Journal of Clinical Microbiology, December 1999, p. 4170-4173, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Mycotic Keratitis Due to Curvularia
senegalensis and In Vitro Antifungal Susceptibilities of
Curvularia spp.
Josep
Guarro,1,*
Tiyomi
Akiti,2
Roberto
Almada-
Horta,3
L. A.
Morizot
Leite-Filho,4
Josepa
Gené,1
Sueli
Ferreira-Gomes,3
Carme
Aguilar,1 and
Montserrat
Ortoneda1
Unitat de Microbiologia, Facultat de Medicina
i Ciències de la Salut, Universitat Rovira i Virgili, 43201 Reus,
Spain,1 and Laboratorio de Micologia,
Hospital Universitário Clementino Fraga Filho, Universidade
Federal do Rio de Janeiro,2 Laboratorio
Almada Horta,3 and Instituto Benjamin
Constant,4 Rio de Janeiro, Brazil
Received 7 June 1999/Returned for modification 10 July
1999/Accepted 18 August 1999
 |
ABSTRACT |
A case of mycotic keratitis due to Curvularia
senegalensis is reported. This case represents the third known
reported infection caused by this rare species. Fungal hyphae were
detected in corneal scrapings, and repeated cultures were positive for
this fungi. The patient was presumed cured after a corneal transplant
and treatment with itraconazole, but the infection recurred and the patient is waiting for a keratoplasty. The in vitro antifungal susceptibilities of the case strain and another 24 strains belonging to
seven species of Curvularia were tested for six antifungal agents. With the exception of flucytosine, and occasionally
fluconazole, the other drugs assayed (amphotericin B, miconazole,
itraconazole, and ketoconazole) were highly effective in vitro.
| |
TEXT |
Keratitis caused by filamentous
fungi, such as Fusarium, Aspergillus,
Acremonium, and Curvularia species, is relatively
frequent (11, 21) and is usually induced by trauma. We
report a case of human keratitis due to Curvularia
senegalensis without known injury or trauma. C. senegalensis causing fungal keratitis has only been reported once
before, approximately 20 years ago (10).
Case report.
A 38-year-old Brazilian housewife presented at
the Institute Benjamin Constant, Rio de Janeiro, Brazil, on 14 July
1997. She complained of a painful, inflammation in her right eye, which was similar to conjunctivitis. The problem appeared spontaneously. One
week earlier, she had noticed a white spot on the cornea and visited an
ophthalmologist, who treated her with a combination of antibiotics and
corticosteroid eye drops and sent her to the institute. She had
previously suffered from pulmonary tuberculosis, and the treatment had
ended 4 months before, but she had no history of preceding ocular
trauma or previous eye disease. Examination revealed a visual acuity of
20/50 (acuity tested by patient counting fingers held up by examination
1 m away) and the presence of a corneal ulcer with diffuse
infiltration through the corneal parenchyma. On 22 July 1997, she was
examined in the Cornea Department and found to have a corneal abscess
of approximately 8 mm in diameter, and treatment was initiated with 1%
atropine eye drops twice daily. Deep corneal epithelium scrapings were
obtained with a Kimura's spatula for Giemsa and Gram stains and culture.
Microscopic examination of corneal scrapings was positive for fungal
elements (Fig. 1). Scrapings stained with
Giemsa revealed abundant septate, darkly pigmented hyphae. On 12 August
1997, a new examination revealed a worsened eye condition, and
pimaricin (5%) every 4 h was added to the treatment regiment. A
corneal transplant was performed on 25 August 1997, and preoperatory
complementary tests showed that the patient had diabetes mellitus
(glycemia, 360 mg/dl). On 14 August 1997, inflammation recurred in
the anterior chamber with pupillary block and hypopyon and
hyperemic conjunctiva. Treatment with itraconazole (100 mg twice daily)
and systemic corticoids was initiated. By 11 November 1997, the patient
had improved substantially, and there was no more inflammation. She was
discharged. On 9 December 1997, the patient returned, complaining of
visual acuity reduction and, under examination, a uveal tract reaction,
which was characterized by precipitates on the posterior surface of the
cornea, was observed. Pred-fort and atropine drops were prescribed. On
28 January 1998, she had intense conjunctival hyperemia (injection),
smarting, and cataract complication, still with uveal reaction. On 17 March 1998, she presented secondary glaucoma by pupil obstruction. She
did not come back until 26 June 1998, when she showed a fetid yellowish
secretion, a new corneal ulcer in the transplanted cornea, conjunctival
injection (or hyperemia), and eyelid edema. A recurrence of the fungus
was suspected, and ulcer scrapings were collected for new culture. The
patient is still awaiting a new keratoplasty.
