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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

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, phaeohypho-

mycosis, 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 (teleo-

morph 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, ithas been reported clinically from numerous cases

of ocular keratitis and rarely from other ocular

conditions such as endophthalmitis [3/12]. It has

also been substantiated as an agent of onycho-

mycosis [13,14]. Reports of subcutaneous infection to

date have been very rare. An isolation from human

subcutaneous tissue is accessioned in the Common-

wealth Agriculture Bureau International (CABI

Biosciences, Egham, Surrey, UK) collection as

IMI 172565 [15] but no details were published onthis 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

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

Received 28 April 2004; Accepted 9 July 2004

2004 ISHAM DOI: 10.1080/13693780400005916

Medical Mycology December 2004, 42, 543/547

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surgical treatment that was applied successfully in this

case.

Case report

A 50-year-old woman with no known immune defi-

ciencies and otherwise in excellent health sustained aninjury 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 histo-

pathology (Fig. 1). Culture yielded an initially non-

sporulating, 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 CBSFungal 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 pend-

ing 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 peptone-

glucose 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.

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 benomyl-

free 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 con-

fluent, after over 17 weeks of incubation. Pycnidia lined

internally with a dense layer of conidiophores

and paraphyses (Fig. 3). Conidiophores hyaline, cylind-

rical 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 uni-

cellular, later becoming medially uniseptate, brown,Fig. 1 Septate filaments of Lasiodiplodia theobromae in cutaneous

ulcer tissue. Silver stain. 100)/ objective.

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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.

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.

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 grow-

ing 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 phaeohypho-

mycotic lesions are frequently related to mem-

bers of the fungal order Pleosporales, especiallyCochliobolus, Lewia, Pyrenophora and Setosphaeria,

usually seen as anamorphs in Bipolaris, Curvularia,

Alternaria, Drechslera and Exserohilum. These ana-

morphs 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,

Fig. 2 Rapidly growing melanized colony of Lasiodiplodia theobro-

mae, Leonians agar, 7 days.

Fig. 3 Fertile layer within Lasiodiplodia theobromae pycnidia

showing conidiogenous cells, paraphyses, and immature conidia.

40)/ objective.

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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 thisagent [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 myco-

logical 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 iden-

tification of sporulating Coelomycetous isolates with

large ellipsoidal conidia should retain them for several

weeks after fruiting to ensure that maximal opportu-

nity is allowed for conidial production and maturation.

The exact conditions promoting maturation are not

known, but the combination of light and relatively

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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