Available via license: CC BY-NC 4.0
Content may be subject to copyright.
Hepatic phaeohyphomycosis due to a novel dematiaceous fungus,
Pleurostoma hongkongense sp. nov., and importance of antifungal
susceptibility testing
Chi-Ching Tsang
a
*, Ka-Fai Chan
a
*, Walton Chan
a
, Jasper F. W. Chan
a
, Rex K. H. Au-Yeung
b
,
Antonio H. Y. Ngan
a
, Ken P. K. Lin
a
, Susanna K. P. Lau
a
and Patrick C. Y. Woo
a
a
Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong;
b
Department of
Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong
ABSTRACT
Pleurostoma species are wood-inhabiting fungi and emerging opportunistic pathogens causing phaeohyphomycosis. In
this study, we isolated a dematiaceous fungus, HKU44
T
, from the subhepatic abscess pus and drain fluids of a liver
transplant recipient with post-transplant biliary and hepatico-jejunostomy bypass strictures. Histology of the abscess
wall biopsy showed abundant fungal hyphae. The patient survived after a second liver transplant and antifungal
therapy. On SDA, HKU44
T
grew initially as white powdery colonies which turned beige upon maturation. Hyphae
were septate and hyaline. Phialides were monophialidic and laterally located, generally closely associated to a cluster
of conidia which were usually reniform. Phylogenetic analyses showed that HKU44
T
is most closely related to, but
distinct from, Pleurostoma ootheca and Pleurostoma repens. These suggested that HKU44
T
is a novel Pleurostoma
species, for which the name Pleurostoma hongkongense sp. nov. is proposed. Antifungal susceptibility testing showed
that Pleurostoma species possessed high MICs/MECs for fluconazole, 5-flucytosine and the echinocandins; whereas
they exhibited a high strain-to-strain variability to the susceptibilities to the other triazoles. As for amphotericin B,
∼65% of the Pleurostoma strains had low MICs (≤1 µg/mL). DNA sequencing should be performed to accurately
identify fungi with Pleurostoma/Phialophora-like morphologies, so is antifungal susceptibility testing for patients with
Pleurostoma infections.
ARTICLE HISTORY Received 18 June 2020; Revised 23 November 2020; Accepted 16 December 2020
KEYWORDS Liver; phaeohyphomycosis; dematiaceous fungus; Pleurostoma;Pleurostoma hongkongense; novel species
Introduction
Pleurostoma species are emerging human pathogens
causing phaeohyphomycosis. This historic genus was
originally established in 1863 [1]. Following its merging
with the asexual genus Pleurostomophora subsequent
to the abolishment of dual nomenclature of the differ-
ent morphs of the same fungus since 2013, it currently
accommodates a total of five species, namely Pleuros-
toma candollei (type species), Pleurostoma ootheca,
Pleurostoma ochraceum, Pleurostoma repens and Pleur-
ostoma richardsiae [2,3]. In nature, this group of fungi
inhabits woods [4,5]; and they have also been isolated
from soil and sewage [4] and found as contaminants
which cause blueing of ground wood pulp [6]. Infection
may result from close contact with Pleurostoma-con-
taminated plants or direct inoculation of contaminated
materials into the body [7–11]. Very often Pleurostoma
infections are localized and manifest as cutaneous or
subcutaneous lesions and/or nodules [7–9,11–30],
although infections of the bones/joints [9,20,22,31–
35], eyes [10,35–37] and urinary tracts [38] are also
observed. In more rare occasions especially in severely
immunocompromised individuals, disseminated inva-
sive infections affecting the bloodstreams, hearts and
livers could also occur [35,39,40]. The aetiological
agent for most of the cases were Pleurostoma richard-
siae [7–25,28–33,35–40], while Pleurostoma ochra-
ceum [26], Pleurostoma ootheca [27] and Pleurostoma
repens [34] were also reported to cause infections in a
few cases.
In 2017, we isolated a dematiaceous fungus from
the subhepatic abscess pus and drain fluids of a
patient. Microscopic examination of the fungal culture
and preliminary internal transcribed spacer (ITS)
region sequencing showed that it is a Pleurostoma
species, although matrix-assisted laser desorption/ion-
ization–time-of-flight mass spectrometry (MALDI–
TOF MS) failed to identify this fungus due to the
© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
CONTACT Patrick C. Y. Woo pcywoo@hku.hk Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 19/F,
Block T, Queen Mary Hospital Compound, Pokfulam, Hong Kong; Susanna K. P. Lau skplau@hku.hk Department of Microbiology, Li Ka Shing Faculty
of Medicine, The University of Hong Kong, 19/F, Block T, Queen Mary Hospital Compound, Pokfulam, Hong Kong
*These authors contributed equally to this study.
Supplemental data for this article can be accessed at https://doi.org/10.1080/22221751.2020.1866955.
Emerging Microbes & Infections
2021, VOL. 10
https://doi.org/10.1080/22221751.2020.1866955
absence of interpretable protein mass spectra. Further
morphological and molecular characterizations
showed that this fungus is closely related to, but dis-
tinct from, all the known Pleurostoma species. There-
fore, we propose a new species, Pleurostoma
hongkongense sp. nov., to describe this fungus. Anti-
fungal susceptibilities of this novel species and the
known Pleurostoma species were also performed to
help guide patient management.
Materials and methods
Patient and strains
Clinical specimens, including pus from a subhepatic
abscess as well as drain fluids, were collected from the
patient, handled according to standard protocols,
directly inoculated on Sabouraud dextrose agars (SDA;
Difco, BD Diagnostics Systems, USA) supplemented
with chloramphenicol (50 µg/mL; Calbiochem, USA)
and incubated at 30°C to obtain the case isolate
HKU44
T
. Fungal materials were harvested directly
from a single colony for subsequent subcultures. Clinical
data werecollected by retrieving and analysing thehospi-
tal record of the patient, and the use of these data was
approved by the Institutional Review Board of The Uni-
versity of Hong Kong/Hospital Authority Hong Kong
West Cluster. The ex-type strains of Pleurostoma ochra-
ceum, Pleutostoma repens and Pleurostoma richardsiae,a
reference strain of Pleurostoma ootheca as well as 23
reference strains of Pleurostoma richardsiae originating
from clinical sources were obtained from the American
Type Culture Collection (ATCC), USA; the Medical
Mycology Research Center (MMRC) of Chiba Univer-
sity (IFM), Japan; the National Collection of Pathogenic
Fungi (NCPF), Public Health England (PHE), UK; the
UAMH Centre for Global Microfungal Biodiversity,
Dalla Lana School of Public Health, University of Tor-
onto, Canada as well as the Westerdijk Fungal Biodiver-
sity Institute (CBS), the Netherlands (Table S1). The
quality control strains for susceptibility testing Aspergil-
lus flavus ATCC 204304, Aspergillus fumigatus ATCC
204305 and Candida parapsilosis ATCC 22019
T
were
obtained from ATCC; Candida albicans CNM-CL
F8555, Pichia kudriavzevii (synonym: Candida krusei)
CNM-CL-3403 and Aspergillus flavus CNM-CM-1813
were obtained from Statens Serum Institut (SSI), Den-
mark; and Pichia kudriavzevii NRRL Y-413 (=ATCC
6258) was obtained from the Agricultural Research Ser-
vice (ARS) Culture Collection (NRRL), Department of
Agriculture, USA.
Phenotypic characterizations
The fungal isolate HKU44
T
and other Pleurostoma ex-
type/reference strains were cultured on malt extract
agar (MEA; Oxoid, UK), potato dextrose agar (PDA;
BD Diagnostic Systems) and SDA (all supplemented
with chloramphenicol [50 µg/mL]) for examination of
growth rates and colony characteristics. Images of the
fungal colonies were captured using the digital camera
α5100 (Sony, Japan). For light microscopy, a slide cul-
ture was prepared by first placing an SDA block measur-
ing 1 cm × 1 cm × 1 cm onto a glass slide. Each surface
of the agar block was inoculated with the conidia of
HKU44
T
(3 × 10
4
colony forming units) and a cover
slip was then placed on top of the agar block. The slide
culture was incubated at 25°C for 7 days after which
the cover slip with the attaching fungal materials,
mounted and stained with lactophenol cotton blue
(BD Diagnostics System), was transferred on a fresh
glass slide. Microscopic characteristics were observed
using the microscope BX43 (Olympus, Japan) under
an original magnification of 1000× and digital images
were obtained by the attached camera DP80 (Olympus)
and the software cellSens Dimension (Olympus).