Portions of the scrapings obtained on both occasions were inoculated on
plates of Sabouraud dextrose agar (with and without penicillin [20
U/ml], streptomycin [40 U/ml], and cycloheximide [0.5 mg/ml]),
blood agar, and brain heart infusion agar. On all plates, an apparently
identical grayish black mold developed with numerous thick-walled,
septate conidia, borne on simple, club-shaped conidiophores. Both
conidia and mycelium were dematiaceous. The fungal isolates were
referred to the Microbiology Unit of the Rovira i Virgili University in
Reus, Spain, for identification and an antifungal susceptibility study.
Morphological study.
The isolates were subcultured on potato
carrot agar (PCA) and oatmeal agar (OA), and incubated at ca. 25°C in
the dark. After 7 days on PCA, the colonies were dark brown and velvety
but loose cottony at the center and 60 to 62 mm in diameter. On OA, the macroscopic characteristics of the fungus were similar to those on PCA,
but it grew more rapidly, reaching 65 to 68 mm in diameter after 7 days. Sporulation was abundant on both media. Conidiophores usually
grew directly on the substrate; they were simple or branched, straight
or flexuous, smooth walled, up to 130 µm long and 4 to 6 µm wide
(Fig. 2A). Conidia had three to five
septa (mostly four septa) and were dark brown, with subhyaline or pale
brown cells at each end, ellipsoidal or broadly fusiform, slightly
curved, 22 to 31 µm long by 11 to 14 µm wide at the broadest part
(Fig. 2B). On the basis of these characteristics, the isolates were identified as C. senegalensis (Fig.
3). Curvularia geniculata, another species involved in some cases of keratomycosis (6), also has conidia, mostly with four septa, but they are markedly geniculate with the central cell usually very different from the rest
(dark brown and swollen). The other reported pathogenic species of
Curvularia, i.e., C. brachyspora, C. clavata, C. lunata, C. pallescens, and
C. verruculosa, usually have conidia with three septa, which
also differ from those of C. senegalensis mainly by their
color, shape, size, or ornamentation. The two clinical isolates of
C. senegalensis were kept in the Mycology Laboratory of the
Faculty of Medicine in Reus, Spain, as FMR 6319 (first isolate) and FMR
6666 (second isolate). A living culture of the former has been
deposited in the Centraalbureau voor Schimmelcultures, Baarn, The
Netherlands, with the accession no. CBS 102171.
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FIG. 2.
C. senegalensis FMR 6319. (A) Conidiophores
bearing conidia photographed with phase-contrast optics. Magnification,
×384. (B) Conidia photographed with Nomarski optics. Magnification,
×1,200.
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|
Antifungal susceptibility testing.
One of the case isolates
(FMR 6319) and four additional isolates of C. brachyspora,
three of C. clavata, four of C. geniculata, three
of C. lunata, four of C. pallescens, two of
C. senegalensis, and four of C. verruculosa from
very diverse sources were tested to determine their susceptibility to
antifungal drugs (Tables 1 and
2). Tests were accomplished by a
previously described microdilution method (16) performed,
where possible, according to the National Committee for Clinical
Laboratory Standards' guidelines for filamentous fungi (14)
by using RPMI 1640 medium buffered to pH 7 with 0.165 M
morpholinepropanesulfonic acid (MOPS), an inoculum of 4 × 102 to 4.5 × 104 CFU/ml, an incubation
temperature of 30 or 35°C, reading the results after 2 or 3 days (48 or 72 h), and an additive drug dilution procedure.
