Fatal Trichoderma harzianum infection in a leukemic pediatric patient

Fatal Trichoderma harzianum infection in a leukemic

pediatric patient

A. SERDA KANTARCIOGLU*, TIRAJE CELKAN$, AYHAN YUCEL*, YUZURU MIKAMI%, SEBUH KURUGOGLU§,

HIROKI MITANI% & KEMAL ALTAS*

*Department of Microbiology and Clinical Microbiology, Cerrahpasa Medical Faculty, Istanbul, Turkey, $Department ofPediatrics Hematology-Oncology, Cerrahpasa Medical Faculty, Istanbul, Turkey, %Medical Mycology Research Center,Chiba University, Chiba, Japan, and §Department of Radiology, Cerrahpasa Medical Faculty, Istanbul, Turkey

We report the repeated isolation for Trichoderma.harzianum, a rare opportunistic

pathogen from three sets of each of the following clinical samples; blood serum,

skin lesions, sputum and throat of a pediatric ALL patient with neutropenia. The

definition of invasive fungal infection requires evidence of the presence of fungal

elements in tissue samples, in addition to the isolation of suspected etiologic agent

in culture. However, invasive procedures are not always applicable due to several

factors, as for example in our case, the poor general status of the individual patient

or thrombocytopenia. The present paper also emphasizes the problems encoun-

tered in obtaining appropriate samples and diagnosing invasive fungal disease in

immunocompromised patient populations, including those with hematological

malignancy. Three cases involving T. harzianum, including this one, have been

described thus far in the literature. All were fatal and the fungus was resistant to

antifungal therapy. A critical review of the other two cases of Trichoderma

infections in humans is provided.

Keywords Trichoderma harzianum, Trichoderma human infection, Trichoderma

antifungal susceptibility, mycoses in pediatric patients, fungal infections in

leukemic patients

Introduction

Many organisms that were previously considered to be

contaminants or harmless colonizers when isolated

from human clinical specimens have emerged as major

causes of morbidity and mortality, especially in im-

munocompromised hosts. The information obtained in

such cases could be valuable in monitoring future

patients with suspected invasive mycoses. Trichoderma

species are saprophytic fungi commonly found in soil,

which have been recently reported as being among

emerging fungal pathogens [1,2]. The aim of our paper

is to report the isolation of an uncommon mould,

Trichoderma harzianum from a pediatric patient with

hematological malignancy. In addition, we discuss the

problems in diagnosing and obtaining supporting

evidence of invasive fungal diseases in immunocom-

promised patient populations including those with

hematological malignancy.

Case report

Clinical course

On 15 February 2005 common acute lymphoblastic

leukemia (ALL) was diagnosed in a previously healthy

9 year-old boy. There was no history of primary

immunodeficiency or familial malignancy. Bone mar-

row studies revealed 94% of malignant cells of French-American-British L1 morphology, but the central

nervous system (CNS) not involved. Treatment was

started according to the ALL-BFM 95 protocol

Correspondence: A Serda Kantarcioglu, Department of

Microbiology and Clinical Microbiology, Cerrahpasa Medical

Faculty, Istanbul University, 34303 Cerrahpasa, Istanbul, TR-

Turkey. Tel & Fax: �90 212 248 46 06; E-mail: s.kantarcioglu@

superonline.com

Received 23 December 2007; Final revision received 8 August 2008;

Accepted 13 August 2008

– 2009 ISHAM DOI: 10.1080/13693780802406225

Medical Mycology March 2009, 47, 207�215

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consisting of prednisolone, vincristine, daunorubicin,

L-asparaginase and intrathecal methotrexate. The boyresponded well to chemotherapy, and 3% of blasts

were found in the bone marrow on day 15 of therapy.

