Journal de Mycologie Médicale (2008) 18, 234—236

CASE REPORT/CAS CLINIQUE

Isolation of Trichoderma atroviride from a livertransplant

Isolement de Trichoderma atroviride à partird’un greffon hépatique

S. Ranque a,*, D. Garcia-Hermoso b, A. Michel-Nguyen a, H. Dumon a

a Parasitology-Mycology Laboratory, AP—HM Timone, 264, Saint-Pierre street, 13385 Marseille cedex 5, Franceb French National Reference Centre for Mycoses and Antifungals (CNR de la mycologie et des antifongiques), Institut Pasteur,Paris, France

Received 20 June 2008; received in revised form 8 September 2008; accepted 9 September 2008Available online 31 October 2008

KEYWORDSTrichoderma atroviride;Liver transplant

* Corresponding author.E-mail address: stephane.ranque@

1156-5233/$ — see front matter # 20doi:10.1016/j.mycmed.2008.09.002

Abstract Six species of Trichoderma have been associated with 24 human infections to date.This is the first report of Trichoderma atroviride, cultured from a liver biopsy specimen of a livertransplant recipient. In the settings of permissive environmental conditions, we may anticipatethat emergent Trichoderma spp. infections will continue to develop.# 2008 Elsevier Masson SAS. All rights reserved.

MOTS CLÉSTrichoderma atroviride ;Greffe hépatique

Résumé À ce jour, six espèces de Trichoderma ont été impliquées dans 24 infectionshumaines. Pour la première fois, Trichoderma atroviride a été isolé d’une biopsie du foiepostmortem chez un patient greffé hépatique. Il est prévisible que l’émergence des infections àTrichoderma spp. se poursuive parallèlement à l’augmentation régulière du nombre de patients àrisque.# 2008 Elsevier Masson SAS. All rights reserved.

Case report

A 49-years-old man underwent a liver transplantation for thetreatment of an hepatocellular carcinoma in the context of

ap-hm.fr (S. Ranque).

08 Elsevier Masson SAS. All rights

alcoholic cirrhosis. He was administered methylpredniso-lone, tacrolimus, ganciclovir, amoxicillin/clavulanate, andmetronidazole. Biliary tract hemorrhages and cholestasisprompted a reintervention in the early postoperativecourse. On day-7 after transplantation, the patient suddenlydeveloped respiratory distress. Liver function tests yielded:alanine aminotransferase (ALT), 262 UI/l; aspartate amino-transferase (AST), 126 UI/l; lactic acid dehydrogenase (LDH),

reserved.

Trichoderma atroviride in a liver biopsy 235

292 UI/l; gammaglutamyl transferase (GGT), 262 UI/l; alka-line phosphatase (ALP), 198 UI/l; direct bilirubin, 19 mmol/l;albumine, 16 g/l; prothrombin time, 72%. On day-12, a pro-babilistic treatment with 800 mg/day oral fluconazole wasstarted. On day-13, a thrombotic microangiopathy possiblytriggered by tracrolimus was suspected because of persis-tently elevated direct bilirubin and LDH levels, anaemia,thrombopenia, and the presence of schizocytes. Tacrolimuswas thus replaced by cyclosporine. On day-15, a CT-scanshowed a normal liver vasculature. Histology of a liver biopsyspecimen revealed ischemic necrosis. Biological abnormali-ties persisted and fluconazole was replaced by voriconazole(400 mg bid) on day-16. Plasmapheresis was performed onday-17 because of an elevated (385 mmol/l) direct bilirubinlevel. On day-19, the patient developed acute renal failure,hypotension, oliguria, metabolic acidosis, and died in apicture of massive hemolysis. A postmortem liver biopsywas performed. A unique sample was sent to the MedicalMycology laboratory. Histological examination showedextensive ischemic necrosis with no direct evidence of afungal pathogen. Pure culture of T. atroviride grew at theinocluation site on Sabouraud’s dextrose agar plate withchloramphenicol and gentamicin of a biopsy specimen cultu-red at 30 8C.

This T. atroviride isolate (National Reference Centre forMycology and Antifungals [NRCMA] No 200501312) was iden-tified with respect to morphological [3] and molecular cri-teria. Its antifungal susceptibility pattern was tested usingthe EUCAST in vitro microdilution method in liquid medium.

Morphological identification

Macroscopic features

Macroscopic features were dense and woolly colonies thatgrew within five days, initially whitish, rapidly becoming darkgreen.

