Pleurostomophora ochracea, a Novel Agent of Human Eumycetoma with Yellow Grains

Abstract

The first yellow-grain fungal mycetoma, in a 60-year-old man from Central Sudan, is reported. Morphological and phylogenetic
analysis of the ribosomal small subunit (SSU), large subunit (LSU), internal transcribed spacer (ITS), β-tubulin (BT2), actin (ACT1), and elongation factor (TEF1) genes revealed that the isolate deviated from any known agent of mycetoma; it clustered in the genus Pleurostoma (anamorph genus, Pleurostomophora) in the order Calosphaeriales. The novel species, here named Pleurostomophora ochracea, is characterized by phenotypic features. The species proved to be highly susceptible to itraconazole, ketoconazole, posaconazole,
and voriconazole, but not to fluconazole. The fungus was inhibited by caspofungin at 8 μg/ml, while no inhibition was found
with 5-flucytosine (MIC > 64 μg/ml). Compared to other members of the genus Pleurostomophora, P. ochracea is slow growing, with a relatively high optimum growth temperature (36 to 37°C). This is the first case of a yellow-grain
fungal mycetoma; yellow grains are otherwise of bacterial nature. Our case emphasizes that identification of mycetoma agents
by the color of the grain only is not sufficient and may lead to inappropriate therapy.

Full text

Pleurostomophora ochracea, a Novel Agent of Human Eumycetoma
with Yellow Grains
Najwa A. Mhmoud,
a
Sarah Abdalla Ahmed,
b,c,d
Ahmed H. Fahal,
a
G. Sybren de Hoog,
b,c,e
A. H. G. Gerrits van den Ende,
b
and Wendy W. J. van de Sande
f
Mycetoma Research Centre, University of Khartoum, Khartoum, Sudan
a
; Centraalbureau voor Schimmelcultures KNAW Fungal Biodiversity Centre, Utrecht, Netherlands
b
;
Institute of Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, Netherlands
c
; Faculty of Medical Laboratory Sciences, University of Khartoum,
Khartoum, Sudan
d
; Peking University Health Center, Beijing, China, and Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China
e
; and Erasmus MC,
University Medical Centre Rotterdam, Department of Medical Microbiology and Infectious Diseases, Rotterdam, Netherlands
f
The first yellow-grain fungal mycetoma, in a 60-year-old man from Central Sudan, is reported. Morphological and phylogenetic
analysis of the ribosomal small subunit (SSU), large subunit (LSU), internal transcribed spacer (ITS), ␤-tubulin (BT2), actin
(ACT1), and elongation factor (TEF1) genes revealed that the isolate deviated from any known agent of mycetoma; it clustered in
the genus Pleurostoma (anamorph genus, Pleurostomophora) in the order Calosphaeriales. The novel species, here named Pleu-
rostomophora ochracea, is characterized by phenotypic features. The species proved to be highly susceptible to itraconazole,
ketoconazole, posaconazole, and voriconazole, but not to fluconazole. The fungus was inhibited by caspofungin at 8 ␮g/ml,
while no inhibition was found with 5-flucytosine (MIC >64 ␮g/ml). Compared to other members of the genus Pleurosto-
mophora,P. ochracea is slow growing, with a relatively high optimum growth temperature (36 to 37°C). This is the first case of a
yellow-grain fungal mycetoma; yellow grains are otherwise of bacterial nature. Our case emphasizes that identification of myce-
toma agents by the color of the grain only is not sufficient and may lead to inappropriate therapy.