From the beginning of chemotherapy, the patient was

neutropenic and piperacillin�tazobactam was adminis-

tered for a period of 14 days for febrile neutropenia

and defervescence was achieved within the following

2 days. The prednisone response at day 8 of induction

therapy was good, only 4% (300/mm3) blast weredetected in peripheral blood samples. He completed

the first month of induction therapy with minimal

complainments, which included abdominal pains ac-

companies by non-bloody but soft stools from which

no microorganisms were recovered in culture. A

reciprocal t(9;22) fuses the BCR (breakpoint cluster

region) gene from chromosome 22 to the ABL

(Abelson) gene from chromosome 9. The fusionprotein is a constitutive protein kinase that alters

signaling pathways that control the proliferation,

survival, and self-renewal of hematopoietic stem cells,

positivity of it reflecting poor prognosis. A target type

skin rash was noticed on day 39 of therapy which was

considered to be drug-related after dermatologic

consultation. The rash consisted of small pustules,

most commonly on the extremities, as well as wide-spread, erythematous indurated elements, sometimes

progressing to necrosis resembling ecthyma gangreno-

sum especially in canule entrance of the skin (Fig. 1A

and B). A profound tremor was noted on the second

day of using high risk blocks (22 March 2005)

including high doses of MTX (5 g/m2), cyclopho-

sphamide, L-asparaginase and vincristine. Puncture

of lumbar fluid applied for intratecal therapy revealedno abnormalities, normal biochemistry values and no

cells. Fever recurred within 6 days of the initiation of

high risk protocol and as a result antimicrobial

therapy was initiated with piperacillin�tazobactam

and liposomal amphotericin B (AMB; conventional

AMB deoxycholate is not available in Turkey pre-

sently) at 1mg/kg/day for 2days, 3 mg/kg/day for

3 days, increasing doses to 5 mg/kg /day. At thattime the patient had been neutropenic with an

absolute neutrophil count of B500 since the initiation

of therapy (45 day). Unfortunately the clinical condi-

tion deteriorated rapidly over the next 2 days. He

developed respiratory insufficiency, and Cullen, Gray-

Turner signs. His clinical symptoms with target lesion

in skin, tremors reflecting CNS involvement and the

lesions in the canule entrance caused us to consider adiagnosis of fusariosis. The following 10 days were

critical for the patient. Pancreatitis was revealed in

ultrasound and computed tomography (CT) imaging

of the abdomen, but biochemistry values were in

normal range for amylase. A CT scan of the lungs was

performed and revealed infiltrates suggesting a mould

infection (Fig. 2 A and B). No etiological micro-

organism was isolated in blood, urine and stool

cultures. Aspergillus galactomannan antigen (GMN)

could not be detected in two serum samples. The

routine microbiological culture studies performed by

the clinical laboratory were negative for fungi, and no

pathogens could be detected in the specimens taken

from the patient. Detailed mycological examination

was requested from Deep Mycosis Laboratory. Anti-

biotic and antimycotic therapy was switched to

meropenem, teicoplanin and AMB liposomal (5 mg/

kg/day). In addition granulocyte colony-stimulating

factor was given at a dosage of 5 g/kg/day and he was

given granulocyte suspension for two consecutive days.

Seventeen days later his clinical condition improved

with the cessation of chemotherapy due to the

Fig. 1 Clinical appearance of skin lesions at different stages of

evolution: (A) an erythematous subcutaneous nodule, and (B) a

necrotic lesion with progressive central necrosis and black scar with

an erythematous halo surrounding the lesion.

– 2009 ISHAM, Medical Mycology, 47, 207�215

208 Kantarcioglu et al.

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continued neutropenia. After albumin and FDP

replacements for pancreatitis and neutropenia im-

provement, amylase and D-dimer values tended to

increase (amylase from 8�181 u/l normal range: 0�90

and D-dimer from 48�581, normal range: 50�228 mg/

l). With the correction of neutropenia, blasts were

detected on peripheral blood which forced us to

suspend hiss ALL treatment for an extended period

of time. As previous high risk block therapy consisting

of high doses of MTX caused severe mucositis and

toxicity, HR 3 block which does not include MTX was

chosen for the next cycle of treatment. The next cycle

of chemotherapy consisting of oral dexamethasone

and intravenous etoposide and cytarabine, (skipping

L-asparaginase because of unequivocal pancreatitis)

