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Journal of Clinical Microbiology, June 2000, p. 2434-2437, Vol. 38, No. 6
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Phaeoisaria clematidis as a Cause
of Keratomycosis
Josep
Guarro,1,2,*
Luiz A.
Vieira,3
Denise
De
Freitas,3
Josepa
Gené,1,2
Luis
Zaror,4
Ana Luisa
Hofling-Lima,3
Olga
Fischman,5
Cecilia
Zorat-Yu,3 and
M.
José
Figueras1
Unitat de Microbiologia, Facultat de Medicina
i Ciències de la Salut, Universitat Rovira i Virgili,
43201-Reus,1 and Institut d'Estudis
Avançats, Universitat Rovira i Virgili,
Tarragona,2 Spain; Disciplina de
Oftalmología3 and Disciplina
de Biología Celular,5 UNIFESP/EPM,
São Paulo, Brazil; and Instituto de
Microbiología Clínica, Universidad Austral de Chile,
Valdivia, Chile4
Received 28 December 1999/Returned for modification 15 February
2000/Accepted 14 March 2000
 |
ABSTRACT |
We report the first case of human infection by Phaeoisaria
clematidis. This fungus caused a corneal ulcer in a Brazilian man who had previously suffered an eye injury. Diagnosis was established by
positive direct examination and repeated cultures. The isolate was
clearly resistant in vitro to the six antifungal agents tested.
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TEXT |
Mycotic keratitis, a medical
curiosity about 2 or 3 decades ago, is now a common manifestation, and
its increasing frequency is of considerable concern in present-day
medicine. Numerous fungi which were considered nonpathogenic to human
beings have been isolated frequently as causal organisms from cases of
keratitis. The genera that most frequently cause keratitis are
Fusarium, Aspergillus, and Candida;
Fenelon and Kennedy (4) listed 20 other genera that also
have been involved in this type of infection. However, in recent years,
a number of other genera have been reported, such as Phoma,
Dichotomophthoropsis, Rhizoctonia,
Cephaliophora, Lasiodiplodia,
Colletotrichum, Cladorrhinum, and
Metarhizium (1). Here we report the first case of
human infection caused by Phaeoisaria clematidis. To our
knowledge, this is the first report of keratitis or of any other
infection caused by this organism in animals.
Case report.
A 47-year-old man was seen in the Ophthalmology
Department of the Escuela Paulista de Medicina, São Paulo,
Brazil. He complained of severe pain, redness, and the appearance of a
white spot in the left eye. Approximately 2 months earlier he had
suffered an injury with a broom. He was given local treatment in the
form of gentamicin and dexamethasone, which initially produced a slight improvement. However, 1 month later, the above symptoms began. Clinical
examination revealed visual acuities of 1 in the right eye and 0.6 in
the left eye. The left eye showed conjunctival hyperemia, epithelial
erosion, and a deep central corneal ulcer, measuring 1 by 1.5 mm, with
irregular borders and a stromatic infiltrate. The right eye did not
show any abnormality. Results of other local and systemic examinations
were negative.
Deep corneal scrapings were collected with a sterile scalpel blade for
direct mounts and cultures. Direct examination of lactophenol wet
mounts of the scrapings revealed abundant septate and pigmented hyphal
fragments. Corneal samples were directly inoculated onto Sabouraud
glucose agar (Difco Laboratories, Detroit, Mich.) and potato dextrose
agar (PDA; Difco) by making a series of C-shaped cuts on the medium.
Cultures were incubated at 25, 30, and 35°C. After 3 days, numerous
small colonies of a dematiaceous mold appeared on all cultures. Results
of routine bacteriological cultures were negative.
Treatment was started with hourly topical 5% natamycin, ofloxacin
every 6 h for 4 days, 1% atropine every 8 h for 8 days,
and
oral ketoconazole at 400 mg/day. After 3 weeks, the condition
of the
eye worsened, with an increase in the corneal edema and
hypopyon.
Antifungal therapy was discontinued, and more corneal
scrapings were
collected for new cultures. The patient was treated
for presumptive
bacterial infection with local antibiotics (cephalothin
at 50 mg/ml and
gentamicin at 14 mg/ml hourly for 9 days). The
cultures were again
negative for bacteria and positive for the
same mold. Antibacterial
treatment was therefore discontinued.
A contact lens was prescribed,
and treatment was started with
antifungal drugs (0.5% amphotericin B
topically and ketoconazole
at 400 mg/day orally) supplemented with
ofloxacin every 6 h. Amphotericin
B was used for 2 weeks. The
lesion improved slightly, and topical
dexamethasone (0.05%) every
8 h for 8 days was added. The ulcer
healed, a leukomatous opacity
formed, and the corticoid was discontinued.
At present, the patient is
awaiting therapeutic
keratoplasty.
Cultures obtained on the two occasions yielded molds with identical
morphological characteristics. One isolate was sent to
the Microbiology
Unit of the Rovira i Virgili University, Reus,
Tarragona, Spain, for
identification and antifungal susceptibility
testing.