Mycetoma is a chronic granulomatous, progressive, subcuta-
neous inflammatory infection (12). It is caused by true fungi
or by bacteria, and hence, it is classified as eumycetoma or actino-
mycetoma, respectively (1,3). The triad of a painless subcutane-
ous mass, multiple sinuses, and seropurulent discharge contain-
ing grains is pathognomic of mycetoma (5,14). Eumycetoma is
more common than actinomycetoma throughout the world, and
in Sudan, it accounts for 70% of all mycetoma cases (5). A variety
of fungi are implicated in causing eumycetoma (Table 1). In Su-
dan, eumycetoma is usually caused by the fungus Madurella my-
cetomatis, which forms black grains in human tissue (1,15). The
most common fungal pathogens causing eumycetoma produce
either black grains or white grains (5,10). While yellow grains
have been encountered in cases of actinomycetoma, they have
never been reported before in eumycetoma. The available diag-
nostic tools for mycetoma are limited and are mostly of a pheno-
typic nature (1). A first indication is usually obtained from the
color of the grain: black grains are produced by fungi and yellow
grains by bacteria, while white grains can be of bacterial or fungal
nature. Culture media used for diagnosis are based upon this first
indication. Here, we present a case of yellow-grain mycetoma,
which was initially misdiagnosed as actinomycetoma based on the
color of the grain and on cytology. In this report, we show that
cultural and molecular techniques, in combination with an up-
dated taxonomy, can be successfully used to identify the yellow-
grain-producing fungus Pleurostomophora ochracea as a new caus-
ative agent of human mycetoma.
CASE REPORT
The patient was a 60-year-old male farmer from central Sudan. In
2006, he presented to the Mycetoma Clinic at the Mycetoma Re-
search Centre of the University of Khartoum, Khartoum, Sudan,
with a massive, recurrent right-foot swelling. His condition
started 20 years prior to presentation with a small, painless right-
foot swelling, which had gradual onset and progress and was pain-
ful. He developed multiple sinuses, and the seropurulent dis-
charge contained yellow grains. The patient underwent four
surgical excisions under general and spinal anesthesia done else-
where. He had neither a history of local trauma nor a family his-
tory of mycetoma. The social and geographic history and drug use
were not contributory to his problem. On examination, he looked
well, not pale and neither jaundiced nor cyanosed. He had a nor-
mal pulse rate (75/min), respiratory rate (16/min), blood pressure
(120/80), and temperature (37°C). Also, his renal and hepatic
function tests, as well as his full blood count, were within normal
limits. Local examination revealed a painless massive mass involv-
ing the dorsal and plantar parts of the right foot. There were mul-
tiple active and healed sinuses, and the seropurulent discharge
contained yellow grains (Fig. 1). The foot X-ray confirmed a mas-
sive soft tissue swelling and revealed a periosteal reaction and bone
destruction (Fig. 1). Fine-needle aspiration for cytology was taken
from the lesion and showed an intense inflammatory infiltrate
with grains typical of actinomycetes. The patient was started on
streptomycin (1 g per day) and cotrimoxazole (945 mg twice a
day) and was followed up regularly in the outpatient clinic. How-
ever, there was no clinical response to this treatment. He was then
switched to amikacin sulfate (15 mg/kg of body weight twice a
Received 4 June 2012 Returned for modification 20 June 2012
Accepted 25 June 2012
Published ahead of print 3 July 2012
Address correspondence to Wendy W. J. van de Sande, w.vandesande
@erasmusmc.nl.
N.A.M. and S.A.A. contributed equally to this article.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.01470-12
September 2012 Volume 50 Number 9 Journal of Clinical Microbiology p. 2987–2994 jcm.asm.org 2987

day) and cotrimaxozole (945 mg twice a day) with no clinical
improvement. He underwent surgical debulking to improve the
response to medical treatment. Numerous yellow grains were col-
lected from discharging sinuses, which were hard in consistency
and irregular in size. Culturing of the grains revealed fungal
growth, indicating that the patient was suffering from a eumyce-
toma rather than an actinomycetoma. Unfortunately, changing
the treatment accordingly was not possible; the patient was lost at
follow-up and was never seen again.
MATERIALS AND METHODS
Clinical specimen. A clinical specimen was collected from the patient
when he presented with yellow-grain mycetoma at the Mycetoma Re-
search Centre, University of Khartoum, Khartoum, Sudan, on 27 October
2008. After obtaining written consent from the patient, an aspirate with
visible yellow grains was taken from the sinuses. The yellow grains were
collected for further identification.