was started at 20 April 2005. Concomitantly the

antimycotic treatment with liposomal AMB at a

dose of 5 mg/kg/day was continued. The block was

completed with no problem on 26 April 2005. A

bloody stool was found 3 days after the end of block,

which was followed with a severe neutropenia (total

leucocytes 100/mm3 with no polymorphonuclear lym-

phocytes-PNL). A rapid clinical deterioration was

noted with the beginning of neutropenia. Intractable

convulsions and respiratory insufficiency worsened the

clinical picture. The CT taken in order to exclude

intracranial hemorrhage revealed no pathology. Thor-

acic and abdominal scan was also taken which

revealed intestinal edema, tyflitis. CT scan performed

after 4 weeks of liposomal ABM treatment revealed

that antimycotic treatment resulted in a marked

decrease of both size and number of pulmonary

infiltrates. The blood culture taken during last febrile

neutropenia period was positive for Klebsiella with an

intermediate resistance to the given antibiotic regimen

(piperacillin�tazobactam). Unfortunately the clinical

condition deteriorated rapidly over the next hours and

he succumbed � despite the use of ventilation.

MYCOLOGY STUDY

Materials and methods

Isolation and identification of the strain

Tissue samples were not obtained due to the patient’sthrombocytopenia. Three sets each of whole blood, pus

from skin lesions, sputum and pharyngeal specimens

were obtained on three separate days (12 April 2005, 15

April 2005, 20 April 2005). The blood samples (5 ml)

were recovered in anti-coagulant free sterile vacuum

tubes. Deep cough spontaneous sputum samples (not

saliva) were collected early in the morning (first morning

sputum) in appropriate sterile containers for freshstudies and their quality evaluated at under 10 squamous

epithelial cells content per low-power microscope field.

Pus samples were taken from skin lesions by pressing

gently on the areas surrounding the lesions, while

smears from throat were obtained using sterile swabs

avoiding contamination with oral flora organisms.

Regarding the patient’s previous negative results for

fungi in hemocultures, aseptically prepared sera fromblood samples were studied through direct microscopy

of unstained or lactophenol cotton blue (LCB) wet

mounts and inoculating a portion of the sear into

Sabouraud dextrose broth (SDB) medium. After in-

cubation at 30 and 378C for 5 days, 3 to 5 drops from

broth cultures were streaked onto Sabouraud dextrose

agar (SDA) and brain-heart infusion agar (BHIA) and

incubated at 25, 30 and 378C for 10 days. Ehrlich-Ziehl-Neelsen (EZN), methylene blue and Giemsa stained

slides were used in the direct microscopic examination

of specimens other than blood. The specimens were

inoculated onto SDA, BHIA and cooked sheep’s blood

agar and incubated at 25, 30 and 378C for 10 days. Oat

meal agar (OA), potato dextrose agar (PDA), potato

sucrose agar, Czapek Dox agar, malt extract agar

cultures were used as differential identification mediaand incubated both at 288C in darkness and at

fluctuating room temperatures under diffuse day light

for 5 and 8 days. The isolated fungi were examined

using classical mycological techniques based on growth

rate, as well as macroscopic and microscopic character-

istics of pure colony. LCB was used for microscopic

examination of culture slides. Phenotypically identified

isolates from serum, pus and sputum samples (numbers1, 2 and 3 respectively) were sent to Research Center for

Pathogenic Fungi and Microbial Toxicoses, Chiba

University, Chiba, Japan for molecular identification.

Fig. 2 Ground-glass nodular densities were seen on parenchymal

window in both lung parencyhmes.


Trichoderma harzianum isolated from a fatal pediatric case 209

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Environmental samples from patient’s room and

deep mycoses laboratory were collected from indoorwalls and furniture surfaces with several sterile swabs

and from indoor air by the settle Petri dish method

(SDA plates, 12 h) and were studied in the same

manner as clinical samples.

