Medical Mycology December 2004, 42, 543 /547

Case Report

Subcutaneous phaeohyphomycosis caused by Lasiodiplodia theobromae and successfully treated surgically

Richard C. Summerbell*, S. Krajden$, R. Levine$ & M. Fuksa$ *Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands and $St. Josephs Health Centre, Toronto, Canada

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While visiting Jamaica, a 50-year-old woman stumbled on an outdoor wooden staircase and sustained an injury to the right leg. The wound was cleaned topically and the patient was given antibacterial therapy. Five weeks later, in Canada, she presented with an ulcer at the injury site. An excisional biopsy showed copious broad, septate, melanized fungal filaments penetrating into tissue. Culture yielded a nonsporulating melanized mycelium. The isolate was strongly inhibited by cycloheximide and benomyl but grew at 378C. After 16 weeks cultivation on modified Leonians agar at 258C, it developed pycnidia characteristic of Lasiodiplodia theobromae , a common tropical phytopathogen mainly known previously as a rare agent of keratitis and onychomycosis in humans. The patient was not given antifungal chemotherapy, and the ulcer, which had been broadly excised in the biopsy procedure, ultimately resolved after treatment with saline compresses. The six-month follow-up showed no sign of infection. This case, interpreted in light of previously reported cases, shows that on rare occasions L. theobromae is able to act as an agent of subcutaneous phaeohyphomycosis and that, when this occurs, debridement alone may be sufficient to eradicate it. Keywords Botryodiplodia , Botryosphaeria rhodina , Lasiodiplodia , phaeohyphomycosis, tropical medicine

Introduction
Phaeohyphomycosis is a well recognized category of fungal diseases caused by species producing dark walled, melanized filaments in human tissue. Lasiodiplodia ( 0/Botryodiplodia ) theobromae (teleomorph Botryosphaeria rhodina ) is a species of mainly tropical and subtropical origin often isolated as a wound pathogen of woody plants and as an agent of fruit rot and wood staining [1,2]. Additionally, it has been reported clinically from numerous cases

Received 28 April 2004; Accepted 9 July 2004 Correspondence: Richard C. Summerbell, Senior Researcher, Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. Tel: '/31 30 212 2688; Fax: '/31 30 251 2097; E-mail: summerbell@cbs.knaw.nl

of ocular keratitis and rarely from other ocular conditions such as endophthalmitis [3 /12]. It has also been substantiated as an agent of onychomycosis [13,14]. Reports of subcutaneous infection to date have been very rare. An isolation from human subcutaneous tissue is accessioned in the Commonwealth Agriculture Bureau International (CABI Biosciences, Egham, Surrey, UK) collection as IMI 172565 [15] but no details were published on this isolation. An apparently iatrogenic case involving an infection of a medical inoculation site on the buttock of a Cambodian who later moved to Australia was documented by Maslen et al . [16]. The present study documents L. theobromae as the agent of subcutaneous infection in a Canadian traveller to Jamaica, and discusses the purely
DOI: 10.1080/13693780400005916

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surgical treatment that was applied successfully in this case.

Case report
A 50-year-old woman with no known immune deficiencies and otherwise in excellent health sustained an injury to the anterior aspect of her right leg when she slipped on a banana peel and stumbled on an exterior wooden staircase while visiting Jamaica. The wound was cleaned topically and the patient was prescribed cloxacillin. Five weeks later, after her return to Canada, she presented with an ulcer at the injury site. A widely excisional biopsy was taken, and copious broad, septate melanized fungal hyphae were demonstrated in histopathology (Fig. 1). Culture yielded an initially nonsporulating, dark pigmented mycelium ultimately identified as Lasiodiplodia theobromae . The isolate was deposited in the American Type Culture Collection (Manassas, VA, USA) as ATCC 76087 and in the CBS Fungal Biodiversity Centre (Centraalbureau voor

Schimmelcultures, Utrecht, The Netherlands) as CBS 339.90. Partly because of the comprehensive nature of the excision, antifungal chemotherapy was deferred pending identification and analysis of the significance of the culture. The patient was prescribed topical soaks with normal saline in sterile compresses 3 to 4 times per diem. The ulcer resolved without further intervention. No sign of infection was seen at a six-month follow-up examination.