Fungal isolation. Grains collected from the sinus were washed twice in
physiological saline and inoculated into Lowenstein-Jensen (LJ) medium
(Hi Media Laboratories, Mumbai, India). The grain was cultured for 3 to
4 weeks at 37°C. This initial inoculation on LJ medium was guided by the
color of the grain: yellow grains were considered to be of bacterial origin.
After a week, the color of the grains converted from yellow to black, and
the grain was subcultured on Sabouraud’s dextrose agar (SDA) (Hi Me-
dia). The culture was identified morphologically as a fungal species. For
identification, the isolate was shipped to the Department of Medical Mi-
crobiology and Infectious Diseases, Erasmus MC, Rotterdam, Nether-
lands, and to the Centraalbureau voor Schimmelcultures (CBS) KNAW
Fungal Biodiversity Centre, Utrecht, Netherlands, and deposited in the
CBS collection under number CBS 131321.
Morphology and physiology. Colony characteristics and growth
morphology were studied by inoculating the isolate onto plates of malt
extract agar (MEA) (Oxoid, United Kingdom), oat meal agar (OA)
(homemade at CBS, Netherlands), corn meal agar (CMA) (homemade at
CBS, Netherlands), and potato dextrose agar (PDA) (Oxoid, United King-
dom) and incubating the plates in the dark at 30°C for up to 4 weeks.
Microscopic mounts in lactic acid with cotton blue were made from cul-
tures grown on a PDA plate and slide cultures after 2 weeks of incubation
at 30°C. The slides were examined and measured with a light microscope
(Nikon Eclipse 80i), and pictures were taken using a camera attached to
the microscope (Nikon; digital-sight DS-5 M). A minimum of 10 mea-
surements per structure were taken after processing in Adobe Photoshop
CS3, and the average was calculated. Cardinal growth temperatures were
determined on PDA, and the cultures were incubated in the dark for 4
weeks at temperatures ranging from 15°C to 40°C at 3°C intervals. The
ability of the isolate to assimilate carbohydrate sources was determined
with the API 20C AUX system (bioMérieux, Marcy l’Etoile, France). Prior
to the carbohydrate assimilation test, the homogenized fungal suspension
was prepared by ultrasonic treatment as published previously (4,36) and
then was adjusted to a 2 McFarland standard with the medium provided.
TABLE 1 Agents of mycetoma in mammals and their phylogenetic
positions
a
Name Order Grain
Acremonium kiliense Hypocreales White
Acremonium potronii Hypocreales White
Acremonium recifei Hypocreales White
Cylindrocarpon cyanescens Hypocreales White
Cylindrocarpon destructans Hypocreales White
Fusarium falciforme Hypocreales White
Fusarium verticillioides Hypocreales White
Fusarium solani Hypocreales White
Phialemonium obovatum Hypocreales White
Aspergillus flavus Eurotiales White
Aspergillus hollandicus Eurotiales White
Aspergillus nidulans Eurotiales White
Bipolaris spicifera Pleosporales Black
Corynespora cassiicola Pleosporales Black
Curvularia geniculata Pleosporales Black
Curvularia lunata Pleosporales Black
Leptosphaeria senegalensis Pleosporales Black
Leptosphaeria thompkinsii Pleosporales Black
Madurella grisea Pleosporales Black
Pseudochaetosphaeronema larense Pleosporales Black
Pyrenochaeta mackinnonii Pleosporales Black
Pyrenochaeta romeroi Pleosporales Black
Neotestudina rosatii Pleosporales White/black
Madurella mycetomatis Sordariales Black
Madurella pseudomycetomatis Sordariales Black
Madurella fahalii Sordariales Black
Madurella tropicana Sordariales Black
Phaeoacremonium parasiticum Diaporthales White
Phaeoacremonium krajdenii Diaporthales White
Exophiala jeanselmei Chaetothyriales Black
Pseudallescheria boydii Microascales White
Trichophyton sp. Onygenales White
Microsporum canis Onygenales White
a
Adapted from de Hoog et al. (10).