Latex agglutination (LA) test

Aspergillus GMN antigen test was performed with the

LA kit (Pastorex Aspergillus, Sanofi Diagnostics Pas-

teur, France) on two serum samples obtained on two

different days.

Molecular analyses of r DNA region

The methods used for DNA extraction were the same

as those described by Tamura et al. [3]. The PCR

primers for ITS amplification and DNA sequencing

were ITS5(5?-GGAAGTAAAAGTCGTAACAAGG-

3?) and ITS4(5?-TCCTCCGCTTATTGATATGC-3?).Amplifications reactions were performed by the pre-viously described method [3]. The PCR products were

purified with a PCR product pre-sequencing kit (U.S.

Biochemical Corp., Cleveland, Ohio, USA), and were

then sequenced using the BigDye Terminator Cycle

Sequencing Reaction Kit (Applied Biosystems, Foster

City, CA, USA) on an ABI PRISM 3130 Genetic

Analyzer (Applied Biosystems), according to the man-

ufacturer’s instructions.

Antifungal susceptibility tests

In vitro susceptibility studies of the isolate against

conventional and two novel triazole antifungal agentswere conducted in accord with the Clinical Laboratory

Standards Institute (CLSI, formerly NCCLS) M38-A

reference broth macrodilution method for filamentous

fungi [4]. The tested antifungal agents were amphoter-

icin B (AMB- Bristol-Meyers Squibb, Wallingford,

Conn; 0.03 to 16 mg/ml), fluconazole (FLZ � Pfizer

Pharmaceuticals, Istanbul, Turkey; 0.125�128 mg/ml),

itraconazole (ITZ � Janssen Pharmaceuticals, Beerse,Belgium; 0.03�16 mg/ml), voriconazole (VRZ � Pfizer

Pharmaceuticals, Istanbul, Turkey; 0.03�16 mg/ml),

posaconazole (PSZ � Schering-Plough Research Insti-

tute, Istanbul, Turkey; 0.015�8 mg/ml), ketoconazole

(KTZ � Milen, Istanbul, Turkey; 0.03�16 mg/ml),

miconazole (MCZ � Selectchemie AG, Zurich, Switzer-

land; 0.03�16 mg/ml), and terbinafine (TRB � Novartis,

Basel, Switzerland; 0.03�128 mg/ml). All antifungalagents were provided as assay powders and dissolved

in water (FLZ), polyethylene glycol (PSZ), or dimethyl

sulphoxide (the remaining agents). Antibiotic medium

3 (Oxoid, Hampshire, England) was used for testing

AMB, and RPMI-1640 (Sigma Chemical Co., St.Louis, MO) with L-glutamine but without sodium

bicarbonate was used for azoles and TRB [4,5]. All

media were supplemented with 2% glucose and buffered

with 0.165 M morpholinepropanesulfonic acid (MOPS;

Sigma). Inoculum suspensions were composed of a

mixture of conidia and hyphae as described in M38-A

protocol, with final concentrations of approximately

0.4�104 to 5�104 CFU/ml. Inoculum quantificationwas performed by inoculating 10 ml of the adjusted

suspension onto SDA plates incubated at 308C and

observing them daily for growth. To control the final

inoculum concentrations, the colonies were counted as

soon as possible after the observation of visible growth

on the SDA cultures to determine the viable number of

CFU/ml. Paecilomyces variotii ATCC 22319 (American

Type Culture Collection, Manassas, VA, USA) andCandida parapsilosis ATCC 22019 with known mini-

mum inhibitory concentrations (MICs) were used for

quality control. Growth and sterility control tubes were

included as part of the quality control. Prior to

antifungal susceptibility test, preliminary experiments

were performed with 1 ml samples of the final inoculum

suspensions incubated at 308 and 358C and growth was

monitored from 24 h to four days to determine theoptimal incubation temperature. MICs were deter-

mined by visual inspection at the first 48-h interval

when growth was observed in the drug-free control

tube [4,6,7]. The MIC endpoints were determined as

the lowest drug concentration causing 100% reduction

in growth (MIC-0) for all tested antifungals [4,5,7].

