F/NI/U
SAU
nkn
own
Brain
and
bone
biopsy
18Im
mu
nosu
ppression/liver
transplan
trecipien
t
Fonsecaeam
onophoraC
BS
100430P
rimary
cerebral(?)Skin
lesions
absent
Absen
tH
yphae
and
fun
galcell-like
forms
10/M/black
African
/Con
goU
nkn
own
Au
topsy48
Imm
un
ocompeten
t/anem
ia,eosin
ophilia,an
dfi
larialparasite
infection
Fonsecaeam
onophoraC
BS
115830Secon
darycerebral(?)
Skinlesion
sabsen
t;pu
lmon
arygran
ulom
abefore
cerebralinfection
(*)
Absen
tH
yphae
inbrain
and
inpu
lmon
arygran
ulom
a
28/M/w
hite/B
razilP
robableen
trypoin
t,knife
wou
nd
inth
erigh
tin
guin
alarea16
yrsprior
Brain
abscess16
Imm
un
ocomprom
ise/sch
istosomiasis, C
hagas
disease,macrocytic
anem
iaFonsecaea
monophora
CB
S117542
Prim
arycerebral
Skinlesion
sabsen
tA
bsent
Hyph
aein
brain48/F/N
I/bornin
Jamaica,livin
gin
USA
Un
know
nB
rainB
iopsy19
Imm
un
osuppression
/renal
transplan
trecipien
t
Fonsecaeapugnacius
CB
S139214
Secondary
cerebralSu
bcutan
eous
skinlesion
sgradu
allydissem
inatin
gto
the
hem
ifacialarea,n
ose,chin
,brain
Presen
tin
skin,
scatteredin
cranialbon
e
Hyph
aein
brain52/M
/wh
ite/Brazil
Skintrau
matic
infection
Brain
abscessdrain
ageT
his
study
Imm
un
ocompeten
t
aM
,male;F,fem
ale;(?),natu
reofin
itiallesionu
nkn
own
;(*),histopath
ologicaldiagnosis
with
out
fun
galstrainisolation
;NI,n
otin
cluded
amon
greported
data.
A Novel Agent of Disseminated Chromoblastomycosis
August 2015 Volume 53 Number 8 jcm.asm.org 2683Journal of Clinical Microbiology
on October 4, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
Azevedo CMPS, de Hoog GS. 2014. Environmental siblings of blackagents of human chromoblastomycosis. Fungal Divers 65:47– 63. http://dx.doi.org/10.1007/s13225-013-0246-5.
6. de Hoog GS, Nishikaku AS, Fernández-Zeppenfeldt G, Padín-GonzálezC, Burger E, Badali H, Richard-Yegres N, Gerrits van den Ende AHG.2007. Molecular analysis and pathogenicity of the Cladophialophora car-rionii complex, with the description of a novel species. Stud Mycol 58:219 –234. http://dx.doi.org/10.3114/sim.2007.58.08.
7. Xi L, Sun J, Lu C, Liu H, Xie Z, Fukushima K, Takizawa K, NajafzadehMJ, de Hoog GS. 2009. Molecular diversity of Fonsecaea (Chaetothyriales)causing chromoblastomycosis in southern China. Med Mycol 47:27–33.http://dx.doi.org/10.1080/13693780802468209.
8. González GM, Rojas OC, González JG, Kang Y, de Hoog GS. 2013.Chromoblastomycosis caused by Rhinocladiella aquaspersa. Med MycolCase Rep 2:148 –151. http://dx.doi.org/10.1016/j.mmcr.2013.08.001.
9. Tanabe H, Kawasaki M, Mochizuki T, Ishizaki H. 2004. Species identi-fication and strain typing of Fonsecaea pedrosoi using ribosomal RNA geneinternal transcribed spacer regions. Nihon Ishinkin Gakkai Zasshi 45:105–112. http://dx.doi.org/10.3314/jjmm.45.105.
10. Najafzadeh MJ, Gueidan C, Badali H, Gerrits van den Ende AHG, Xi L,de Hoog GS. 2009. Genetic diversity and species delimitation in the op-portunistic genus Fonsecaea. Med Mycol 47:17–25. http://dx.doi.org/10.1080/13693780802527178.