FIG 1 Clinical presentation of the patient. (A) The patient had a mycetoma on the right foot, with massive, recurrent foot swelling. (B) On the foot X-ray, a
massive soft tissue swelling, a periosteal reaction, and bone destruction are seen.
Mhmoud et al.
2988 jcm.asm.org Journal of Clinical Microbiology

Finally, 100 ␮l of this inoculum was used to fill the cupules of the test strips
as directed by the manufacturer. The ability of the isolate to hydrolyze
casein, gelatin, and starch was determined by culturing the strains on
selective plates according to the manufacturer’s instructions. The ability
to produce urease was determined by culturing on urea agar (urea base
concentrate [Difco] with 1.5% [wt/vol] agar). Sodium chloride tolerance
was determined by comparing the growth after 2 weeks on SDA slants
containing 0, 0.5, 5, 10, and 30% NaCl.
DNA extraction, amplification, and sequencing. Genomic DNA was
extracted by scraping material off Sabouraud plates, freezing it in liquid
nitrogen, and grinding it with a mortar and pestle. DNA was extracted
from the resulting pulp with the Promega Wizard Kit (Promega) accord-
ing to the manufacturer’s instructions. The ribosomal DNA (rDNA) in-
ternal transcribed spacer (ITS) gene and partial sequences of the actin
(ACT1), ␤-tubulin (BT2), and elongation factor 1␣(TEF1) genes, as well
as the 18S rDNA gene (small subunit [SSU]) and 28S rDNA gene (large
subunit [LSU]), were amplified and sequenced. The primer pairs for the
genes were ITS4 and ITS5 (39), ACT-512F and ACT-783R (9), T1 and
BT2b (18,28), EF2 and EF728F (9,23), NS1 and NS24 (17,39), and LRoR
and LR5 (38), respectively. Additional primers used for sequencing of the
SSU included NS2, NS3, NS6, and NS7 (39).
Alignment and phylogenetic analysis. A consensus sequence was
computed from the forward and reverse sequences with SeqMan from the
Lasergene package (DNAstar, Madison, WI). Sequences retrieved from
GenBank are listed in Fig. 2 and in Table 2. Sequences were aligned with
Multiple Sequence Comparison by Log-Expectation (MUSCLE) using the
EMBL-EBI Web server (http://www.ebi.ac.uk/Tools/msa/muscle/). An
alignment was constructed for the complete ITS (ITS1-5.8S-ITS2), in-
cluding 30 strains representing 12 species. A combined 28S LSU and 18S
SSU alignment for 39 strains from 36 fungal species was constructed. To
investigate the phylogenetic relationship of the newly isolated fungus and
related taxa, maximum-likelihood, maximum-parsimony, and Bayesian
analyses were used for both alignments. Maximum likelihood with 500
bootstraps using the Tamura-Nei model and maximum-parsimony anal-
ysis were conducted in MEGA v. 5.05 (33,34). Bayesian analysis was done
using MrBayes v. 3.1.2 software. All trees were constructed by the out-
group method and edited in MEGA v. 5.05.
Antifungal susceptibility. Antifungal susceptibilities for eight anti-
fungal drugs were determined in triplicate by using the colorimetric Sen-
sititre YeastOne method (Trek Diagnostic Systems, East Grinstead,
United Kingdom) as described previously (36). In short, the isolate was
cultured for 10 days in RPMI 1640 medium supplemented with L-glu-
tamine (0.3 g/liter) and 20 mM morpholinepropanesulfonic acid at 37°C.
Mycelia were harvested by 5 min of centrifugation at 2,158 ⫻gand
washed once with sterile saline. After sonication (20 s at 28-␮m maximum
power; Soniprep, Beun de Ronde, Netherlands) of the hyphal suspension,
Tween 60 was added at 0.05% (vol/vol), and the transmissions were ad-
justed to 70% at 660 nm (Novaspec II; Pharmacia Biotech). The inocu-
lated plates were incubated at 37°C for 7 days. The drug concentrations
used ranged from 0.016 ␮g/ml to 8 ␮g/ml for amphotericin B, itracona-
zole, ketoconazole, and voriconazole; from 0.25 ␮g/ml to 128 ␮g/ml for
fluconazole; and from 0.125 ␮g/ml to 64 ␮g/ml for 5-flucytosine.