Antifungal susceptibility tests of the isolate were

repeated twice to assess the reproducibility.

Results

Isolation

Direct microscopic examination of all pus, sputum, and

throat samples stained with EZN, methylene blue and

Giemsa techniques revealed thick walled subgloboseindividual fungal cells (Fig. 3). Thin septate hyphae

were observed in pus and serum specimens along with

individual fungal structures (Fig. 4). Cultures on SDA

showed white mycelial growth after 4 days of incuba-

tion at 308C. The same fungus was isolated as the

only microorganism from each of three sera, pus and

sputua samples and were transferred onto differential

media for further examination. The same fungus grewfrom throat samples along with pharyngeal flora

bacteria and these cultures were discarded. The isolate

first produced whitish-green floccose colonies, which



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became green due to abundant conidiation especially in

daylight, forming concentric white and green rings.

Colony reverse on PDA plates was colorless when

grown in diffuse day light, but a lemon yellow diffusible

pigment was evident when grown in dark. Conidio-

phores pyramidally branched, i.e., long, repeatedly

branched lateral branches below the apex, with short

branches near the end of the hypha. Conidiogenous

cells (phialides) were flask-shaped, phialoconidia were

subglobose, broadly ellipsoidal to cylindrical, subhya-

line, smooth-walled and accumulated in balls at the

apex. Smooth, thick walled terminal or subglobose

intercalary chlamydoconidia either single or in triplets

were abundant especially on PDA and OA media

(Fig. 5). The isolate was phenotypically identified as

Trichoderma spp. by comparing its morphological

characteristics with the standard descriptions given by

De Hoog & Guarro [1] and Summerbell [2]. Thick

walled non-budding round to oval individual fungal

cells seen in direct microscopic examination of the

specimens were retrospectively recognized as possible

chlamydospores which are a remarkable microscopic

feature of the fungus in culture. Trichoderma spp. were

not recovered from environmental surfaces and air

samples obtained from patient’s room and the deep

mycoses laboratory. Living cultures of the case isolate

have been preserved in the collection of Medical

Mycology Research Center (former Research Centerfor Pathogenic Fungi and Microbial Toxicoses), Chiba

University, Chiba, Japan as IFM 54705 (No 1), IFM

54706 (No 2) and IFM 54707 (No 3).

Latex agglutination (LA) test

Aspergillus GMN antigen results were negative in twosera samples tested.

Molecular genetic study

BLAST search program using ITS region sequences

of the 3 fungal isolates revealed that the three isolates

(No.1, 2 and 3 strains) of the fungus showed similar-

ity value of 100% to Trichoderma harzianum (Gene-Bank accession numbers AB282750 [IFM 54705],

AB282751 [IFM 54706], AB282752 [IFM 54707]).

Therefore these three strains were identified as T.

harzianum on the basis of these results and their

morphologic features. This identification was con-

firmed by identification tool established for Tricho-

derma on www.isth.info [8�10].

Antifungal susceptibility test results

MIC’s were as follows: AMB�16 mg/ml; ITZ, MCZ,

KTZ, VRZ, PSZ, TRB B0.03; and FLZ 0.125 mg/

ml. The isolate was found to be resistant in vitro to

AMB and susceptible to other antifungal agents

tested. Quality control strains showed MICs withinacceptable limits. Repeated test results showed exactly

the same MIC values against all tested antifungal

agents.Fig. 4 Direct microscopy of pus smear showing thin septate hyphae

along with individual fungal cells (Giemsa stained preparation,�100)

Fig. 5 Chlamydospores in culture preparation (Lactophenol cotton

blue,�40).

Fig. 3 Direct microscopy of pus smear showing individual fungal

cells (Giemsa stained preparation, �100)



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Discussion

Reported human cases with Trichoderma species are

limited in number [11,12]. The first known human case

involved its inadvertent introduction in an immuno-

competent host with contaminated intravenous fluid

[14]. Trichoderma species are responsible for localized

or disseminated infections including continuous ambu-

latory peritoneal dialysis-associated peritonitis, pul-

monary mycetoma, lung, liver and brain abscesses in

immunocompromised patients with a hematological

malignancies or solid organ transplants [12,15�20].