11. Najafzadeh MJ, Sun J, Vicente VA, Xi L, Gerrits van den Ende AHG, deHoog GS. 2010. Fonsecaea nubica sp. nov., a new agent of human chro-moblastomycosis revealed using molecular data. Med Mycol 48:800 – 806.http://dx.doi.org/10.3109/13693780903503081.
12. De Hoog GS, Zeng JS, Harrak MJ, Sutton DA. 2006. Exophiala xenobi-otica sp. nov., an opportunistic black yeast inhabiting environments richin hydrocarbons. Antonie Van Leeuwenhoek 90:257–268. http://dx.doi.org/10.1007/s10482-006-9080-z.
13. Badali H, Gueidan C, Najafzadeh MJ, Bonifaz A, Gerrits van den EndeAHG, de Hoog GS. 2008. Biodiversity of the genus Cladophialophora.Stud Mycol 61:175–191. http://dx.doi.org/10.3114/sim.2008.61.18.
14. Li DM, de Hoog GS. 2009. Cerebral phaeohyphomycosis—a cure at whatlengths? Lancet Infect Dis 9:376 –383. http://dx.doi.org/10.1016/S1473-3099(09)70131-8.
15. Kantarcioglu AS, de Hoog GS. 2004. Infections of the central nervoussystem by melanized fungi: a review of cases presented between 1999and 2004. Mycoses 47:4 –13. http://dx.doi.org/10.1046/j.1439-0507.2003.00956.x.
16. Nóbrega JP, Rosemberg S, Adami AM, Heins-Vaccari EM, da Silva LacaC, de Brito T. 2003. Fonsecaea pedrosoi cerebral phaeohyphomycosis(chromoblastomycosis): first human culture-proven case reported in Bra-zil. Rev Inst Med Trop Sao Paulo 45:217–220. http://dx.doi.org/10.1590/S0036-46652003000400008.
17. Surash S, Tyagi A, de Hoog GS, Zeng JS, Barton RC, Hobson RP. 2005.Cerebral phaeohyphomycosis caused by Fonsecaea monophora. Med My-col 43:465– 472. http://dx.doi.org/10.1080/13693780500220373.
18. Takei H, Goodman JC, Powell SZ. 2007. Cerebral phaeohyphomyco-sis caused by Cladophialophora bantiana and Fonsecaea monophora: re-port of three cases. Clin Neuropathol 26:21–27. http://dx.doi.org/10.5414/NPP26021.
19. Koo S, Klompas M, Marty FM. 2010. Fonsecaea monophora cerebralphaeohyphomycosis: case report of successful surgical excision and vori-conazole treatment and review. Med Mycol 48:769 –774. http://dx.doi.org/10.3109/13693780903471081.
20. Najafzadeh MJ, Sun J, Vicente VA, Klaassen CH, Bonifaz A, Gerrits vanden Ende AHG, Menken SBJ, de Hoog GS. 2011. Molecular epidemiol-ogy of Fonsecaea species. Emerg Infect Dis 17:464 – 469. http://dx.doi.org/10.3201/eid1703.100555.
21. Silva JP, de Souza W, Rozental S. 1998. Chromoblastomycosis: a retro-spective study of 325 cases on Amazonic Region (Brazil). Mycopathologia143:171–175. http://dx.doi.org/10.1023/A:1006957415346.
22. Vicente VA, Attili-Angelis D, Pie MR, Queiroz-Telles F, Cruz LM,Najafzadeh MJ, de Hoog GS, Zhao J, Pizzirani-Kleiner A. 2008. Envi-ronmental isolation of black yeast-like fungi involved in human infection.Stud Mycol 61:137–144. http://dx.doi.org/10.3114/sim.2008.61.14.
23. Sun J, Najafzadeh MJ, Gerrits van den Ende AH, Vicente VA, Feng P,Xi L, de Hoog GS. 2012. Molecular characterization of pathogenic mem-bers of the genus Fonsecaea using multilocus analysis. PLoS One 7:e41512.http://dx.doi.org/10.1371/journal.pone.0041512.
24. Sun J, Najafzadeh MJ, Vicente VA, de Hoog GS. 2010. Rapid detection
of pathogenic fungi using loop-mediated isothermal amplification, exem-plified by Fonsecaea agents of chromoblastomycosis. J Microbiol Methods80:19 –24. http://dx.doi.org/10.1016/j.mimet.2009.10.002.