Morphological study.
For identification purposes, this
clinical isolate was subcultured on PDA, potato carrot agar (PCA;
20 g of potato, 20 g of carrot, 18 g of agar, 1,000 ml
of tap water [homemade]), and oatmeal agar (OA; 30 g of oat
flakes, 1 g of MgSO4 · 7H2O,
1.5 g of H2KPO4, 15 g of agar, 1,000 ml of tap water [homemade]) and incubated at ca. 25°C in the dark.
After 10 days, the colonies on PDA were velvety, umbonate, mouse gray
to brown, flat, and whitish at the edge, with a dark olivaceous
reverse, and attained 9 to 11 mm in diameter. On PCA and OA, the
colonies were very similar and developed more rapidly than on PDA,
attaining a diameter of 12 to 14 mm after 10 days (Fig.
1). They were flat, brownish gray, and
cottony at first, with thin fascicles at the center, and then became
brown and powdery, with a dark brown reverse. The fungus sporulated
profusely, and numerous synnemata (bundles of conidiophores sporulating
in the apical part) (Fig. 2) appeared
after 15 to 20 days of incubation on all the media.
The microscopic characteristics of the isolate were determined by
making wet mounts with acid lactic, which were then examined
under a
light microscope (Leitz Dialux 20). Synnemata were erect,
cylindrical,
or slightly clavate, were up to 1,500 µm long and
25 to 80 µm wide
near the base, and had a dark brown stipe composed
of parallel,
smooth-walled, septate, branched hyphae 2 to 3 µm
in width and
conidiogenous cells that usually covered the upper
two-thirds to
one-third of the stipe. Conidiogenous cells (Fig.
3) that also arose from undifferentiated
hyphae were denticulate,
smooth walled, pale brown, subhyaline toward
the apex, cylindrical
or clavate, and 10 to 20 µm long by 2 to 3 µm
wide when young
but with age often considerably longer and geniculate.
Denticles
were terminal and lateral, scattered, or grouped, more or
less
cylindrical, and up to 1 µm long. Conidia were aseptate, smooth
walled, subhyaline, ellipsoidal or narrowly clavate, often slightly
curved, and 5 to 10 µm long by 2 to 3 µm wide. Chlamydospores
(Fig.
4) were abundantly produced from
submerged and superficial
mycelium. They were usually lateral, sessile,
thick and smooth
walled, dark brown, subglobose, ovoid or ellipsoidal,
11 to 14
µm long by 8 to 10 µm wide, aseptate (rarely with one or
two septa),
and up to 17 µm long. Moreover, after approximately 2 weeks, fruiting
body initials (up to 60 µm in diameter) of a probable
ascomycete
were detected on all the media, but they did not ripen under
different
culture conditions.
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FIG. 3.
P. clematidis denticulate conidiogenous cells
and conidia viewed by scanning electron microscopy. Magnification,
×11,000.
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FIG. 4.
P. clematidis terminal and lateral
chlamydospores, conidiogenous cells, and conidia viewed by
phase-contrast microscopy. Magnification, ×640.
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On the basis of the above characteristics, the isolate was tentatively
identified as
P. clematidis, although curved conidia
had
never before been described for this species, nor had its
teleomorph.
The only
Phaeoisaria species with curved or at least
in part
slightly curved conidia are
P. curvata (
2),
P. triseptata (
6), and
P. sparsa var.
cubensis (
7). However, the latter
two species
differ from our strain because they have septate conidia,
and the
conidia of the former are larger, are always curved, and
never produce
synnemata. In addition, the only known species with
a teleomorph is the
Phaeoisaria state of
Peroneutypella echidna (Ascomycota, Diatripales) (
3), but its conidia are smaller
(5 to 5.5 µm
long).
To achieve ripe fruiting body initials and verify the stability of the
conidial morphology, we inoculated a conidial suspension
of the isolate
onto sterilized plant material by the technique
described by Gené
and Guarro (
5). Under these conditions,
the fruiting body
initials did not ripen after 2 months of incubation;
curved conidia
were practically absent, and the other microscopic
characteristics were
very similar to those described above. This
clinical strain was
therefore identified as
P. clematidis. Living
cultures of
the strain have been kept in the culture collection
of the Faculty of
Medicine, Rovira i Virgili University, Reus,
Spain, as FMR 6274 and
have also been deposited in the Commonwealth
Agricultural Bureaux
International Bioscience, Egham, United Kingdom
(IMI 381458), and in
the Centraalbureau voor Schimmelcultures,
Baarn, The Netherlands (CBS
102276).
Antifungal susceptibility testing.
The fungal isolate was
tested to determine its susceptibility to amphotericin B, miconazole,
itraconazole, ketoconazole, fluconazole, and flucytosine (Table
1). Tests were carried out by a
previously described microdilution method (10) mainly
according to the guidelines recommended for molds by the National
Committee for Clinical Laboratory Standards (8); we used
RPMI 1640 medium buffered to pH 7.0 with 0.165 morpholinepropanesulfonic acid (MOPS), an inoculum of 4.7 × 104 CFU/ml, an incubation temperature of 30°C, a
second-day reading (48 h), and an additive drug dilution procedure.