Nucleotide sequence accession numbers. The GenBank accession
numbers for isolate CBS 131321 amplified genes are as follows: 28S rRNA,
JX073274; 18S rRNA, JX073269; ␤-tubulin, JX073271; actin, JX073275;
and elongation factor, JX097097.
MycoBank accession number. The MycoBank accession number for
P. ochracea is MB800514.
RESULTS
Phylogeny. A BLAST search with the ITS, 18S SSU, and 28S LSU
sequences did not yield identity with any known fungus. There-
fore, the sequences were used to determine the higher-order phy-
logeny for our isolate. The alignment of LSU and SSU consisted of
1,909 characters, of which 1,520 were constant, 62 were parsi-
mony uninformative, and 327 were parsimony informative. In-
trons were found in SSUs of three strains from the data set, and
they were deleted from the alignment. Maximum-parsimony
analysis of the combined data set resulted in 8 most parsimonious
trees (length of the tree ⫽1,109; consistency index [CI] ⫽
0.500451; retention index [RI] ⫽0.759444; rescaled consistency
index [RCI] ⫽0.380065). The SSU and LSU phylogenetic analyses
showed that the reported pathogenic isolate was closely related to
the family Pleurostomataceae, with high bootstrap support with all
algorithms used (1.00, 100, and 100) (Fig. 2) in the order
Calosphaeriales.
The ITS alignment consisted of 600 characters, of which 342 were
constant, 242 were parsimony informative, and 16 were parsimony
uninformative. Maximum-parsimony analysis resulted in 217 most
parsimonious trees (length ⫽356; CI ⫽0.688202; RI ⫽0.904475;
RCI ⫽0.622462). ITS analysis confirmed that the species belonged to
the Pleurostomataceae, in which it formed a well-supported clade with
the genus Pleurostoma (0.99, 93, and 94) (Fig. 3). Moreover, analysis
of the actin, ␤-tubulin, and elongation factor of the newly isolated
strain and a representative isolate from each species currently attrib-
uted to the Pleurostomataceae confirmed that the isolate was appro-
priately placed in this family (data not shown) as a member of the
anamorph genus Pleurostomophora.
Physiology. The minimum growth temperature for the isolate
was 15°C, and the maximum was above 40°C, with an optimum at
36 to 37°C (Fig. 4). Measurements were also taken after 8 days of
TABLE 2 Calosphaeriales strains
Strain no. Name ITS Source
STE-U 6181 Calosphaeria africana EU367445 Plant
CBS 120870 Calosphaeria africana EU367444 Plant
CBS 115999 Calosphaeria pulchella EU367451 Plant
SM05 Calosphaeria pulchella HM237300 Plant
LM06 Calosphaeria pulchella HM237298 Plant
DC04 Calosphaeria pulchella HM237299 Plant
SS07 Calosphaeria pulchella HM237297 Plant
CBS 127681 Jattaea discreta HQ878587 Plant
CBS 119343 Jattaea leucospermi EU552127 Plant
STE-U 6401 Jattaea mookgoponga EU367450 Plant
CBS 120867 Jattaea mookgoponga EU367449 Plant
CBS 120871 Jattaea prunicola EU367446 Plant
STE-U 6400 Jattaea prunicola EU367448 Plant
STE-U 6399 Jattaea prunicola EU367447 Plant
CBS 294.39 Pleurostomophora repens AF083195 Plant
CBS 131321 Pleurostomophora ochracea (JX073270) Human
CBS 115329 Pleurostoma ootheca HQ878590 Plant
IFM5 54325 Pleurostomophora richardsiae AB364704 Human
CBS 302.62 Pleurostomophora richardsiae AB364698 Plant
CBS 506.90 Pleurostomophora richardsiae AB364702 Human
CBS 295.39 Pleurostomophora richardsiae AB364697 Plant
IFM 4926 Pleurostomophora richardsiae AB364694 Human
PC1 Pleurostomophora richardsiae AB364693 Human