The clinical importance of the genus Trichoderma was

comprehensively reviewed by Kredics et al. [21]. Two

recent pulmonary infections were attributed to Tricho-

derma citrinoviride [22] and the teleomorph state

Hypocreaceae [23]. The prognosis for any type of

Trichoderma infection is poor, with 10 deaths among

25 cases [11,12,19�22]. T.harzianum ability to produce

extracellular aspartic proteases, hydrolytic enzymes,

sidefores, high reproductive capacity and its efficient

utilization of nutrients including aminoacids as carbon

and nitrogen sources [21,24,25] allow it to survive

under unfavorable conditions, including growth at

elevated temperatures up to 408C [21]. Studies on the

ecophysiology of the genus Trichoderma have shown

that species belonging to the Longibrachiatum section

of the genus (including T. citrinoviride and T. long-

ibrachiatum) have higher optimum growth temperatures

[24]. Because growth at elevated temperatures is one of

the virulence factors of fungi, it is not surprising, that

most of the strains involved in Trichoderma infections

belong to the Longhibrachiatum section of the genus.

All of the clinical strains were able to grow at

physiological pH, which can promote their growth as

facultative human pathogens [26]. Other factors, like

the hydrophobicity of conidia, melanic or carotenoid

pigments and mycotoxins are also among the possible

virulence factors of opportunistic fungal pathogens

[27]. However, the role and significance of these factors

in human infections are not sufficiently understood and

in a comparative study of potential virulene factors [28]

there were no significant differences in the examined

features between strains derived from clinical or soil

samples. Studies dealing with the taxonomic positions

of clinical Trichoderma isolates [23,29] have shown that

some of them had been misidentified. Although six

species of the genus have been reported as human

pathogens, only the involvement of T. longibrachiatum,

T. citrinoviride and T. harzianum could be confirmed as

of yet by molecular methods. A series of originally

misidentified clinical Trichoderma isolates were reiden-

tified as T. longibrachiatum. T. longibrachiatum is the

most commonly recovered from cases of invasive

infections [11,18], while T. harzianum has only beenassociated with two human cases, i.e., a fatal peritonitis

in a patient receiving peritoneal dialysis [22] and a

postmortem diagnosis of a systemic infection in a renal

transplant patient [17]. The present case is the third

ever reported, but only the second it which the identity

of the etiologic agent was confirmed by molecular

methods. The latter procedures were not employted in

the case reported by Guiserix et al. [22]. Our patientrisk factors that probably contributed to the invasive

fungal infection included severe neutropenia, receiving

antibiotics, corticosteroids and chemotherapy for ALL.

Despite the fact that the standard definition of

invasive fungal infections requires microscopic visuali-

zation of fungal elements in tissue samples and isolation

of fungi on culture, obtaining such samples is not always

possible due to several factors including poor generalstatus of the individual patient or thrombocytopenia.

Therefore, the definition of an invasive fungal infection

may have to rely on a combination of less-specific

clinical, laboratory and radiological data [30]. In our

case, a definitive diagnosis of a radiologically demon-

strable lung involvement and/or ulceronecrotic skin

lesions was not considered feasible, because of the

patient’s thrombocytopenia. However, in our case,microscopic examination of Gram, EZN and Giemsa

stained sputum smears revealed thin septate hyphae

before the recovery of the fungus in culture. These

findings supported studying a series of fresh first

morning sputum specimens collected on three different

days. Moreover, examination of stained smears prepared

from pus samples from skin lesions obtained on different

days revealed the presence of a fungus with similarmorphologic features as seen in sputum samples. How-

ever, serum Aspergillus GMN tests were twice negative.