Measurement was made for 40–60 structures for each
microscopic character. HKU44
T
was also microscopi-
cally examined by scanning electron microscopy, per-
formed following a simplified protocol [41]. Briefly,
polycarbonate filter membranes with a pore size of
0.8 µm were placed on tap water agar (1.5% agarose
[w/v; BD Diagnostic Systems]). Strain HKU44
T
was
then inoculated on the membranes and was incubated
at 25°C for 14 days. Upon colony maturation, the culture
was fixed using formalin vapour by adding 1 mL of 37%
formaldehyde (in water, v/v; Sigma Aldrich, USA) onto
the lid of the inverted Petri dish which was then securely
sealed and incubated overnight. Membranes with fixed
fungal materials were then collected and washed with
absolute ethanol (Merck, Germany) twice and then
sent to the Electron Microscope Unit of The University
of Hong Kong for sample processing, where the fixed
samples were dried by the critical point dryer CPD 030
(Bal-Tec, Liechtenstein) using liquid carbon dioxide as
the transitional fluid, mounted on aluminium stubs
and then coated with a thin layer (10–20 nm) of palla-
dium for electrical conduction by the cool sputter coater
SCD 005 (Bal-Tec). Coated specimens were then exam-
ined using the field emission scanning electron micro-
scope S-4800 (Hitachi High-Technologies, Japan).
DNA extraction, ITS, partial 28S nrDNA, partial
18S nrDNA and partial β-tubulin gene
sequencing, sequence identity analyses and
phylogenetic analyses
Extraction of fungal DNA, polymerase chain reaction
(PCR), and sequencing of the ITS, partial 28S nuclear
ribosomal DNA (nrDNA), partial 18S nrDNA and
partial β-tubulin gene for the case isolate HKU44
T
and the ex-type/reference strains of other Pleurostoma
species were carried out following our previous publi-
cation [42] with the primer pairs ITS1/ITS4 [43] for
82 C.-C. TSANG ET AL.
the ITS, NL1/NL4 [44] for partial 28S nrDNA, NS1/
NS4 [43] for partial 18S nrDNA as well as TUB2-F/
TUB2-R [45] for partial β-tubulin gene. The DNA
sequences obtained, together with those of other clo-
sely related species/strains accessioned in the DDBJ/
ENA/GenBank databases, were then compared by
pairwise alignment using BioEdit 7.2.0 [46]. These
sequences were also analysed by multiple sequence
alignment using MUSCLE 3.8 [47]. After end-trim-
ming, divergent or poorly aligned regions of the
DNA sequences were removed using Gblocks 0.91b
[48,49] with relaxed parameters. Test for substitution
model and phylogenetic tree reconstruction for each
DNA marker were performed by the maximum likeli-
hood method using MEGA 6.0.6 [50]. Phylogenetic
analyses included 531, 560, 822 and 397 nucleotide
positions of the ITS, partial 28S nrDNA, partial 18S
nrDNA and partial β-tubulin gene sequences, respect-
ively. Phylogenetic trees were also reconstructed using
the concatenated sequences of the four DNA markers
(total 2340 nucleotide positions) of HKU44
T
and other
Pleurostoma strains by the maximum likelihood
method as described above and by the Bayesian evol-
utionary analysis using BEAST 1.10.4 [51]. For Baye-
sian inference, ten million generations were run with
trees sampled every 1000th generation to yield
10,000 trees. The trees, after a 10% burn-in, were sum-
marized as a single tree using TreeAnnotator 1.8.0 by
choosing the tree with the maximum sum of posterior
probabilities (maximum clade credibility) and viewed
using FigTree 1.4.0.
In vitro antifungal susceptibility test
The in vitro susceptibilities against 11 antifungal drugs,
including amphotericin B (Cayman Chemical, USA),
anidulafungin (SelleckChem, USA), caspofungin (Tar-
getMol, USA), micafungin (TargetMol), fluconazole
(TargetMol), isavuconazole (TargetMol), itraconazole
(TargetMol), posaconazole (Sigma-Aldrich), ravuco-
nazole (Sigma-Aldrich), voriconazole (TargetMol)
and flucytosine (Sigma-Aldrich), were determined by
the microbroth dilution method according to the guide-
lines by the European Committee on Antimicrobial
Susceptibility Testing (EUCAST) (test range: 0.0156–
8 µg/mL for itraconazole and posaconazole; 0.0312–
16 µg/mL for other drugs) [52] and our previous publi-
cation [53]. For echinocandins, minimum effective
concentration (MEC) endpoints were recorded as the
lowest drug concentrations in which abnormal, short
and branched hyphal clusters were observed; whereas
for the other antifungal agents, minimum inhibitory
concentration (MIC) endpoints yielding no visible fun-
gal growth by eyes were recorded. Strains Aspergillus
flavus ATCC 204304 and CNM-CM-1813, Aspergillus
fumigatus ATCC 204305, Candida albicans CNM-CL
F8555, Candida parapsilosis ATCC 22019
T
as well as
Pichia kudriavzevii CNM-CL-3403 and NRRL Y-413
were used as quality controls.
Data availability
A dried specimen of the case isolate HKU44
T
(the holo-
type) was deposited to UAMH whereas living cultures
of HKU44
T
were deposited to UAMH and the Culture
Collection of Switzerland (CCOS). The ITS, partial 28S
nrDNA, partial 18S nrDNA and partial β-tubulin gene
sequences of HKU44
T
and the ex-type/reference strains
of other Pleurostoma species were deposited in the
DDBJ/ENA/GenBank databases (Table S1).
Results
Patient
A 65-year-old Chinese man was admitted in March
2017 for management of post-liver transplant biliary
stricture. The patient had hepatitis B virus-related
liver failure and living donor liver transplantation
was performed in November 2015. Biliary stricture
developed and right hepatico-jejunostomy bypass
was performed in August 2016. The patient developed
stricture of the hepatico-jejunostomy bypass and was
admitted in March 2017 for a second hepatico-jeju-
nostomy bypass. Post-operatively, the patient devel-
oped increasing abdominal pain. Computed
tomography examination of the abdomen revealed a
4.3 cm × 0.8 cm collection with gas pocket and rim
enhancement was observed around the tubal drains
in the upper abdomen (Figure 1). Laparotomy was
performed in May 2017 and a loculated abscess with
5 mL of pus was observed in the subhepatic space.
Drainage of the abscess was performed. Biopsy of
the abscess wall showed abundant fungal hyphae
(Figure 2). Bacterial and fungal cultures of the pus
recovered Klebsiella pneumoniae, Enterococcus faeca-
lis, Enterobacter cloacae and a dematiaceous fungus
(strain HKU44
T
). Despite antimicrobial treatment
including the use of anidulafungin, the same dema-
tiaceous fungus was repeatedly recovered from the
drain fluid eight more times. The patient developed
progressive liver failure and cadaveric liver transplan-
tation was performed again in July 2017. Post-opera-
tively, amphotericin B and voriconazole were given
for 16 days, followed by voriconazole maintenance
therapy for three months. The function of the graft
was normal and the dematiaceous fungus was not iso-
lated from the patient again up to the time of writing,
three years after the second liver transplantation.
Phenotypic characterizations
The colony macroscopy of HKU44
T
varied greatly
when cultured on different media. On MEA, colonies
EMERGING MICROBES AND INFECTIONS 83
were grey with creamy mycelial tufts on the surface.
The reverse was beige (Figure 3(a,d)). On SDA,
HKU44
T
initially grew white but developed a beige
complexion on day 5. The colony surface was powdery
but the outer colony border was floccose (Figure 3(b,
e)). The reverse surface maintained the same color-
ation. Meanwhile on PDA, the colony surface was
flat and creamy white. As the colony matured, a yellow
pigment or hue appeared to diffuse from the initial
point of inoculation, but remained confined to the
mycelial mat (Figure 3(c,f)). Furthermore, short
white creamy mycelial tufts began to emerge from
the outer ring of the growing colony and later grew
inwards as the colony matured. No diffusible pigment
was observed for the fungus when cultured on all the
three media tested. The growth rate of HKU44
T
on the
three media tested also differed. After 7 days of incu-
bation at 25°C, HKU44
T
grew significantly the fastest
on PDA, attaining a colony diameter of approximately
50 mm; whereas on SDA and MEA the fungus grew
slower, attaining colony diameters of around 35 mm
and 31 mm, respectively (Figure S1(a)). In addition,
growth of HKU44
T
was influenced by temperature.