CBS 483.80 Pleurostomophora richardsiae AB364701 Human
IFM 41579 Pleurostomophora richardsiae AB364695 Human
CBS 270.33 Pleurostomophora richardsiae AY729811 Human
CBS 406.93 Pleurostomophora richardsiae AB364703 Plant
CBS 631.94 Phaeoacremonium aleophilum AF266647 Plant
CBS 222.95 Phaeoacremonium inflatipes AF266655 Plant
STE-U 5963 Togninia minima EU128019 Plant
CBS 113648 Togniniella acerosa EU367453 Plant
CBS 113726 Togniniella acerosa EU367452 Plant
Yellow-Grain Fungal Mycetoma
September 2012 Volume 50 Number 9 jcm.asm.org 2989

incubation on PDA to compare the growth pattern of the isolate
with those of other Pleurostomophora species. The colony diame-
ter reached only 1 to 2 mm when grown at 20°C. The isolate
decomposed casein in the first week. Starch was hydrolyzed; some
plates showed a narrow clear zone around the colony, and in oth-
ers, a zone extending up to 2.4 cm was observed. Gelatin hydroly-
sis was positive using both the tube and plate techniques. Urease
was strongly positive within 3 days. The isolate showed some sen-
sitivity to cycloheximide and was sensitive to salt, exhibiting some
inhibition with 0.5%, poor or no growth with 5%, and no growth
with 10% and 30% NaCl.
Antifungal susceptibility. The MICs of the strain tested are
shown in Table 3. Our strain was highly susceptible to the azoles
itraconazole, ketoconazole, posaconazole, and voriconazole. Low
MICs were also found for amphotericin B. The only azole for
which a high MIC was found (128 ␮g/ml) was fluconazole. The
fungus was inhibited by caspofungin at 8 ␮g/ml, but not by 5-flu-
cytosine (MIC ⬎64 ␮g/ml).
Taxonomy. Pleurostomophora ochracea Mhmoud, Abdalla
Ahmed, Fahal, de Hoog, van de Sande, sp. nov. MycoBank acces-
sion number MB800514. Etymology: named after its formation of
yellow grains in human tissue.
After 1 week of growth on all media tested (PDA, MEA, OA, and
CMA), the colony surface and reverse were typically creamy white to
pale yellow and felty, initially with some mycelial tufts. On OA and
CMA, colonies turned dark olivaceous brown to nearly black after 1
month of incubation. No diffusible pigment was produced in any
medium (Fig. 5). Vegetative hyphae were branched, septate, and hy-
aline to pale brown, turning dark brown after longer incubation. Hy-
phae were 1.2 to 3.2 ␮m wide. Hyphal walls were smooth or verrucu-
lose. Conidiophores were absent. Conidiogenous cells were variable
and poorly differentiated, and phialidic. Three distinct types of phi-
FIG 2 Phylogram of two loci (SSU and LSU) obtained by Bayesian analysis, maximum likelihood, and maximum parsimony (values of ⬎0.8 for Bayesian
probability and ⬎80% for maximum likelihood and maximum parsimony are shown with thick branches). Leotia lubrica and Crinula caliciiformis were
used as the outgroup. The portion of the phylogram relating to Calosphaeriales is shaded. The names of families within the order and the new isolate are
shown in bold.
Mhmoud et al.
2990 jcm.asm.org Journal of Clinical Microbiology

alides were observed. Type I phialides were short adelophialides with-
out basal septa, occasionally wider at the base, and were (0.4) 1.0 to
5.0 (6.5) ␮m (where the parenthetical values are the minimum and
maximum sizes, respectively) by (1) 1.0 to 2.0 (3) ␮m. Type II phi-
FIG 4 Colony diameters after 1 month of incubation at various temperatures
ranging from 15 to 36°C at 3°C intervals, including 10°C, 37°C, and 40°C.