Microscopic features

Microscopic features included: conidiophores with branchingpattern at right angles; phialides (8—10 mm � 2 mm), flask-shaped often curved in whorls of two, three or four verti-

Figure 1 Slide culture of Trichoderma atroviride. A. Flask-shapeconidia (� 1000).Culture sur lame de Trichoderma atroviride. A. Verticilles de phialidparoi lisse (� 1000).

cillate; conidia (3.5 mm � 4 mm) dark green sub-globose,short ellipsoidal, smooth-walled; chlamydospores were pre-sent (Fig. 1).

Molecular identification

A 861 bp DNA sequence of our isolate shared 100% identitywith the AF278796 sequence of T. atroviride strain ATCC36042.

Antifungal susceptibility testing

This T. atroviride isolate exhibited the following MIC (mg/l):

� a

d

es

mphotericin B: 1;

� 5 -fluorocytosine: � 64; � fl uconazole: � 64; � it raconazole: � 8; � v oriconazole: 8; � c aspofungin: 0.5.

Discussion

The susceptible hosts’ reservoir of opportunistic moldswidens concomitantly to the increasing number of severelyimmunocompromised patients such as transplant recipients.With an increasing number of invasive infections reports,molds of the genus Trichoderma can be added to the growinglist of emerging human pathogens [1]. Trichoderma spp. arefree-living filamentous fungi that are common in soil and rootecosystems. T. atroviride (teleomorph: Hypocrea atroviri-dis) is an opportunistic, avirulent plant symbiont used as abiological control agent because it parasitizes a variety ofphytopathogenic fungi [4]. We report on the first isolation ofT. atroviride from a human patient.

A positive culture obtained by a sterile procedure from anormally sterile and clinically as well as radiologically abnor-mal site is consistent with the diagnosis of T. atroviridesystemic infection. Yet, the absence of hyphae in the histo-logical examination of the patient’s liver goes againstthe pathological implication of this mold. However, a

phialides in verticils (� 400). B. Sub-globose smooth-walled

en forme de bouteille (� 400). B. Conidies sub-globuleuses à

236 S. Ranque et al.

contamination of the patient’s sample is unlikely becauseTrichoderma spp. are seldom isolated from our hospital’senvironment and T. atroviride has not been previously cultu-red in our laboratory. There was no definitive evidence infavor of a disseminated infection in this patient, and theoverall pathological importance of this mold infection canonly be conjectured with respect to the complex clinicalpresentation of this patient who ultimately died. A retro-spective analysis of the patient’s file revealed no particularexpositional risk to Trichoderma spp. or any other mold otherthan immunosuppression.

In a comprehensive review of the literature, Chouakiet al. found 22 human infections caused by Trichodermaspp [1]. Recently, De Miguel at al. reported another case ofTrichoderma viride infection [2]. Including one personalobservation of a fatal Trichoderma longibrachiatum (NRCMANo 200300360) cholecystitis and peritonitis treated withvoriconazole and caspofungin in a 70-year-old man whounderwent splenectomy to diagnose a Castelman’s diseaseand the present T. atroviride infection, brings to 24 thereports of Trichoderma spp. infections in humans to date. Sixspecies: Trichoderma harzianum, Trichoderma koningii, Tri-choderma longibrachiatum, Trichoderma pseudokoningii,Trichoderma citrinoviride, and T. viride have been identifiedas etiologic agents of infections in immunocompromised

hosts or in continuous ambulatory peritoneal dialysisassociated peritonitis [1]. In this report, a seventh species,T. atroviride, is implicated for the first time. We may anti-cipate that emergent Trichoderma spp. infections will conti-nue to develop in the settings of permissive environmentalconditions, selective antifungal pressure and an expandingpopulation of immunocompromised hosts.

References

[1] Chouaki T, Lavarde V, Lachaud L, Raccurt CP, Hennequin C.Invasive infections due to Trichoderma species: report of 2cases, findings of in vitro susceptibility testing, and review ofthe literature. Clin Infect Dis 2002;35:1360—7.

[2] De Miguel D, Gomez P, Gonzalez R, Garcia-Suarez J, Cuadros JA,Banas MH, et al. Nonfatal pulmonary Trichoderma viride infec-tion in an adult patient with acute myeloid leukemia: report ofone case and review of the literature. Diagn Microbiol Infect Dis2005;53:33—7.

[3] Gams W, Bissett J. Morphology and identification ofTrichoderma. In: Kubicek CP, Harman GE, editors. Trichodermaand Gliocladium, vol.1: basic biology, taxonomy and genetics.London: Taylor & Francis Ltd; 1998. p. 278.

[4] Harman GE, Howell CR, Viterbo A, Chet I, Lorito M. Trichodermaspecies-opportunistic, avirulent plant symbionts. Nat Rev Micro-biol 2004;2:43—56.