On SDA after 7 days of incubation, growth of
HKU44
T
at 25°C and 30°C was comparable, with col-
ony diameters of around 30–40 mm. When the temp-
erature increased to 35–37C°, the fungus grew
significantly faster, attaining colony diameters of
around 60 mm (Figure S1(b)).
Microscopically, hyphae were septate and hyaline
and hyphal walls were verruculose (Figure 4). Phia-
lides appeared to be monophialidic, but were usually
closely associated to a cluster of conidia and were
usually laterally located rather than terminally on
hyphae (Figure 4). Several distinguishable phialide
structures were observed including swollen elongate-
ampulliform phialides with constricted bases (Figure
4(a,d,g)), relatively long type I-like adelophialides
which lacked basal septa (Figure 4(b,e,h)) as well as
shorter and indistinct projection-like conidiophores
associated with spherical to ellipsoidal conidia (Figure
4(c,f,i)). Collarettes were usually inconspicuous,
though slightly cupped or tubular collarettes could
be observed for the longer and larger conidiophores
(Figure 4(e,g,h)). Conidia sported a wide variety of
shapes and sizes including spherical, oblong-elliptical,
Figure 1. Computed tomography image of the upper abdomen. A small fluid collection with gas pocket was found and rim
enhancement was observed around the tubal drains.
84 C.-C. TSANG ET AL.
Figure 2. Photomicrographs of the biopsied liver abscess wall tissue. (a) Abundant fungal hyphae, a small amount of necrotic
tissue, brown bile pigments as well as a few acute inflammatory cells were observed (Periodic acid-Schiffstaining, original mag-
nification 200×). (b) The fungal hyphae were highly septate (Grocott methenamine-silver staining, original magnification 400×).
EMERGING MICROBES AND INFECTIONS 85
allantoid and most commonly reniform. Notably, sub-
spherical and oblong shaped conidia tended to associ-
ate more frequently with shorter conidiophores
(Figure 4(c,f,i)), while reniform and allantoid conidia
were found congregated to the other two types of con-
idiophores (Figure 4(a–h)).
Molecular characterizations
PCR of the ITS, partial 28S nrDNA, partial 18S
nrDNA and partial β-tubulin gene of HKU44
T
and
the reference strains yielded DNA products with
lengths of about 600, 600, 1100 and 500 bp, respect-
ively. Pairwise alignment showed that the partial ITS
sequence of HKU44
T
possessed an 88.9% identity to
Pleurostoma ootheca CBS 115329, an 88.6% identity
to Pleurostoma repens CBS 294.39
T
, an 87.3% identity
to Pleurostoma richardsiae CBS 270.33
T
and an 86.5%
identity to Pleurostoma ochraceum CBS 131321
T
. The
partial 28S nrDNA sequence of HKU44
T
possessed a
98.8% identity to Pleurostoma ootheca CBS 115329, a
98.2% identity to Pleurostoma repens CBS 294.39
T
,a
97.9% identity to Pleurostoma richardsiae CBS
270.33
T
and a 95.9% identity to Pleurostoma ochra-
ceum CBS 131321
T
. The partial 18S nrDNA sequence
of HKU44
T
possessed a 99.5% identity to Pleurostoma
richardsiae CBS 270.33
T
, a 99.4% identity to Pleuros-
toma ootheca CBS 115329 and Pleurostoma repens
CBS 294.39
T
and a 99.0% identity to Pleurostoma
ochraceum CBS 131321
T
. The partial β-tubulin gene
sequence of HKU44
T
possessed an 80.1% sequence
identity to Pleurostoma repens CBS 294.39
T
, a 79.1%
sequence identity to Pleurostoma ootheca CBS
115329, a 78.8% identity to Pleurostoma richardsiae
CBS 270.33
T
and a 76.6% sequence identity to Pleuros-
toma ochraceum CBS 131321
T
. Phylogenetic analyses
showed that HKU44
T
occupied a unique phylogenetic
position in the ITS, partial 28S nrDNA, partial 18S
nrDNA and partial β-tubulin gene trees (Figure S2)
by the maximum-likelihood method as well as in the
concatenated sequence trees by both the maximum-
likelihood method and Bayesian evolutionary analysis
(Figure 5). Although HKU44
T
is most closely related
to Pleurostoma ootheca and Pleurostoma repens,itis
distinct from all the Pleurostoma species character-
ized, suggesting it is a novel Pleurostoma species.
In vitro antifungal susceptibilities
The in vitro susceptibilities of HKU44
T
and other
Pleurostoma strains to 11 different antifungal agents
are listed in Table 1. HKU44
T
possessed high MICs/
MECs (>8 µg/mL) to all antifungal agents tested
except amphotericin B (0.5 µg/mL). As for other
Pleurostoma strains, all of them had high MICs to
flucytosine and fluconazole (≥4 µg/mL) and most
Figure 3. Growth of Pleurostoma hongkongense HKU44
T
on various culture media after 7 days of incubation at 25°C. (a, d) Malt
extract agar (MEA). (b, e) Sabouraud dextrose agar (SDA). (c, f) Potato dextrose agar (PDA). Brightness was adjusted individually for
each panel.
86 C.-C. TSANG ET AL.
had high MECs (≥2 µg/mL) to the echinocandins,
except Pleurostoma ochraceum CBS 131321
T
and
Pleurostoma richardsiae NCPF 2765 which had low
MECs (0.5 and 1 µg/mL, respectively) to caspofungin.
In addition to fluconazole, in general these Pleutos-
toma strains possessed high MICs to other triazole
agents as well. Only ten of them possessed low MICs
(≤1 µg/mL) to posaconazole, seven to itraconazole,
four to voriconazole and three to each of isavucona-
zole and ravuconazole. On the contrary, the majority
of other Pleutostoma strains tested possessed low
MICs (≤1 µg/mL) to amphotericin B, including Pleur-
ostoma repens CBS 294.39, Pleurostoma ochraceum
CBS 131321
T
,Pleurostoma ootheca CBS 115329 and
14 strains of Pleurostoma richardsiae.
Taxonomy
Pleurostoma hongkongense, C.-C. Tsang, K.-F. Chan,
W. Chan, J.F.W. Chan, R.K.H. Au-Yeung, A.H.Y.
Ngan, K.P.K. Lin, S.K.P. Lau, P.C.Y. Woo, sp. nov.
Mycobank accession number: MB 835850.
Etymology: of or belonging to Hong Kong, the
place where the holotype was isolated.
Colonies on MEA grey, with creamy mycelial tufts
and a beige reverse, reaching ∼31 mm in diameter
after 7 days of incubation at 25°C. Colonies on SDA
initially white, turning beige after 5 days of incubation,
powdery, with a floccose border and same-coloured
reverse, reaching ∼30–40 mm in diameter after 7
days of incubation at 25–30°C, and around 60 mm
in diameter at 35–37C°. On PDA, colonies flat and
Figure 4. Microscopic features of Pleurostoma hongkongense HKU44
T
. Arrows indicate notable structures. (a–c) Scanning electron
microscopy photographs of notable phialide and conidia features. Scale bars = 5 μm. (d–i) Bright-field microscopy of correspond-
ing features using a slide culture preparation. Slides were prepared via wet mount and stained with lactophenol cotton blue (orig-
inal magnification 1000×). Scale bars = 10 μm.
EMERGING MICROBES AND INFECTIONS 87
creamy white, with a yellow hue at the centre and
short, white creamy mycelial tufts emerging from the
rims upon maturation, reaching ∼50 mm in diameter
after 7 days of incubation at 25°C.
Hyphae 1.3–6.5 µm (x= 2.5 ± 0.9 µm, n = 58) wide,
septate and hyaline, with verruculose walls. Phialides
1.5–11.0 µm × 0.7–3.0 µm (x= 4.7 ± 2.7 µm × 1.7 ±
0.5 µm, n = 42), monophialidic, usually laterally
located and closely associated to a cluster of conidia.