FIG 3 Phylogenetic tree resulting from Bayesian analysis, maximum likelihood, and maximum parsimony for the ITS gene (values of ⬎0.8 for Bayesian
probability and ⬎80% for maximum likelihood and maximum parsimony are shown with thick branches). Togninia minima,Phaeoacremonium inflatipes, and
Phaeoacremonium aleophilum were used as the outgroup. Light-gray circles, strains isolated from plants; dark-gray circles, human-pathogenic strains.
TABLE 3 Antifungal susceptibility of P. ochracea
Antifungal agent MIC (␮g/ml)
Amphotericin B 1
Itraconazole 0.25
Ketoconazole 0.25
Fluconazole 128
Voriconazole 0.5
Posaconazole 1
5-Flucytosin ⬎64
Caspofungin 8
Yellow-Grain Fungal Mycetoma
September 2012 Volume 50 Number 9 jcm.asm.org 2991

alides were elongate-ampulliform, swollen at the base and tapered
toward the apex, (6.2) 7 to 8.6 (8.7) by (1.3) 1.0 to 2.0 (2.5) ␮m. Type
III phialides were (10.7) 11.6 to 17.9 (20.0) by (1.3) 1.7 to 2.4 (2.6)
␮m, (sub)cylindrical to elongate-ampulliform (Fig. 5). Conidia were
smooth-walled and aggregate at the tip of the phialides; most conidia
were hyaline, and some were brown. Two types were observed: either
small and subspherical to ellipsoidal, (2.0) 2.2 to 3.0 (3.5) by (1.3) 1.6
to 2.0 (2.3) ␮m, or larger and allantoid, (3.5) 4 to 6 (6.2) by (1.2) 1.7
to 2.5 (2.6) ␮m(Fig. 5). Teleomorph unknown. Holotype: dried cul-
ture in CBS Herbarium H-20972; ex-type strain CBS 131321, isolated
by N. A. Mhmoud and A. H. Fahal from human yellow-grain myce-
toma, Khartoum, Sudan.
DISCUSSION
Mycetoma is a chronic progressive disease characterized by sup-
purative, swollen lesions and sinuses and can be caused by both
bacteria and fungi (2,15). Based on the color of the grain, fungal
mycetomata can be divided into two large groups: those causing
black or white grains. Black-grain mycetoma is mainly associated
with M. mycetomatis,Madurella grisea,Leptosphaeria senegalensis,
FIG. 5. P. ochracea growth and morphology. Shown are colonies after 2 weeks of incubation at 37°C. (A) PDA. (B) MEA. (C) OA. (D, E, and F) Different-shape
conidia. (G, H, and I) Type I phialides. (J, K, and L) Type II phialides. (M, N, and O) Type III phialides. All scale bars, 10 ␮m.
Mhmoud et al.
2992 jcm.asm.org Journal of Clinical Microbiology

Pyrenochaeta romeroi, and Exophiala jeanselmei; white-grain my-
cetoma is usually caused by Pseudallescheria boydii,Acremonium
kiliense, and other, occasional agents (5)(Table 1). Yellow-grain
eumycetoma, to our knowledge, has never been reported. In areas
of high endemicity, like the Sudan, 70% of all mycetoma cases are
eumycetoma, caused by M. mycetomatis, a species producing
black grains (5,15). The remaining 30% of mycetoma cases are of
bacterial origin, and many of these are characterized by the pres-
ence of yellow grains. With the limited availability of diagnostic
techniques in routine laboratories in areas of high endemicity, the
color of grains has decisive diagnostic value (15). In this commu-
nication, we report on the first authenticated case of human eu-
mycetoma infection caused by a yellow-grain-producing fungus.
We identified the causative agent by multigene phylogenetic
analysis. The novel pathogenic isolate clustered in the ascomycete
order Calosphaeriales. The small order Calosphaeriales comprises
only four genera separated into two families: the family Calospha-
eriaceae contains three genera, viz.,Calosphaeria,Jattaea, and Tog-
niniella, while our fungus clustered in the Pleurostomataceae, con-
taining only a single teleomorph genus, Pleurostoma, and a single
anamorph genus, Pleurostomophora (30). Our fungus was para-
phyletic to Pleurostoma ootheca and Pleurostomophora repens (Fig.