MICs and minimum fungicidal concentrations were very high,
demonstrating the inefficacy of all the drugs tested.
Species of
Phaeoisaria are easily recognized by the erect
synnemata bearing numerous denticulate conidiogenous cells in the
upper
region (
2). However, when they are grown in cultures,
these
typical structures are sometimes not produced.
P. clematidis is a soilborne fungus, ubiquitous, cosmopolitan, and a well-known
plant
pathogen. It has been isolated on dead plant material and
is very
common in the tropics (
3). Using a murine model, Okeke
et
al. (
9) demonstrated that this species was able to survive
passage in mice and proliferate in tissue, although infections
of the
cornea were not studied. The fungus produced mainly localized,
subcutaneous nodular lesions and visceral effects, depending on
the
means of inoculation. The tissue form comprised short septate
hyphae
and brown, thick-walled chlamydoconidium-like
cells.
In the present case, the fungus may have been introduced at the time of
injury. It is more likely, however, that it was introduced
later to the
wound by airborne spores and that topical corticoids
and antibiotics
facilitated its penetration. In the treatment
of the fungal infection,
both antifungal and antibacterial drugs
were used with corticoids, but
there is no clear evidence that
either of them had any real effect. On
the other hand, the antifungal
agents used last (amphotericin B and
ketoconazole), which seemed
to be the ones that produced the corneal
healing, were clearly
inefficient in vitro. It is unfortunate that the
patient received
a topical steroid prior to proper diagnosis and
treatment. Topical
steroids exacerbate infections and should never be
considered
in keratomycosis or bacterial keratitis unless there is
secure
knowledge that the correct antimicrobial agent is already being
given. Although some authors have used corticosteroids as adjunctive
therapy, these probably have no place in the treatment of fungal
keratitis and are more likely to result in deeper penetration
of the
infections (
4).
In conclusion,
P. clematidis is a saprophytic fungus which
has been demonstrated, both experimentally and in this study, to
be
pathogenic for animals, including humans. It should be added
to the
list of opportunistic fungi that can infect an immunocompetent
host.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unitat de
Microbiologia, Departament de Ciències Mèdiques
Bàsiques, Facultat de Medicina i Ciències de la Salut,
Universitat Rovira i Virgili, Carrer Sant Llorenç 21, 43201-Reus,
Spain. Phone: 34 977759359. Fax: 34 977759322. E-mail:
umb{at}fmcs.urv.es.
| |
REFERENCES |
| 1.
|
de Hoog, G. S., and J. Guarro (ed.).
1995.
Atlas of clinical fungi.
Centraalbureau voor Schimmelcultures, Baarn, The Netherlands.
|
| 2.
|
de Hoog, G. S., and M. C. Papendorf.
1976.
The genus Phaeoisaria.
Persoonia
8:407-414.
|
| 3.
|
Deighton, F. C.
1974.
Four synnematous hyphomycetes.
Trans. Br. Mycol. Soc.
62:243-252.
|
| 4.
|
Fenelon, L. E., and S. M. Kennedy.
1996.
Fungal diseases in ophthalmology, p. 221-223.
In
C. C. Kibbler, D. W. R. Mackenzie, and F. C. Odds (ed.), Principles and practice of clinical mycology. John Wiley & Sons Ltd., Chichester, England.
|
| 5.
|
Gené, J., and J. Guarro.
1996.
A new Chaetomium from Thailand.
Mycol. Res.
100:1005-1009.
|
| 6.
|
Holubová-Jechová, V.
1988.
Studies on Hyphomycetes from Cuba. VII. Seven new taxa of dematiaceous Hyphomycetes.
Ceska Mykol.
42:23-30.
|
| 7.
|
Mercado-Sierra, A.,
M. J. Figueras, and J. Gené.
1997.
New or rare hyphomycetes from Cuba. VIII. Species of Lylea, Phaeoisaria, Arxiella, Graphium, Periconia, and Ramichloridium.
Mycotaxon
63:369-375.
|
| 8.
|
National Committee for Clinical Laboratory Standards.
1998.
Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi; proposed standard M38-P.
National Committee for Clinical Laboratory Standards, Wayne, Pa.
|
| 9.
|
Okeke, C. N.,
H. C. Gugnani, and W. I. B. Onuigbo.
1991.
Potential pathogenicity of Cladosporium tenuissimum, Phaeoisaria clematidis and Ramichloridium subulatum in a mouse model.
Mycopathologia
114:65-70[Medline].
|
| 10.
|
Pujol, I.,
J. Guarro,
C. Llop,
L. Soler, and J. Fernández.
1996.
Comparison study of broth macrodilution and microdilution antifungal susceptibility tests for the filamentous fungi.
Antimicrob. Agents Chemother.
40:2106-2110[Abstract].
|
Journal of Clinical Microbiology, June 2000, p. 2434-2437, Vol. 38, No. 6
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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