Methods
The isolate was subcultured on to Sabouraud peptoneglucose agar with 100 mg/ml cycloheximide, 100 mg/ml chloramphenicol, and 50 mg/ml gentamicin, on to the fungal sporulation medium Leonians agar [19] as modified by R.F. Cain (fide D.W. Malloch [17]) to include 1.0 g L(1 yeast extract [17; full recipe online at http://www.botany.utoronto.ca/ResearchLabs/ MallochLab/Malloch/Moulds/Cultivation.html] and on to the same medium amended with 2, 4 and 10 mg/ml benomyl [18]. All media were incubated at 258C. in the dark. For induction of sporulation, subcultures on modified Leonians agar were incubated in the light at room temperature, 218C.

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Results
Isolates maintained on modified Leonians agar for 1 month at 258C and then 3 months at room temperature produced pycnidia of L. theobromae near the beginning of their fifth month of incubation. Subcultures on Sabouraud agar with cycloheximide showed strong inhibition. The fungus was also strongly inhibited by benomyl, with radial growth reduced 88.2% by 10 mg/ml benomyl as compared to benomylfree modified Leonians control.

Description of the etiologic agent

Colony fast growing, beginning pale, soon deep grey, deeply lanose (Fig. 2), producing stromatic aggregates of ostiolate, setose pycnidia, sometimes partially confluent, after over 17 weeks of incubation. Pycnidia lined internally with a dense layer of conidiophores and paraphyses (Fig. 3). Conidiophores hyaline, cylindrical or tapering, short, arising directly from the pseudoparenchymatous inner wall of the pycnidia. Conidiogenous cells holoblastic, cylindrical or with an ampulliform swelling near the apex (Fig. 3). Conidia at first hyaline, broadly ellipsoidal, smooth, and unicellular, later becoming medially uniseptate, brown,
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Fig. 1 Septate laments of Lasiodiplodia theobromae in cutaneous ulcer tissue. Silver stain. 100 )/ objective.

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Fig. 2 Rapidly growing melanized colony of Lasiodiplodia theobromae , Leonians agar, 7 days.

asperulate and prominently longitudinally striate, with striae remaining separate at the poles (Fig. 4), mostly 20 /30 )/10 /15 mm. Paraphyses hyaline, cylindrical or narrowly tapering, to 50 mm long.

Fig. 3 Fertile layer within Lasiodiplodia theobromae pycnidia showing conidiogenous cells, paraphyses, and immature conidia. 40 )/ objective.

Discussion
The great majority of case reports involving L. theobromae have been from ocular keratitis. The present case and that of Maslen et al . [16], taken together, indicate that throughout its distribution range, this pantropically distributed fungus presents a hazard in that it may cause subcutaneous ulcer subsequent to traumatic inoculation. In the present case, the resolution of the infection after surgical debridement and application of saline compresses may indicate a relatively low virulence. It is also possible that surgery alone constituted successful therapy only because this was an incipient lesion with inoculum still localized. However, the only other well documented case of subcutaneous infection by this organism, that of Maslen et al. [16], documented a relatively long-established infection for which treatment was also effected purely by drainage and debridement.
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These results suggest that surgery may be further investigated as a sole therapy in future cases of localized lesions caused by this organism, which produces no known type of disseminable element, such as yeast, conidia or fission cells, in host tissue. Assessing the likely significance of the fungus growing from the lesion was at first problematic because fruiting did not occur for over 4 months. The patients manifest improvement meant that molecular testing was difficult to justify in terms of cost-effectiveness. Fast growing, dematiaceous mycelia from phaeohyphomycotic lesions are frequently related to members of the fungal order Pleosporales, especially Cochliobolus, Lewia, Pyrenophora and Setosphaeria, usually seen as anamorphs in Bipolaris, Curvularia, Alternaria, Drechslera and Exserohilum. These anamorphs share the common feature that conidia, when produced, appear to be poroconidial: they are produced as blastoconidia from a ring of darkened cell wall material suggestive of, and long interpreted as,

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dilute media (especially cellulosic media such as oatmeal, cornmeal or pablum cereal agar) are generally salutary to the fruiting of such organisms. Leonians agar was originally developed [19] to promote the fruiting of pycnidial fungi and is equally or more useful for this purpose in the modified form used in this study. In situations calling for rapid identification of L. theobromae , molecular study is probably necessary, and reference may be made to several well documented ribosomal and elongation factor alpha sequences available on GenBank [2].

Acknowledgements
Thanks to Judy Clent and Myrna de Castro for technical assistance. We honour the memory of our colleague and friend, the late Julius Kane, who was part of the investigation team for this case.