3). Because of the absence of sexual sporulation, we prefer to clas-
sify the new species in the anamorph genus Pleurostomophora.
The majority of causative agents of mycetoma belong to the
orders Sordariales (particularly the genus Madurella) and Pleospo-
rales (particularly coelomycetes) (Table 1). The order Calospha-
eriales is a new addition to the list of orders harboring potential
agents. The order was described by Barr in 1983 (6,7); recent
taxonomic revision showed that the Togniniaceae telemorph of
Phaeoacremonium had some affinity to Diaporthales (27), but in
our study, the family was found as a separate cluster (Fig. 2), which
is in agreement with the results of Réblová (29). Based on SSU and
LSU data, and unlike previous publications (21,29), the Pleuros-
tomataceae showed affinity to the Diaporthales, whereas the
Calosphaeriaceae formed a separate, well-supported clade (Fig. 2).
The order Calosphaeriales, comprising only 10 species, partly at
large mutual phylogenetic distances, is still poorly defined. Sup-
posedly related fungi, such as Phaeoacremonium, were found to be
unalignable for the species-level genes used in this study, i.e., with
degrees of variability equal to or larger than that of the ITS.
Most members of the cluster Pleurostoma-Pleurostomophora
are wood-inhabiting fungi (8,37). Two cases of phaeohyphomy-
cosis caused by P. repens have been reported (20,26). However,
reexamination of both pathogenic strains revealed misidentifica-
tions, with the correct name being Phaeoacremonium krajdenii
(26). Pleurostomophora richardsiae is the only confirmed human-
pathogenic species in the order Calosphaeriales. It is known as an
environmental fungus but has been reported as the causative
agent of subcutaneous granuloma, phaeohyphomycosis, a chro-
moblastomycosis-like infection, and a bone infection (19,22,24,
32,35).
Pleurostomophora ochracea and P. richardsiae both produce
different types of phialides and conidia, but in P. richardsiae it is
noticeably more pronounced (37). In P. ochracea, all three types of
phialides are able to produce differently shaped conidia. All spe-
cies of Pleurostomophora and the teleomorph genus Pleurostoma
grow at temperatures ranging between 10 and 40°C, but P. ochra-
cea has a relatively high optimum growth temperature (36°C ver-
sus 30°C) and a low growth rate. This is expressed after 8 days at
20°C, where colony diameters of P. ochracea are only 1 to 2 mm,
while P. richardsiae reaches 18 to 19 mm, P. ootheca 30 to 32 mm,
and P. repens 59 to 61 mm (37).
The clinical isolate of P. ochracea in this study appeared to be
highly susceptible, in vitro, to ketoconazole and itraconazole,
compounds that are commonly used in the treatment of subcuta-
neous infections, including mycetoma by pigmented fungi (16,
21,25,31). Of all azole antifungals tested, fluconazole was the least
effective. Once it was established that the causative agent was a
fungus, our patient was advised to take ketoconazole at 400 mg/
day, according to the guidelines of the Mycetoma Research Centre
in Khartoum, Sudan. However, the patient refused treatment and
did not return to the center, and follow-up was impossible.
As noted in most cases of mycetoma, it is difficult to explain
how our patient acquired his infection. He did not report any type
of injury. Since the patient is a farmer by occupation, he is in daily
contact with soil, thorns, wood, and other trauma-causing ob-
jects; even minor trauma might have facilitated the introduction
of the fungus into his foot. The present case report shows that
proper species identification in the diagnosis of mycetoma re-
mains troublesome and that identification based exclusively on
the color of the grains may be erratic (11,13). Molecular identifi-
cation is recommended to ascertain appropriate therapy.
ACKNOWLEDGMENT
Wendy W. J. van de Sande is supported by a postdoctoral fellowship from
the Netherlands Organization for Scientific Research (NWO, VENI-
Grant 916.11.178).
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