25. Najafzadeh MJ, Sun J, Vicente VA, de Hoog GS. 2011. Rapid identificationof fungal pathogens by rolling circle amplification using Fonsecaea as amodel. Mycoses 54:e577– e582. http://dx.doi.org/10.1111/j.1439-0507.2010.01995.x.
26. Najafzadeh MJ, Badali H, Illnait-Zaragozi MT, de Hoog GS, Meis JF.2010. In vitro activities of eight antifungal drugs against 55 clinical isolatesof Fonsecaea spp. Antimicrob Agents Chemother 54:1636 –1638. http://dx.doi.org/10.1128/AAC.01655-09.
27. Feng P, Lu Q, Najafzadeh MJ, Gerrits van den Ende AHG, Sun J, Li R,Xi L, Vicente VA, Lai W, Lu C, de Hoog GS. 2014. Cyphellophora and itsrelatives in Phialophora: biodiversity and possible role in human infection.Fungal Divers 65:17– 45. http://dx.doi.org/10.1007/s13225-012-0194-5.
28. de Hoog GS, Gerrits van den Ende AHG. 1998. Molecular diagnostics ofclinical strains of filamentous basidiomycetes. Mycoses 41:183–189. http://dx.doi.org/10.1111/j.1439-0507.1998.tb00321.x.
29. Masclaux F, Guého E, de Hoog GS, Christen R. 1995. Phylogeneticrelationships of human-pathogenic Cladosporium (Xylohypha) species in-ferred from partial LS rRNA sequences. J Med Vet Mycol 33:327–338.http://dx.doi.org/10.1080/02681219580000651.
30. Glass N, Donaldson G. 1995. Development of primer sets designed foruse with the PCR to amplify conserved genes from filamentous Ascomy-cetes. Appl Environ Microbiol 61:1323–1330.
31. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6:molecular evolutionary genetics analysis version. Mol Biol Evol 30:2725–2729. http://dx.doi.org/10.1093/molbev/mst197.
32. Katoh K, Toh H. 2008. Recent developments in the MAFFT multiplesequence alignment program. Brief Bioinform 9:286 –298. http://dx.doi.org/10.1093/bib/bbn013.
33. Swofford D. 2003. PAUP*. Phylogenetic analysis using parsimony. Ver-sion 4. Sinauer Associates, Sunderland, MA.
34. de Hoog GS, Vicente VA, Najafzadeh MJ, Harrak MJ, Seyedmousavi S.2011. Waterborne Exophiala species causing disease in coldblooded ani-mals. Persoonia 27:46 –72. http://dx.doi.org/10.3767/003158511X614258.
35. Hillis DM, Bull JJ. 1993. An empirical test of bootstrapping as a method forassessing confidence in phylogenetic analysis. Syst Biol 42:182–192. http://dx.doi.org/10.1093/sysbio/42.2.182.
36. Seyedmousavi S, Guillot J, de Hoog GS. 2013. Phaeohyphomycoses,emerging opportunistic diseases in animals. Clin Microbiol Rev 26:19 –35.http://dx.doi.org/10.1128/CMR.00065-12.
37. Lirng JF, Tien RD, Osumi AK, Madden JF, McLendon RP, Sexton D.1995. Cerebral phaeohyphomycosis complicated with brain abscess: a casereport. Zhonghua Yi Xue Za Zhi (Taipei) 55:491– 495.
38. Horré R, de Hoog GS. 1999. Primary cerebral infections by melanized fungi:a review. Stud Mycol 43:176–193. http://www.fungalbiodiversitycentre.com/publications/1043/content/pdf/176-193.pdf.
39. Abramo F, Bastelli F, Nardoni S, Mancianti F. 2002. Feline cutaneousphaeohyphomycosis due to Cladophialophora bantiana. J Feline Med Surg4:157–163. http://dx.doi.org/10.1053/jfms.2002.0183.
40. Eliesi L, Balandraud V, Boulouha L, Crespeau F, Guillot J. 2003. Fatalsystemic phaeohyphomycosis in a cat due to Cladophialophora bantiana. JVet Med A Physiol Pathol Clin Med 50:50 –55. http://dx.doi.org/10.1046/j.1439-0442.2003.00501.x.