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References
Fig. 4 Mature, longitudinally striate Lasiodiplodia theobromae didymoconidia, face and end view. 100 )/ objective. 1 Punithalingam E. Botryodiplodia theobromae . CMI Descriptions of Pathogenic Fungi and Bacteria, No. 519. Kew, Surrey: Commonwealth Mycological Institute, 1976. 2 Denman S, Crous PW, Groenewald JG, et al . Circumscription of Botryosphaeria species associated with Proteaceae based on morphology and DNA sequence data. Mycologia 2003; 95: 294 /307. 3 Puttana ST. Mycotic infections of the cornea. J All-India Ophthalmol Soc 1967; 15: 11 /18. 4 Laverde S, Moncada LH, Restrepo A, Vera CL. Mycotic keratitis: 5 cases caused by unusual fungi. Sabouraudia 1973; 11: 119 /123. 5 Valenton MJ, Rinaldi MG, Butler EE. A corneal abscess due to the fungus Botryodiplodia theobromae . Can J Ophthalmol 1975; 10: 416 /418. 6 Rebell G, Forster RK. Lasiodiplodia theobromae as a cause of keratomycoses. Sabouraudia 1976; 14: 155 /170. 7 Slomovic AR, Forster RK, Gelender H. Lasiodiplodia theobromae panophthalmitis. Can J Ophthalmol 1985; 20: 225 /8. 8 Gomes DJ, Huq F, Sharif A. Mycotic corneal ulcer in Bangladesh. Bangl Med Res Council Bull 1988; 14: 49 /57. 9 Thomas PA, Kuriakose T, Kirupashanker MP, Maharajan VS. Use of lactophenol cotton blue mounts of corneal scrapings as an aid to the diagnosis of mycotic keratitis. Diagn Microbiol Infect Dis 1991; 14: 219 /224. 10 Thomas PA, Garrison RG, Jansen T. Intrahyphal hyphae in corneal tissue from a case of keratitis due to Lasiodiplodia theobromae . J Med Vet Mycol 1991; 29: 263 /267. 11 Gonawardena SA, Ranasinghe KP, Arseculeratne SN, Seimon CR, Ajello L. Survey of mycotic and bacterial keratitis in Sri Lanka. Mycopathologia 1994; 127: 77 /81. 12 Borderie VM, Bourcier TM, Poirot JL, et al . Endophthalmitis after Lasiodiplodia theobromae corneal abscess. Graefes Arch Clin Exp Ophthalmol 1997; 235: 259 /261. 13 Restrepo A, Arango M, Velez H, Uribe L. The isolation of Botryodiplodia theobromae from a nail lesion. Sabouraudia 1976; 14: 1 /4. 14 Velez H, Diaz F. Onychomycosis due to saprophytic fungi. Mycopathologia 1985; 91: 87 /92.

a sunken pore. Within this group, Alternaria alternata and A. infectoria in particular, in our observation, include numerous isolates from human lesions growing in a nonsporulating form in culture over a prolonged period of time. A valuable clue that an isolate from phaeohyphomycosis is not one of these fungi comes from demonstrating sensitivity to benomyl, as was seen with L. theobromae isolated from this case. L. theobromae is well known to be susceptible to this agent [1,18], while Pleosporales with poroconidial anamorphs are not [18]. Though benomyl has recently been removed from the worldwide market by its manufacturer, most biosystematically interested mycological laboratories and many medical mycological reference laboratories possess stocks that, due to the microgram quantities used, are likely to be available for a number of years to come. Once L. theobromae sporulates, it can be confused with no other fungus, particularly when conidia have matured as striate didymoconidia. Isolates with only immature conidia may be confused with numerous Coelomycetes, such as Cryptosporiopsis species, and laboratory workers wishing to do morphological identification of sporulating Coelomycetous isolates with large ellipsoidal conidia should retain them for several weeks after fruiting to ensure that maximal opportunity is allowed for conidial production and maturation. The exact conditions promoting maturation are not known, but the combination of light and relatively

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15 Punithalingam E. Sphaeropsidales in culture from humans. Nova Hedwigia 1979; 31: 119 /158. 16 Maslen MM, Collis T, Stuart R. Lasiodiplodia theobromae isolated from a subcutaneous abscess in a Cambodian immigrant to Australia. J Med Vet Mycol 1996; 34: 279 /283. 17 Malloch D. Moulds / their isolation, cultivation and identication . Toronto, Canada: University of Toronto Press, 1981.

18 Summerbell RC. The benomyl test as a fundamental diagnostic method for medical mycology. J Clin Microbiol 1993; 31: 572 / 577. 19 Leonian CH. A study of some factors promoting pycnidium formation in some Sphaeropsidales. Amer J Bot 1924; 11: 19 /50.

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