Three types of phialide structures were observed,
including swollen elongate-ampulliform phialides
with constricted bases, relatively long type I-like ade-
lophialides which lacked basal septa as well as shorter
and indistinct projection-like conidiophores associ-
ated with spherical to ellipsoidal conidia. Collarettes
inconspicuous, though slightly cupped or tubular col-
larettes were observable for the longer and larger con-
idiophores. Conidia 0.3–11.0 µm × 1.5–3.7 µm (x=
2.6 ± 0.4 µm × 2.7 ± 0.6 µm, n = 49), most commonly
reniform, occasionally spherical, oblong-elliptical or
Figure 5. Phylogenetic tree showing the relationship of Pleurostoma hongkongense HKU44
T
to its closely related species within
the genus Pleurostoma. The tree was inferred from the concatenated sequence data of the internal transcribed spacer (ITS) region,
partial β-tubulin gene, partial 18S nrDNA and partial 28S nrDNA by the maximum likelihood method with the substitution model
T92 (Tamura 3-parameter model) + G (gamma-distributed rate variation) + I (estimated proportion of invariable sites). The scale
bar indicates the estimated number of substitutions per base. Only nodes that were well supported by the maximum-likelihood
method (≥70% bootstrap support) have their bootstrap support, calculated from 1,000 replicates, shown; and all these nodes
were also well supported by the Bayesian inference method (posterior probabilities ≥0.99). The DDBJ/ENA/GenBank nucleotide
accession numbers for Jattaea algeriensis STE-U 6201
T
are EU367446 (ITS), EU367466 (β-tubulin), EU367462 (18S nrDNA) and
EU367456 (28S nrDNA).
88 C.-C. TSANG ET AL.
allantoid. Subspherical and oblong shaped conidia
usually associated with shorter conidiophores and
reniform and allantoid conidia usually congregated
to the other two types of conidiophores.
Holotype: UAMH 12185. Ex-type culture: HKU44
T
(= CCOS 1933
T
= UAMH 12185
T
).
Pleurostoma hongkongense may be phenotypically
difficult to differentiate from other Pleurostoma species
due to the high variability observed in the conidiophore
and conidia characteristics of each species (Table 2).
Furthermore, colony age and maturity are also the con-
founding factors for microscopic identification. The
asexual morphs of members of Pleurostoma are mono-
phialidic and heavily sporulating. Pleurostoma richard-
siae is likely the easiest to differentiate, as its conidia are
most commonly spherical and aggregate as slimy
masses at the apices of distinct flared collarettes (Figure
S3(a)). The wide saucer shaped collarette observed in
Pleurostoma richardsiae is not observed in other Pleur-
ostoma species, which usually display either cupped or
slightly rounded collarettes. Pleurostoma repens can be
differentiated through its relatively long, branched and
elongated conidiophores. In addition, spherical conidia
have never been observed in Pleurostoma repens.Pleur-
ostoma ootheca were observed to have the shortest
phialides. In addition, spherical conidia were found
to be tightly bunched at the apices of these short phia-
lides (Figure S3(b)). Pleurostoma ochraceum and Pleur-
ostoma hongkongense share remarkably similar
phialide and hyphal structures, but a large noticeable
difference is the degree of melanization of conidia.
Pleurostoma ochraceum has distinctly melanized and
thick-walled spherical conidia (Figure S3(c)). The
asexual morph of P. candollei is not known [2].
Discussion
We report the isolation of HKU44
T
from the subhepa-
tic abscess as well as repeatedly from the drain fluid
eight more times in a liver transplant recipient, who
received prednisolone and tacrolimus that had sup-
pressed his immune system, rendering him susceptible
to invasive fungal infections. The clinical significance
of the fungus was evident by the invasion of the fungus
to the patient’s tissue as demonstrated by the presence
of abundant fungal hyphae as well as necrotic tissue
and acute inflammatory cells in histological sections
of the biopsied specimen. Culture of the abscess pus
and drain fluid specimens also showed colonies at
the primary inoculum on all agar plates inoculated
with the specimens, supporting that the isolate was
from the clinical specimens, instead of due to environ-
mental contamination. Sequencing of four different
DNA regions commonly used for phylogenetic
studies, including the ITS, partial 28S nrDNA, partial
18S nrDNA and partial β-tubulin gene [54], and phy-
logenetic analyses showed that HKU44
T
stood out as a
unique branch distinct from the other Pleurostoma
Table 1. Minimum inhibitory concentrations (MICs) or minimum effective concentrations (MECs) of different antifungal agents
against Pleurostoma species.
a
Species Strain
MICs/MECs (µg/mL)
Triazoles Echinocandins Others
FLC ISA ITC POS RAV VRC AFG CAS MFG 5FC AMB
P. hongkongense HKU44
T
>16 >16 >8 >8 >16 16 >16 16 >16 >16 0.5
P. repens CBS 294.39
T
16 1 0.5 0.5 1 0.25 4 2 16 >16 0.25
P. richardsiae ATCC 58041 >16 4 >8 1 4 2 >16 16 >16 >16 1
ATCC 58116 >16 >16 >8 >8 >16 >16 >16 16 >16 >16 2
CBS 270.33
T
>16 1 0.5 0.25 1 2 4 8 >16 >16 0.5
CBS 271.66 >16 2 0.5 2 2 4 >16 16 >16 >16 1
CBS 406.93 >16 >16 >8 >8 >16 >16 >16 16 >16 >16 2
CBS 483.80 >16 >16 >8 >8 >16 >16 >16 >16 >16 >16 2
CBS 506.90 >16 4 8 1 8 2 >16 4 >16 >16 2
IFM 4926 >16 4 1 1 4 2 >16 16 >16 >16 1
IFM 41579 >16 4 >8 8 4 4 2 8 >16 >16 1
IFM 63001 >16 >16 >8 >8 >16 >16 >16 16 >16 >16 2
IFM 63541 >16 >16 8 8 16 >16 4 8 2 >16 1
UAMH 4377 >16 2 1 1 2 1 >16 16 >16 >16 1
UAMH 5052 >16 >16 >8 >8 >16 >16 >16 8 >16 >16 1
UAMH 5056 >16 2 1 1 4 4 >16 16 >16 >16 0.5
UAMH 5058 >16 2 >8 >8 >16 1 4 4 >16 >16 1
UAMH 8335 >16 >16 >8 >8 >16 8 >16 8 >16 >16 2
UAMH 8654 >16 >16 >8 >8 >16 >16 >16 16 >16 >16 2
UAMH 10082 >16 16 >8 >8 >16 16 >16 8 >16 >16 2
UAMH 10452 >16 16 8 8 16 16 >16 8 >16 >16 1
NCPF 2707 >16 8 >8 8 4 8 >16 2 >16 >16 1
NCPF 2752 >16 4 2 1 4 4 >16 16 >16 >16 2
NCPF 2765 >16 >16 >8 >8 >16 >16 >16 1 >16 >16 4
NCPF 2961 >16 8 >8 8 4 2 >16 8 >16 >16 1
NCPF 7565 >16 >16 >8 >8 >16 >16 >16 4 >16 >16 1
P. ochraceum CBS 131321
T
4 0.5 0.25 0.25 0.5 0.5 4 0.5 4 >16 1
P. ootheca CBS 115329 >16 16 >8 1 8 8 >16 8 >16 >16 1
a
AFG, anidulafungin; AMB, amphotericin B; CAS, caspofungin, FLC, fluconazole; ISA, isavuconazole; ITC, itraconazole; MFG, micafungin; POS, posaconazole;
RAV, ravuconazole; VRC, voriconazole; 5FC, flucytosine.
EMERGING MICROBES AND INFECTIONS 89
species in the concatenated trees (Figure 5) as well as
all the individual DNA marker trees (Figure S2),
despite being the most closely related to Pleurostoma
ootheca and Pleurostoma repens. HKU44
T
also pos-
sessed morphological features different to those of
other known Pleurostoma species (Figure 4 and Figure
S3). All these results supported that HKU44
T
should
belong to a novel Pleurostoma species distinct from
any other member of this genus, and we propose a
novel species, Pleurostoma hongkongense, to accom-
modate HKU44
T
.