41. Campbell CK, Al-Hedaithy SSA. 1993. Phaeohyphomycosis of the braincaused by Ramichloridium mackenziei sp. nov. in Middle Eastern coun-tries. J Med Vet Mycol 31:325–332. http://dx.doi.org/10.1080/02681219380000391.
42. Sutton DA, Slifkin M, Yakulis R, Rinaldi MG. 1998. US case report ofcerebral phaeohyphomycosis caused by Ramichloridium obovoideum (R.mackenziei): criteria for identification, therapy, and review of other knowndematiaceous neurotropic taxa. J Clin Microbiol 36:97–102.
43. Al-Abdely HM, Najvar L, Bocanegra R, Fothergill A, Loebenberg D,Rinaldi MG, Graybill JR. 2000. SCH 56592, amphotericin B, or itracona-zole therapy of experimental murine cerebral phaeohyphomycosis due toRamichloridium obovoideum (Ramichloridium mackenziei). AntimicrobAgents Chemother 44:1159 –1162. http://dx.doi.org/10.1128/AAC.44.5.1159-1162.2000.
44. Chang CL, Kim DS, Park DJ, Kim HJ, Lee CH, Shin JH. 2000. Acutecerebral phaeohyphomycosis due to Wangiella dermatitidis accompaniedby cerebrospinal fluid eosinophilia. J Clin Microbiol 38:1965–1966.
45. Bonifaz A, Davoudi MM, de Hoog GS, Padilla-Desgarennes C,
de Azevedo et al.
2684 jcm.asm.org August 2015 Volume 53 Number 8Journal of Clinical Microbiology
on October 4, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
Vázquez-González D, Navarrete G, Meis JF, Badali H. 2013. Severedisseminated phaeohyphomycosis in an immunocompetent patientcaused by Veronaea botryosa. Mycopathologia 175:497–503. http://dx.doi.org/10.1007/s11046-013-9632-5.
46. López Martínez R, Méndez Tovar LJ. 2007. Chromoblastomycosis. ClinDermatol 25:188–194. http://dx.doi.org/10.1016/j.clindermatol.2006.05.007.
47. De Hoog GS, Queiroz-Telles F, Haase G, Fernandez-Zeppenfeldt G,Attili Angelis D, Gerrits van den Ende AHG, Matos T, Peltroche-Llacsahuanga H, Pizzirani-Kleiner AA, Rainer J, Richard-Yegres N,Vicente V, Yegres F. 2000. Black fungi: clinical and pathogenic ap-proaches. Med Mycol 38(Suppl 1):243–250. http://dx.doi.org/10.1080/mmy.38.s1.243.250.
48. Lucasse C, Chardome J, Magis P. 1954. Cerebral mycosis from Cladospo-rium trichoïdes in a native of the Belgian Congo. Ann Soc Belg Med Trop34:475– 478. (In French.)
49. Carrión AL. 1975. Chromoblastomycosis and related infections: new con-cepts, differential diagnosis, and nomenclatorial implications. Int J Der-matol 14:27–32. http://dx.doi.org/10.1111/j.1365-4362.1975.tb00074.x.
50. Shimosaka S, Waga S. 1983. Cerebral chromoblastomycosis complicatedby meningitis and multiple fungal aneurysms after resection of a granu-loma. J Neurosurg 59:158 –161. http://dx.doi.org/10.3171/jns.1983.59.1.0158.
51. Seyedmousavi S, Badali H, Chlebicki A, Zhao J, Prenafe-Boldú FX, deHoog GS. 2011. Exophiala sideris, a novel black yeast isolated from envi-ronments polluted with toxic alkyl benzene and arsenic. Fungal Biol 115:1030 –1037. http://dx.doi.org/10.1016/j.funbio.2011.06.004.
52. Najafzadeh MJ, Vicente VA, Sun J, Meis JF, de Hoog GS. 2011. Fonse-caea multimorphosa sp. nov., a new species of Chaetothyriales isolated fromfeline cerebral abscess. Fungal Biol 115:1066 –1076. http://dx.doi.org/10.1016/j.funbio.2011.06.007.
A Novel Agent of Disseminated Chromoblastomycosis
August 2015 Volume 53 Number 8 jcm.asm.org 2685Journal of Clinical Microbiology
on October 4, 2020 by guest
http://jcm.asm
.org/D
ownloaded from