Although, positive blood cultures have been included

by the European Organization for Research and

Treatment of Cancer/Mycoses Study Group (EORTC/

MSG) as one of the criteria in the diagnosis of invasive

fungal infections [30], cultures in our case remained

negative for fungi. Unfortunately, while blood culturesare sensitive for bacterial pathogens, they are usually a

poor diagnostic tool for invasive mycosis. A single or

even multiple negative blood cultures for fungi does not

exclude disseminated fungal infections. The common

isolation rate of fungi in hemocultures is low [31�33]

and blood cultures are known to be rarely positive for

patients with proven invasive aspergillosis [34] and

candidiasis [35�38]. With Trichoderma species as well,despite documented dissemination, only one case of

positive blood culture has been described in the

literature [12,27]. Therefore, since a fungal infection



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was strongly suspected in the present case, aseptically

prepared sera samples were used for microscopy andmycological culturing and T. harzianum was success-

fully isolated from the patient’s three sera specimens.

Necrotic ulcerated skin lesions that developed on the

extremities were clinically interpreted as possible man-

ifestations of a deep seated, pulmonary Fusarium

infection. Interestingly, similar skin lesions were also

described in two previous cases caused by two different

Trichoderma species [19]. A pediatric patient withaplastic anemia and prolonged neutropenia [39] had a

necrotic cutaneous lesion on his wrist caused by T.

longibrachiatum. In another case [16], ulceronecrotic

skin lesions were described in an adult bone marrow

recipient and T. pseudokoningii was reported as being

recovered from of skin, brain, and lung samples.

However, T. pseudokoningii strain IP 210.92 was sub-

sequently reidentified as T. longibrachiatum [24,27]. Theunusual moniliaceous fungi that comprised the genus

Trichoderma are philogenetically close to species in the

genus Fusarium [1] but comparable data as to the

relative pathogenicity of the two generare are not yet

available. We suggest that in immunocompromised

patients, Trichoderma spp. should be considered in

the differential diagnosis of fungal infections associated

with ulceronecrotic skin lesions similar to those notedin infections caused by Fusarium spp. Regarding the

previous case reports, Trichoderma infections are

characterized by the presence of fine septate hyaline

hyphae in tissue sections. In the present case, despite

the fact that a tissue biopsy could not be obtained due

to the patient’s condition, fine hyaline hyphae were seen

in the series of sera and pus samples. Further, thick

walled, non-budding, subglobose individual fungalcells were observed in direct microscopic examination

of the specimens and retrospectively recognized as

possible chlamydospores which are remarkably similar

to structures microscopically noted when Trichoderma

harzianum is grown in culture. Chlamydospores pre-

sented in tissue specimens were also reported in a

previous case caused by another Trichoderma species,

T. longibrachiatum [40].Trichoderma species form a large group of fungi

difficult to distinguish from and often mistaken for

members of Penicillium or Aspergillus. Because

of the variability of morphological characters of

Trichoderma spp., current molecular techniques have

been demonstrated to be clinically useful for identifica-

tion of Trichoderma species [2]. Accurate species

identification might assist in the accumulation ofreliable epidemiological data on the association with

these opportunistic fungi in human infections. There-

fore, in the present case, the isolate was phenotypically

identified as Trichoderma spp. by comparing the

morphological characteristics [1,2] and species differ-entiation was performed by molecular techniques.

Members of the genus Trichoderma are most com-

monly recovered from soil, especially from water-logged

soil and other water-associated environmental sources

[41�43]. They have been reported to be infrequently

present in the air samples [44] and the detection of

Trichoderma in indoor air has been considered to be an

indicator of moisture-damaged building materials [45].In agreement with these reports, we were not able to

recover Trichoderma from environmental surfaces and

air samples collected from the patient’s room and the

deep mycoses laboratory. Trichoderma species has also

been isolated from grains and foods and in a case

reported by Richter et al. [18] the thought to have been

acquired through the gastrointestinal tract. In another

case involving a patient who had received an allogenicbone marrow transplant for ALL, T. longibrachiatum

was isolated from stool and a perirectal ulcer biopsy

specimen. At autopsy, histological sections from the

lungs, liver, brain and intestinal wall showed infiltration

by septate, branching hyphae and cultures inoculated

with tissue samples were positive for the same fungus

[18]. Myoken et al. [46] reported a case of necrotizing

stomatitis which rapidly disseminated from the oralmucosa to the lungs during neutropenia. Among in-

vasive cases due to Trichoderma species, mucosal inva-

sion in oral cavity [46] and/or intestinal wall [18] was

remarkable. In the present case, although radiologically

demonstrable typhlitis was noted, the aetiology re-

mained unknown. Since autopsy was denied, a possible

relationship could not be demonstrated between the

necrotizing colitis as a risk factor and probable portal ofentrance due to mucosal barrier damage.