In addition to the discovery of a novel Pleurostoma
species, the present case also represents the first case of
Pleurostoma subhepatic abscess in a liver transplant
recipient. Among all the 37 cases of Pleurostoma infec-
tions reported, 33 (89.2%) were caused by Pleurostoma
richardsiae, and the remaining four cases were due to
Pleurostoma hongkongense, Pleurostoma ochraceum,
Pleurostoma ootheca and Pleurostoma repens, respect-
ively. For predisposing factors, more than 70% of
Pleurostoma infections have major underlying diseases
(Table 3). The commonest medical conditions leading
to systemic immunosuppression are diabetes mellitus
(33.3%), malignancies (18.5%) and renal transplant
(18.5%). Of all the 37 cases, 27 (73.0%) had infections
(osteomyelitis, cutaneous/subcutaneous nodules,
masses, lesions, cysts or abscesses, bursitis, cellulitis,
eumycetoma and chromoblastomycosis) of the extre-
mities, with 18 cases (66.7%) involving the lower
limbs only, six (22.2%) infecting the upper limbs
only and three (11.1%) affecting both upper and
lower extremities. The portal of entry of the fungus
is believed to be direct inoculation of the dematiaceous
fungus through the skin as a result of unnoticed minor
trauma. Pleurostoma infection has only been reported
in one other liver transplant recipient (Case 35, Table
3), who was a 57-year-old Thai man with disseminated
phaeohyphomycosis and hepatic artery and portal
vein thrombosis due to Pleurostoma richardsiae [40].
The fungus was recovered from the patient’s blood
as well as the blood clot from the failed liver graft.
Identification of the fungus was achieved through
ITS sequencing. On the other hand, our present case
had surgical site infection with subhepatic space infec-
tion. Although the route of infection cannot be ascer-
tained, we speculate that the fungus has invaded
through the wound of the surgical site and settled in
the subhepatic space.
DNA sequencing should be performed to accu-
rately identify Pleurostoma species from fungal cul-
tures with Pleurostoma-orPhialophora-like
morphologies. Microscopically, Pleurostoma species
are characterized by the possession of Phialophora-
like single, separate, hyaline to pigmented, monophia-
lidic, short conidiophores with inconspicuous or
flaring collarettes producing smooth, hyaline,
dimorphic conidia, either straight to allantoid or
short and ellipsoid, aggregated as slimy masses at the
apices of conidiogenous cells during their asexual life
cycles [55]. However, identification to the species
level is best performed by DNA sequencing of one
or more conserved markers, especially the ITS region
[9–11,25–27,29,30,38,40]. Notably, two early cases
of subcutaneous nodules were originally reported as
Pleurostoma repens-associated based on morphologi-
cal identification of the filamentous fungi isolated
[56,57]. However, subsequent molecular studies
revealed that the two fungal isolates were actually
Phaeoacremonium krajdenii instead [58]. As for
MALDI–TOF MS, although it has been increasingly
used for the rapid identification of filamentous fungi
Table 2. Microscopic features of different Pleurostoma species
during asexual life cycles.
Species Hyphae Phialides Conidia
P. hongkongense 2–6μm wide,
septate,
verruculose,
and hyaline
3–30 μm long,
phialide variety
similar to
P. ochraceum
Microconidia
2–8μm long,
spherical,
ellipsoidal or
oblong;
macroconidia
most
commonly
allantoid and
reniform
P. repens 2–4μm wide,
septate,
smooth and
initially
hyaline,
pale brown
when
matured
6–30 μm long,
inconspicuous,
slightly flared
collarrettes on
elongated
phialides,
conidiophores
often branched
3–6μm long,
cylindrical to
allantoid only
P. richardsiae 2–4μm wide,
septate and
smooth
when
young,
heavily
septated,
thick-walled
and brown
when
matured
10–35 μm long,
flask shaped
with distinct
saucer-shaped
flared collarette
at apex
2–6μm long,
commonly
spherical or
ellipsoidal
P. ochraceum 1.2–3.2 μm
wide,
branched,
septate,
smooth and
verruculose
6–20 μm long,
three types of
phialides
described: type
1
adelophialides,
type 2 elongate-
ampulliform,
and larger,
longer type 3
cyclindrical/
ampulliform
phialides
1.5–6μm long,
melanised
sub-spherical
or ellipsoidal
P. ootheca 1.8–3.3 μm
wide,
septate and
hyaline
6–19 μm long,
inconspiciously
flared
collarettes,
short phialides
3–6μm long,
most
commonly
oblong,
straight, or
allantoid;
shorter and
ellipsoid
conidia also
present
The asexual morph of P. candollei is not known [2]. Data for
P. hongkongense were obtained in this study, data for the other Pleuros-
toma species were cited from the literature [18,26,55].
90 C.-C. TSANG ET AL.
Table 3. Infections caused by Pleurostoma species.
Case
Sex
a
/
Age
(year) Ethnicity Underlying medical conditions Clinical syndrome Isolation source Fungal species Antifungal treatment Outcome Reference
1 M/80 American Congestive heart failure Subcutaneous cystic mass on
proximal interphalangeal
joint of left fifth finger
Cyst fluid P. richardsiae Surgical excision Remission [12]
2 F/50 German Not mentioned Dacryocystitis Lacrimal sac stone P. richardsiae Surgical excision Remission [36]
3 M/79 African
American
None Chronic bursitis on right
elbow
Cystic mass P. richardsiae Surgical excision Remission [31]
4 M/65 American
Puerto
Rican
Diabetes mellitus Right foot sole lesion Purulent fluid from
lesion
P. richardsiae Surgical excision and drainage Remission [13]
5 M/43 American
Jamaican
Diabetes mellitus,
adrenocortical carcinoma,
multiple hepatic metastases
Ulcerative lesion on right leg Lesion discharge P. richardsiae Not mentioned Deceased [13]
6 M/52 American None Subcutaneous lesion on left
index finger
Subcutaneous
mass
P. richardsiae Surgical excision Remission [7]
7 F/70 American Myeloproliferative disease Osteomyelitis of right foot Bone P. richardsiae Debridement of infected bone,
amphotericin B for 1 week followed by
flucytosine for 2.5 weeks
Deceased due to subsequent
disseminated Nocardia
infection
[32]
8 M/54 American Tendinitis, received
methylprednisolone injection
Subcutaneous mass on right
dorsal index finger
Not mentioned P. richardsiae Surgical excision Not mentioned [14]
9 M/30 Malaysian
Indian
Left femur fracture with skeletal
traction and intramedullary
nailing
Cutaneous lesions over left
lower tibia & ankle
Lesion biopsy P. richardsiae Oral griseofulvin and topical naftifine for
three weeks, surgical excision five
months after recurrence
Persisted for two more months
and then lost to follow-up
[15]
10 M/47 French
Senegalese
Hepatitis B virus carrier,
recurrent duodenal ulcer
Cutaneous nodule at right
patella
Cystic mass biopsy P. richardsiae Surgical excision Remission [16,20]
11 M/30 Japanese Non-Hodgkin’s lymphoma,
steroid-induced diabetes
mellitus, osteoporosis
Subcutaneous abscess on
right dorsal foot
Pus P. richardsiae Oral flucytosine, topical amphotericin B &
surgical excision
Deceased due to disseminated
intravascular coagulation
[17]
12 F/60 American Hypertension, diabetes mellitus,
atherosclerotic heart disease,
obesity
Subcutaneous cystic nodule
on left medial foot
Cyst aspirate P. richardsiae Surgical excision Remission [18]
13 M/64 Australian Polymyalgia rheumatica, on
prednisone & azathioprine
therapy
Cutaneous nodules on right
knee, right thigh and
elbows
Skin biopsy P. richardsiae &
Exophiala
jeanselmei
Oral flucytosine for 10 days, then
ketoconazole for 1 week, then
itraconazole for 3 months
Remission [19]
14 M/39 French Renal transplant Knee bursitis Not mentioned P. richardsiae Surgical drainage and antifungal therapy,
then surgical excision after recurrence
Not mentioned [20]
15 F/15 Indian None Cutaneous lesion near
waistline
Skin scrapings &
biopsy
P. richardsiae Topical clotrimazole for 15 days Remission [8]
16 F/50 Indian None Cutaneous lesion near
waistline
Skin scrapings &
biopsy
P. richardsiae Topical clotrimazole for 15 days Remission [8]
17 M/52 British Diabetes mellitus, porcine mitral
valve replacement
Endocarditis & fungaemia Blood, aortic &
porcine mitral
valves
P. richardsiae Aortic and mitral valves replacement,
amphotericin B for nearly 6 weeks
Deceased [39]
18 M/54 British Afro-
Caribbean
Renal transplant, hypertension,
steroid-induced diabetes
mellitus
Cutaneous lesions on right
ring finger & left knee
Pus P. richardsiae Surgical excision Remission [21]
(Continued)
EMERGING MICROBES AND INFECTIONS 91
Table 3. Continued.