The MICs of the six antifungal agents against the clinical isolate (FMR
6319) were as follows: 0.25 µg/ml for amphotericin B, 1 µg/ml for
miconazole and ketoconazole, 0.25 µg/ml for itraconazole, 16 µg/ml
for fluconazole, and 256 µg/ml for 5-fluorocytosine. This is the
greatest number of species of Curvularia that has been
tested so far in a single study for in vitro antifungal susceptibility. Tables 1 and 2 show the antifungal susceptibility results of the 25 isolates. In contrast to the results of You et al. (22), amphotericin B, itraconazole, miconazole, and ketoconazole were highly
effective against almost all the species tested. Generally, C. pallescens displayed the highest MICs. These results are in agreement with those of Bent and Kuhn (2), who tested five strains of Curvularia spp. obtained from sinus aspirates
from allergic fungal sinusitis patients. All five strains were
sensitive to amphotericin B, ketoconazole, and nystatin; two strains
were sensitive to itraconazole, and all were resistant to fluconazole. In addition, Sutton et al. (20) reported C. lunata as sensitive to practically all the antifungals available,
with the exception of flucytosine. However, despite the fact that in
vitro resistance to a particular agent does provide valuable
information in selecting a proper antifungal agent for treatment, these
results should be interpreted with caution because in vitro studies are
only limited approximations of the in vivo situations. Studies of
correlation of in vitro data with clinical outcome are needed for a
more definitive evaluation of the predictive value of MICs for
filamentous fungi.
The genus Curvularia comprises about 30 species. It was
widely studied and monographed by Ellis (8, 9) and Sivanesan (19). Its telomorphs are included in the ascomycete genus
Cochliobolus. Most of the species are pathogens of grains
and plants common to tropical areas. They are also commonly found in
agricultural soils (7). Curvularia spp. are
darkly pigmented fungi with conidia efficiently adapted to aerial
dispersal (7). They are, therefore, habitual components of
air mycobiota and have a worldwide distribution.
Curvularia species had been previously considered
nonpathogens or thought to affect humans only rarely, but these fungi
are now increasingly being reported to cause human disease (3, 22). Seven species of Curvularia have been involved in
human infections. They are morphologically very similar with the
differences mainly in the conidial features (size, number of septa,
shape, and ornamentation) (6). In human pathology,
Curvularia spp. are frequently associated with allergic
sinusitis (12), although several cases of ocular infections
such as keratitis (1, 15) or endophthalmitis (17)
have also been reported. Of the approximately 50 reported cases of
Curvularia infection, approximately a third were keratitis.
C. lunata is the species most commonly found in clinical
specimens. Curvularia infections are usually acquired by
either direct inoculation or inhalation (22). However, in the case reported here, there was no evidence of trauma to explain how
or when the fungus entered the cornea. The only predisposing factor was
that the patient was diabetic and had initially used eye drops
containing steroids. Marcus et al. (13) also reported a case
of Curvularia keratitis without trauma. Fungal keratitis without evidence of trauma has rarely been reported (5).
C. senegalensis was reported more than 20 years ago as the
cause of two cases of keratitis (10). These patients were
treated topically with 5% pimaricin suspensions, but little was
reported about the evolution of the lesions. This species has also been involved in two cases of allergic bronchopulmonary disease
(22). The management of Curvularia infections
usually involves surgical treatment with or without the use of
antifungal agents. Some patients have been successfully treated with
amphotericin B (22). Despite their in vitro efficacy,
therapy with azole agents, such as miconazole and ketoconazole, has
been disappointing due to the frequent recurrence of infection
(22). Itraconazole (18) and terbinafine
(4) have also been used successfully. For localized
infections such as sinusitis or keratitis, surgical treatment alone may
be adequate (22). Our patient appeared to recover
successfully after a cornea transplant and treatment with itraconazole
for 3 months, which was also effective in vitro. However, a recurrence
of the infection was probably due to remaining small hyphal elements
which were not completely eliminated within 3 months of treatment with
itraconazole at a dose of 200 mg/day.
| |
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,
Tarragona, Spain. Phone: 34 977759359. Fax: 34 977759322. E-mail:
umb{at}fmcs.urv.es.
| |
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Journal of Clinical Microbiology, December 1999, p. 4170-4173, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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