The first human case involving T.harzianum was in an

82-year-old, non-insulin-dependent diabetic patient re-

ceiving continuous ambulatory peritoneal dialysis. De-

spite antifungal treatment with ketoconazole and

flucytosine, the outcome was fatal [22]. Guarro et al.

[17] reported another fatal case of systemic infection

caused by T.harzianum and the fungus was recoveredfrom abscess in brain and lung tissue (Strain CBS

102174). The patient had not receive any antifungal

treatment because the fungal infection was not diag-

nosed until postmortem examination. Up to this date,

very limited data are available on the in vitro antifungal

susceptibilities of Trichoderma spp. [5,18,49�51] and

T. harzianum in particular [17,48]. Comparative datasets

concerning the antifungal susceptibilities of T.

harzianum CBS 102174, as well as a series of T.

longibrachiatum isolates using the Etest method mod-

ified for moulds have been described [21,28].



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In studies with the CLSI M38-A macrodilution

method we found that our isolate had a high in vitro

MIC value against AMB (�16 mg/ml), but low MICs

were found with ITZ, KTZ, MCZ, VRZ, PSZ, TRB

(50.03 mg/ml) and FLZ (0.125 mg/ml). In a recent in

vitro study [48], MICs of AMB were reported as 2 mg/

ml for 15 strains of Trichoderma spp., including 13 clin-

ical isolates. Because our patient received empirical

treatment with liposomal AMB treatment due to the

clinical diagnosis of a Fusarium infection, it is difficult

to interpret whether the case isolate had an innate or

acquired resistance to this agent. For VRZ, the low

MIC values reported by Guarro et al. [17] and Kratzer

et al. [48] were in agreement with our in vitro results.

Although, this broad-spectrum antifungal has shown

clinical efficacy in several infections caused by other

uncommon and resistant fungi [17], it was not available

in this country during the time that our patient was

being treated. The FLZ susceptibility of our Tricho-

derma isolate (MIC�0.125 mg/ml) would appear to be

lower than those reported for other clinical isolates

(MIC range from 12.5 and 1024 mg/ml).

Many environmental fungi, including Trichoderma

species, which have low inate pathogenicity are likely to

be contaminants or colonizers in immunocompetent

patients but may cause invasive disease in severely

immunocompromised patients [52]. Unfortunately, the

general signs and symptoms present in invasive fungal

infections are usually atypical and nonspecific reducing

the opportunities to make the correct clinical diagnosis.

Although, the highest level of certainty in diagnosing

an invasive mycosis requires establishing the presence

of fungi in tissue by biopsy or a needle aspirate, such

invasive techniques are not always possible due to the

patients’ conditions. Herein, we describe repeated

isolation of an uncommon fungus, T.harzianum from

a pediatric patient with ALL presenting with progres-

sive pulmonary infiltrations plus ulceronecrotic skin

lesions similar to those seen with deep infections caused

by Fusarium spp. to bring such cases to the attention of

clinicians. To date, three known cases of T. harzianum,

including ours, have been fatal and the resistance of this

species to antifungal therapy is remarkable.

Acknowledgements

We thank Pfizer, Inc and Schering Plough for supplying

antifungal powders.

The present work was partly supported by the

Research Fund of Istanbul University, Project No:

UDP-607/02082005.

Declaration of interest: The authors report no conflicts

of interest. The authors alone are responsible for the

content and writing of the paper.

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This paper was first published online on iFirst on 23 January 2009.



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