Case
Sex
a
/
Age
(year) Ethnicity Underlying medical conditions Clinical syndrome Isolation source Fungal species Antifungal treatment Outcome Reference
19 M/28 French HIV, hepatitis C, intravenous
drug use, tuberculosis,
orthopaedic reconstructive
surgery of right foot
Osteomyelitis Bone aspirates of
left elbow & left
foot
P. richardsiae Amphotericin B and flucytosine followed
by itraconazole
Not mentioned [33]
20 M/41 American Surgical left ankle bone chip
removal
Left ankle osteomyelitis Surgical
debridement
specimens
P. repens Surgical debridement, amphotericin B,
oral ketoconazole for several months
Remission [34]
21 M/36 American Metallic intraocular foreign
body
Right eye endophthalmitis Vitreous samples P. richardsiae Pars plana vitrectomy and ketoconazole,
followed by a repeat vitrectomy with
intraocular lens removal, intravitreal
amphotericin B and miconazole
Recurred after 12 months post-
second vitrectomy
[37]
22 M/77 American None Septic infrapatellar bursitis &
contiguous cellulitis
Bursal & superficial
pustule aspirates,
skin punch
biopsy
P. richardsiae Intravenous amphotericin B for 2 weeks,
followed by oral itraconazole for 3
months
Remission [22]
23 M/45 New
Zealander
Renal transplant, transplant-
related diabetes mellitus
Subcutaneous left foot
abscess
Lesion aspirates,
tissues
P. richardsiae Surgical excision and liposomal
amphotericin B for 1 week, repeat
debridement & liposomal amphotericin
B for 2 weeks followed by itraconazole
for 6 weeks after recurrence
Remission [23]
24 M/59 Japanese Liver cirrhosis, hepatocellular
carcinoma, hepatitis C
Eumycetoma on left foot Lesion biopsy P. richardsiae Surgical excision Remission [24]
25 M/43 Korean None Right shin
chromoblastomycosis
Skin tissue biopsy P. richardsiae Oral itraconazole 3 months, then
combined with terbinafine for 3 months
Remission [25]
26 M/60 Sudanese None Yellow-grain eumycetoma on
right foot
Grains P. ochraceum Surgical debulking Lost to follow-up [26]
27 M/54 Canadian None Subcutaneous nodule on
right patella & prepatellar
bursitis
Prepatellar fluid
aspirate
P. richardsiae Therapy declined by patient Lost to follow-up [9]
28 M/59 Martinican Renal transplant Left ankle abscess Cutaneous tissue P. ootheca Itraconazole for 1.5 months, then
posaconazole for 2 months
Remission [27]
29 M/43 American Alcoholic cardiomyopathy,
porcine mitral valve
replacement, intravenous
drug abuse, on systemic
corticosteroid therapy
Endogenous
endophthalmitis of right
eye associated with
osteomyelitis and
endocarditis
Vitreous biopsy P. richardsiae Intravitreal amphotericin B during initial
diagnostic vitrectomy, topical
natamycin & intravenous amphotericin
B, intravenous voriconazole added later,
followed by repeat vitrectomy and
intravitreal amphotericin B
Deceased due to massive
intracranial haemorrhage
[35]
30 F/75 Dutch Chinese Diabetes mellitus, chronic
kidney disease, rheumatoid
arthritis
Left arm infection Pus and wound P. richardsiae Voriconazole for 6 months, repeated
surgical eradication, discontinuation of
immunosuppression
Deceased due to subsequent
progressive multifocal
leukoencephalopathy due to
Human polyomavirus 2
reactivation
[28]
31 M/78 Japanese Diabetes mellitus, prostate &
bladder cancers,
myelodysplastic syndrome
Subcutaneous cyst on right
crus
Pus P. richardsiae Local heat therapy with disposable
chemical pocket warmers for 2 months,
then combined with intracutaneous
amphotericin B for 3 months
Deceased due to cancers [29]
32 F/35 Indian None Perilacrimal mass P. richardsiae Surgical excision Remission [10]
92 C.-C. TSANG ET AL.
in clinical microbiology laboratories, use of this tech-
nology for Pleurostoma identification is still not poss-
ible since reference protein mass spectra for
Pleurostoma species are still lacking in the databases
by the two major commercial platforms (Bruker Dal-
tonics and Vitek, bioMérieux). Moreover, our attempt
of MALDI–TOF MS analysis using the Bruker Dal-
tonics platform for our case isolate HKU44
T
as well
as other reference Pleurostoma strains also failed in
generating interpretable protein mass spectra for data-
base matching, probably due to difficulties in extract-
ing cellular proteins during formic acid digestion. An
optimal protein extraction protocol for sample prep-
aration and expansion of the reference mass spectral
libraries will be essential before MALDI–TOF MS
could be utilized for the rapid identification of Pleur-
ostoma species.
Treatment of Pleurostoma infections is often
achieved by a combination of surgical excision/debri-
dement of the infected tissue and antifungal therapy.
Of the 37 cases of Pleurostoma infections, 25 (67.6%)
required surgery to remove the infected tissue and
22 (59.4%) involved the administration of antifungals
(Table 3). For the present patient as well as the pre-
vious liver transplant recipient, they were both treated
successfully with a second liver transplant and anti-
fungal agents. As for the choice of antifungal agents,
almost all the Pleurostoma strains tested in the present
study had high MICs/MECs to fluconazole, 5-flucyto-
sine and the echinocandins (Table 1). For amphoteri-
cin B, 18 (64.3%) of the 28 Pleurostoma strains tested,
including our case isolate Pleurostoma hongkongense
HKU44
T
, had MICs of ≤1 µg/mL, which is the general
breakpoint for Candida species, Cryptococcus neofor-
mans, Aspergillus fumigatus and Aspergillus niger
[59]. As for the susceptibilities to the other triazoles,
the results showed a high strain-to-strain variability
(Table 1). In general, a strong Spearman correlation
among the susceptibilities to different triazoles can
be observed (Table S2). In view of the highly variable
antifungal susceptibility profiles for Pleurostoma
species and strains, MICs/MECs should be deter-
mined for deciding the optimal treatment regimen
for individual patients. Remarkably, for the 36 Pleur-
ostoma infection cases previously reported, seven of
the isolates recovered were included in this study for
susceptibility testing (ATCC 58041 [32], CBS 506.90
[8], CBS 131321
T
[26], UAMH 5052 [13], UAMH
5056 [12], UAMH 5058 [13] and UAMH 10082
[23]). Amongst these seven cases, antifungal therapies
were only administered for three of the patients (Case
7, ATCC 58041 [32]; Case 16, CBS 506.90 [8] and Case
23, UAMH 10082 [23]inTable 3). For Case 7, the
patient was treated with the debridement of infected
bone and amphotericin B prescription for one week
followed by flucytosine prescription for two and a
half weeks [32], although susceptibility testing in this
Perilacrimal organised lesion
with transient nasolacrimal
duct obstruction
33 M/72 Turkish Papillary urothelial carcinoma Urinary tract infection Urine P. richardsiae Amphotericin B for 4 days followed by
itraconazole for 14 days
Remission [38]
34 M/78 Spaniard None Subcutaneous cyst nodule on
right dorsal hand
Cyst biopsy P. richardsiae Therapy declined by patient Not mentioned [30]
35 M/57 Thai Hepatitis B virus-related
cirrhosis, liver transplant
Fungaemia associated with
hepatic artery and portal
vein thrombosis
Blood and liver
blood clot
P. richardsiae Second liver transplant, amphotericin B
for 4 weeks
Remission [40]
36 M/74 Singaporean
Chinese
Renal transplant, bronchiectasis Subcutaneous thigh nodules Skin biopsy and
pus
P. richardsiae Surgical excisions, itraconazole for 10
months
Remission [11]
37 M/65 Hong Kong
Chinese
Hepatitis B virus-related liver
failure, liver transplant
Progressive liver failure Subhepatic abscess
pus and drain
fluids
P. hongkongense Second liver transplant, amphotericin B
and voriconazole
Remission Present
case
a
F, female; M, male.
Note: The fungal strains (UTMB 184 = CBS 423.75 [60] and SM 3531 = ATCC 58115 = BB 903 = CBS 110366 [56,57]) isolated from two reported cases of Pleurostoma repens were subsequently reidentified as Phaeoacremonium krajdenii by
molecular studies [58].
EMERGING MICROBES AND INFECTIONS 93
study showed that the isolate ATCC 58041 possessed
MICs of 1 µg/mL and >16 µg/mL for these two anti-
fungals, respectively. Similarly, for Case 23, the patient
was first treated with surgical excision and prescrip-
tion of liposomal amphotericin B for one week. How-
ever, due to recurrence a repeat debridement was
performed and liposomal amphotericin B for two
weeks followed by itraconazole for six weeks was pre-
scribed [23]. Again, susceptibility testing in this study
showed that isolate UAMH 10082 possessed high
MICs of 2 µg/mL and >8 µg/mL for these two antifun-
gals, respectively. For Case 16, the patient was treated
with clotrimazole for 15 days [8]. Susceptibility to this
drug was not tested in the present study and therefore,
the activity of this drug to the fungal isolate (CBS
506.90) remains unknown. As for the present reported
case due to Pleurostoma hongkongense, the patient was
treated with antimicrobials including anidulafungin
before the second liver transplant. However, this was
not successful in curing the infection which may be
partly due to a high MEC for anidulafungin (>16 µg/
mL) for HKU44
T
. After the second liver transplant,
the patient was treated with amphotericin B and vor-
iconazole while HKU44
T
possessed MICs of 0.5 and
16 µg/mL for these two drugs, respectively. As a result,
antifungal susceptibility testing should be performed
for Pleurostoma species to guide patient management
and to avoid the unnecessary use of less or ineffective
drugs.
Acknowledgements
This work was partly supported by the Innovation and
Technology Fund Midstream Research Programme for Uni-
versities (MRP/026/18) of the Innovation and Technology
Commission, the Government of the Hong Kong Special
Administrative Region. Any opinions, findings, conclusions
or recommendations expressed in this material/event (or by
members of the project team) do not reflect the views of the
Government of the Hong Kong Special Administrative
Region, the Innovation and Technology Commission or
the Innovation and Technology Fund Research Projects
Assessment Panel. We are grateful to Mrs Adrien Szekely
of NCPF for providing the NCPF reference strains for
free; Professor Maiken C. Arendrup and Dr Karin
M. Jørgensen of SSI for providing the CNM-CL and
CNM-CM reference strains for free; as well as the curators,
especially Mr Travis W. Adkins, of NRRL for providing the
NRRL reference strains for free. We also thank the Electron
Microscope Unit of The University of Hong Kong which
helped process the samples for scanning electron
microscopy.
Disclosure statement
Patrick C. Y. Woo has provided scientific advisory/labora-
tory services for Gilead Sciences, Incorporated; Inter-
national Health Management Associates, Incorporated;
Merck & Corporation, Incorporated; Micología Molecular
S.L. and Pfizer, Incorporated. The other authors report no
conflicts of interest. The funding sources had no role in
study design, data collection, analysis, interpretation, or
writing of the report. The authors alone are responsible
for the content and the writing of the manuscript. Part of
this study has been presented as an abstract for the 30
th
European Congress of Clinical Microbiology and Infectious
Diseases.
Funding
This work was partly supported by the Innovation and
Technology Fund Midstream Research Programme for Uni-
versities (grant number MRP/026/18) of the Innovation and
Technology Commission, the Government of the Hong
Kong Special Administrative Region. Any opinions,
findings, conclusions or recommendations expressed in
this material/event (or by members of the project team)
do not reflect the views of the Government of the Hong
Kong Special Administrative Region, the Innovation and
Technology Commission or the Innovation and Technology
Fund Research Projects Assessment Panel.
ORCID
Chi-Ching Tsang http://orcid.org/0000-0001-6705-2866
Ka-Fai Chan http://orcid.org/0000-0003-2290-3182
Walton Chan http://orcid.org/0000-0003-0909-8414
Jasper F. W. Chan http://orcid.org/0000-0001-6336-6657
Rex K. H. Au-Yeung http://orcid.org/0000-0002-3137-
0886
Ken P. K. Lin http://orcid.org/0000-0002-0226-8874
Susanna K. P. Lau http://orcid.org/0000-0002-1383-7374
Patrick C. Y. Woo http://orcid.org/0000-0001-9401-1832
References
[1] Tulasne LR, Tulasne C. Selecta Fungorum Carpologia
II. Paris: Imperiali Typographeo Excudebatur; 1863.
[2] Réblová M, Jaklitsch WM, Réblová K, et al.
Phylogenetic reconstruction of the Calosphaeriales
and Togniniales using five genes and predicted RNA
secondary structures of ITS, and Flabellascus tenuiros-
tris gen. et sp. nov. PLoS One. 2015;10(12):e0144616.
[3] Réblová M, Miller AN, Rossman AY, et al.
Recommendations for competing sexual-asexually
typified generic names in Sordariomycetes (except
Diaporthales,Hypocreales, and Magnaporthales).
IMA Fungus. 2016;7(1):131–153.
[4] Schol-Schwarz MB. Revision of the genus Phialophora
(Moniliales). Persoonia. 1970;6(1):59–94.
[5] Barr ME. Notes on the Calosphaeriales. Mycologia.
1985;77(4):549–565.
[6] Melin E, Nannfeldt JA. Researches into the blueing of
ground wood-pulp. Svenska Skogsvårdsför Tidskr.
1934;32:397–430.
[7] Moskowitz LB, Cleary TJ, McGinnis MR, et al.
Phialophora richardsiae in a lesion appearing as a
giant cell tumor of the tendon sheath. Arch Pathol
Lab Med. 1983;107:374–376.
[8] Singh SM, Agrawal A, Naidu J, et al. Cutaneous
phaeohyphomycosis caused by Phialophora richard-
siae and the effect of topical clotrimazole in its treat-
ment. Anton Leeuwenh. 1992;61(1):51–55.
[9] Levenstadt JS, Poutanen SM, Mohan S, et al.
Pleurostomophora richardsiae –an insidious fungus
presenting in a man 44 years after initial inoculation:
94 C.-C. TSANG ET AL.
a case report and review of the literature. Can J Infect
Dis Med Microbiol. 2012;23(3):110–113.
[10] Alam MS, Vaidehi D, Therese KL, et al. Rare
Pleurostomophora richardsiae mass causing transient
nasolacrimal duct obstruction. Ophthal Plast
Reconstr Surg. 2017;33(6):e154–e156.
[11] Tee LY, Tan BH, Tan A-L, et al. Subcutaneous phaeo-
hyphomycosis caused by Pleurostomophora richard-
siae in a renal transplant recipient. JAAD Case Rep.
2020;6(1):66–68.
[12] Schwartz IS, Emmons CW. Subcutaneous cystic gran-
uloma caused by a fungus of wood pulp (Phialophora
richardsiae). Am J Clin Pathol. 1968;49(4):500–505.
[13] Corrado ML, Weitzman I, Stanek A, et al.
Subcutaneous infection with Phialophora richardsiae
and its susceptibility to 5-fluorocytosine, amphotericin
B and miconazole. Sabouraudia. 1980;18(2):97–104.
[14] Reyes FA, Buchman MT. Phialophora richardsiae
infection mimicking a soft tissue mass of a finger. J
Hand Surg Br. 1986;11(2):274.
[15] Suppiah M, Chin CS, Keah KC. Phialophora richard-
siae isolated from a cutaneous lesion. Med J
Malaysia. 1987;42(4):306–308.
[16] Bonnefoy A, Luboinski J, Francoual S, et al.
Phaeohyphomycosis due to Phialophora richardsiae.
Med Mal Infect. 1988;18(4):235–238.
[17] Ikai K, Tomono H, Watanabe S. Phaeohyphomycosis
caused by Phialophora richardsiae. J Am Acad
Dermatol. 1988;19(3):478–481.
[18] Pitrak DL, Koneman EW, Estupinan RC, et al.
Phialophora richardsiae infection in humans. Rev
Infect Dis. 1988;10(6):1195–1203.
[19] Tam M, Freeman S. Phaeohyphomycosis due to
Phialophora richardsiae. Australas J Dermatol.
1989;30(1):37–40.
[20] Guého E, Bonnefoy A, Luboinski J, et al. Subcutaneous
granuloma caused by Phialophora richardiae: case
report and review of the literature Phialophora
richardsiae. Mycoses. 1989;32(5):219–223.
[21] Jumaa PA, Lightowler C, Baker LRI, et al. Cutaneous
infection caused by Phialophora richardsiae treated
successfully by surgical excision in an immunocom-
promised patient. J Infect. 1995;30(3):261–262.
[22] Cornia PB, Raugi GJ, Miller RA. Phialophora richard-
siae bursitis treated medically. Am J Med. 2003;115
(1):77–79.
[23] Yehia M, Thomas M, Pilmore H, et al. Subcutaneous
black fungus (phaeohyphomycosis) infection in renal
transplant recipients: three cases. Transplantation.
2004;77(1):140–142.
[24] Sakayama K, Kidani T, Sugawara Y, et al. Mycetoma of
foot: a rare case report and review of the literature.
Foot Ankle Int. 2004;25(10):763–767.
[25] Son Y-M, Kang H-K, Na S-Y, et al.
Chromoblastomycosis caused by Phialophora richard-
siae. Ann Dermatol. 2010;22(3):362–366.
[26] Mhmoud NA, Ahmed SA, Fahal AH, et al.
Pleurostomophora ochracea, a novel agent of human
eumycetoma with yellow grains. J Clin Microbiol.
2012;50(9):2987–2994.
[27] Amazan E, Desbois N, Fidelin G, et al. First case of
phaeohyphomycosis due to Pleurostoma ootheca in a
kidney transplant recipient in Martinique (French
West Indies). Med Sante Trop. 2014;24(3):323–325.
[28] de Regt MJA, Murk J-L, Schneider-Hohendorf T, et al.
Progressive multifocal leukoencephalopathy and black
fungus in a patient with rheumatoid arthritis without
severe lymphocytopenia. JMM Case Rep. 2016;3(4):
e005053.
[29] Noguchi H, Hiruma M, Matsumoto T, et al.
Subcutaneous cystic phaeohyphomycosis due to
Pleurostomophora richardsiae. J Dermatol. 2017;44
(4):e62–e63.
[30] Cuenca-Barrales C, De Salazar A, Chueca N, et al.
Phaeohyphomycosis due to Pleurostomophora richard-
siae: an uncommon cutaneous fungal infection. J Eur
Acad Dermatol Venereol. 2018;32(10):e376–e377.
[31] Torstrick RF, Harrison K, Heckman JD, et al. Chronic
bursitis caused by Phialophora richardsiae. A case
report. JBJS. 1979;61(5):772–774.
[32] Yangco BG, TeStrake D, Okafor J. Phialophora
richardsiae isolated from infected human bone: mor-
phological, physiological and antifungal susceptibility
studies. Mycopathologia. 1984;86(2):103–111.
[33] Uberti-Foppa C, Fumagalli L, Gianotti N, et al. First
case of osteomyelitis due to Phialophora richardsiae in
a patient with HIV infection. AIDS. 1995;9(8):975–976.
[34] Rossmann SN, Cernoch PL, Davis JR. Dematiaceous
fungi are an increasing cause of human disease. Clin
Infect Dis. 1996;22(1):73–80.
[35] Fox AR, Houser KH, Morris WR, et al. Dematiaceous
fungal endophthalmitis: report of a case and review of
the literature. J Ophthalmic Inflamm Infect. 2016;6
(1):43.
[36] Listemann H. Die kulturelle untersuchung eines tra-
nensteines mit isolierung des pilzes Phialophora
richardsiae. Ernst Rodenwaldt Arc. 1975;2(2):45–52.
[37] Lieb DF, Smiddy WE, Miller D, et al. Case report: fun-
gal endophthalmitis caused by Phialophora richard-
siae. Retina. 2003;23(3):406–407.
[38] Polat SH, Kizilay F, Uyan A, et al. Urinary tract infec-
tion due to Pleurostomophora richardsiae. Med Mycol.
2018;56(Suppl. 2):S91.
[39] Juma A. Phialophora richardsiae endocarditis of aortic
and mitral valves in a diabetic man with a porcine
mitral valve. J Infect. 1993;27(2):173–175.
[40] Sribenjalux W, Chongtrakool P, Chayakulkeeree M.
Disseminated phaeohyphomycosis with hepatic artery
and portal vein thrombosis caused by
Pleurostomophora richardsiae in a liver transplant
recipient: a case report. Transpl Infect Dis. 2019;21
(3):e13075.
[41] Ngan AHY. Atlas of fungi. Hong Kong: June Just
Printing; 2016.
[42] Woo PCY, Ngan AHY, Tsang CCC, et al. Clinical
spectrum of Exophiala infections and a novel
Exophiala species, Exophiala hongkongensis. J Clin
Microbiol. 2013;51(1):260–267.
[43] White T, Bruns T, Lee S, et al. Amplification and direct
sequencing of fungal ribosomal RNA genes for phylo-
genetics. In: Innis M, Gelfand D, Shinsky J, editor.
PCR protocols: a guide to methods and applications.
San Diego: Academic Press; 1990. p. 315–322.
[44] O’Donnell K. Fusarium and its near relatives. In:
Reynolds DR, Taylor JW, editor. The fungal holo-
morph: Mitotic, meiotic and pleomorphic speciation
in fungal systematics. Wallingford: CAB
International; 1993. p. 225–233.
[45] Cruse M, Telerant R, Gallagher T, et al. Cryptic species
in Stachybotrys chartarum. Mycologia. 2002;94
(5):814–822.
[46] Hall TA. Bioedit: a user-friendly biological sequence
alignment editor and analysis program for Windows
95/98/NT. Nucleic Acids Symp Ser. 1999;41:95–98.
EMERGING MICROBES AND INFECTIONS 95
[47] Edgar RC. MUSCLE: multiple sequence alignment
with high accuracy and high throughput. Nucleic
Acids Res. 2004;32(5):1792–1797.
[48] Castresana J. Selection of conserved blocks from mul-
tiple alignments for their use in phylogenetic analysis.
Mol Biol Evol. 2000;17(4):540–552.
[49] Talavera G, Castresana J. Improvement of phylogenies
after removing divergent and ambiguously aligned
blocks from protein sequence alignments. Syst Biol.
2007;56(4):564–577.
[50] Tamura K, Stecher G, Peterson D, et al. MEGA6: mol-
ecular evolutionary genetics analysis version 6.0. Mol
Biol Evol. 2013;30(12):2725–2729.
[51] Drummond AJ, Suchard MA, Xie D, et al. Bayesian
phylogenetics with BEAUti and the BEAST 1.7. Mol
Biol Evol. 2012;29(8):1969–1973.
[52] Arendrup MC, Meletiadis J, Mouton JW. EUCAST
definitive Document E.Def 9.3.1. Method for the deter-
mination of broth dilution minimum inhibitory con-
centrations of antifungal agents for conidia forming
moulds 2017. Available from: http://www.eucast.org/
fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/
EUCAST_E_Def_9_3_1_Mould_testing__definitive.pdf.
[53] Tsang C-C, Tang JYM, Chan K-F, et al. Diversity of
phenotypically non-dermatophyte, non-Aspergillus
filamentous fungi causing nail infections: importance
of accurate identification and antifungal susceptibility
testing. Emerg Microbes Infect. 2019;8(1):531–541.
[54] Tekpinar AD, Kalmer A. Utility of various molecular
markers in fungal identification and phylogeny.
Nova Hedwigia. 2019;109(1–2):187–224.
[55] Vijaykrishna D, Mostert L, Jeewon R, et al.
Pleurostomophora,ananamorphofPleurostoma
(Calosphaeriales), a new anamorph genus morphologi-
callysimilar to Phialophora.StudMycol.2004;50:387–395.
[56] Hironaga M, Nakano K, Yokoyama I, et al.
Phialophora repens, an emerging agent of subcu-
taneous phaeohyphomycosis in humans. J Clin
Microbiol. 1989;27(3):394–399.
[57] Nakano K, Yokoyama I, Kitajima J, et al. A case of sub-
cutaneous granuloma caused by Phialophora repens.
Rinsho Hifuka. 1990;44(1):25–28.
[58] Mostert L, Groenewald JZ, Summerbell RC, et al.
Species of Phaeoacremonium associated with
infections in humans and environmental reservoirs
in infected woody plants. J Clin Microbiol. 2005;43
(4):1752–1767.
[59] European Committee on Antimicrobial Susceptibility
Testing. Overview of antifungal ECOFFs and clinical
breakpoints for yeasts, moulds and dermatophytes
using the EUCAST E.Def 7.3, E.Def 9.3 and E.Def
11.0 procedures. Version 2, valid from 2020-09-24.
2020. Available from: http://www.eucast.org/.
[60] Meyers WM, Dooley JR, Kwon-Chung KJ. Mycotic
granuloma caused by Phialophora repens. Am J Clin
Pathol. 1975;64(4):549–555.
96 C.-C. TSANG ET AL.
