Isolation of Trichoderma atroviride from a livere[1]

CASE REPORT/CAS CLINIQUE

Isolation of Trichoderma atroviride from a liver transplant

Isolement de Trichoderma atrovirideàpartir

d’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,13385Marseille cedex5,France

b French National Reference Centre for Mycoses and Antifungals(CNR de la mycologie et des antifongiques),Institut Pasteur, Paris,France

Received20June2008;received in revised form8September2008;accepted9September2008

Available online31October2008

KEYWORDS

Trichoderma atroviride;

Liver transplant

Abstract Six species of Trichoderma have been associated with24human infections to date.

This is the?rst report of Trichoderma atroviride,cultured from a liver biopsy specimen of a liver

transplant recipient.In the settings of permissive environmental conditions,we may anticipate

that emergent Trichoderma spp.infections will continue to develop.

MOTS CLéS

Trichoderma atroviride;

Greffe hépatique

Résuméàce jour,six espèces de Trichoderma ontétéimpliquées dans24infections

humaines.Pour la première fois,Trichoderma atroviride aétéisoléd’une biopsie du foie

postmortem chez un patient grefféhépatique.Il est prévisible que l’émergence des infectionsà

Trichoderma spp.se poursuive parallèlementàl’augmentation régulière du nombre de patientsà

risque.

Case report

A49-years-old man underwent a liver transplantation for the

treatment of an hepatocellular carcinoma in the context of

alcoholic cirrhosis.He was administered methylpredniso-

lone,tacrolimus,ganciclovir,amoxicillin/clavulanate,and

metronidazole.Biliary tract hemorrhages and cholestasis

prompted a reintervention in the early postoperative

course.On day-7after transplantation,the patient suddenly

developed respiratory distress.Liver function tests yielded:

alanine aminotransferase(ALT),262UI/l;aspartate amino-

transferase(AST),126UI/l;lactic acid dehydrogenase(LDH), Journal de Mycologie Médicale(2008)18,234—236

*Corresponding author.

E-mail address:stephane.ranque@ap-hm.fr(S.Ranque).

doi:10.1016/j.mycmed.2008.09.002

292UI/l;gammaglutamyl transferase(GGT),262UI/l;alka-line phosphatase(ALP),198UI/l;direct bilirubin,19m mol/l; albumine,16g/l;prothrombin time,72%.On day-12,a pro-babilistic treatment with800mg/day oral?uconazole was started.On day-13,a thrombotic microangiopathy possibly triggered by tracrolimus was suspected because of persis-tently elevated direct bilirubin and LDH levels,anaemia, thrombopenia,and the presence of schizocytes.Tacrolimus was thus replaced by cyclosporine.On day-15,a CT-scan showed a normal liver vasculature.Histology of a liver biopsy specimen revealed ischemic necrosis.Biological abnormali-ties persisted and?uconazole was replaced by voriconazole (400mg bid)on day-16.Plasmapheresis was performed on day-17because of an elevated(385m mol/l)direct bilirubin level.On day-19,the patient developed acute renal failure, hypotension,oliguria,metabolic acidosis,and died in a picture of massive hemolysis.A postmortem liver biopsy was performed.A unique sample was sent to the Medical Mycology laboratory.Histological examination showed extensive ischemic necrosis with no direct evidence of a fungal pathogen.Pure culture of T.atroviride grew at the inocluation site on Sabouraud’s dextrose agar plate with chloramphenicol and gentamicin of a biopsy specimen cultu-red at308C.

This T.atroviride isolate(National Reference Centre for Mycology and Antifungals[NRCMA]No200501312)was iden-ti?ed with respect to morphological[3]and molecular cri-teria.Its antifungal susceptibility pattern was tested using the EUCAST in vitro microdilution method in liquid medium. Morphological identi?cation

Macroscopic features

Macroscopic features were dense and woolly colonies that grew within?ve days,initially whitish,rapidly becoming dark green.

Microscopic features

Microscopic features included:conidiophores with branching pattern at right angles;phialides(8—10m m?2m m),?ask-shaped often curved in whorls of two,three or four verti-cillate;conidia(3.5m m?4m m)dark green sub-globose, short ellipsoidal,smooth-walled;chlamydospores were pre-sent(Fig.1).

Molecular identi?cation

A861bp DNA sequence of our isolate shared100%identity with the AF278796sequence of T.atroviride strain ATCC 36042.

Antifungal susceptibility testing

This T.atroviride isolate exhibited the following MIC(mg/l): amphotericin B:1;

5-?uorocytosine:!64;

?uconazole:!64;

itraconazole:!8;

voriconazole:8;

caspofungin:0.5.

Discussion

The susceptible hosts’reservoir of opportunistic molds widens concomitantly to the increasing number of severely immunocompromised patients such as transplant recipients. With an increasing number of invasive infections reports, molds of the genus Trichoderma can be added to the growing list of emerging human pathogens[1].Trichoderma spp.are free-living?lamentous fungi that are common in soil and root ecosystems.T.atroviride(teleomorph:Hypocrea atroviri-dis)is an opportunistic,avirulent plant symbiont used as a biological control agent because it parasitizes a variety of phytopathogenic fungi[4].We report on the?rst isolation of T.atroviride from a human patient.

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

a Figure1Slide culture of Trichoderma atroviride.A.Flask-shaped phialides in verticils(?400).B.Sub-globose smooth-walled conidia(?1000).

Culture sur lame de Trichoderma atroviride.A.Verticilles de phialides en forme de bouteille(?400).B.Conidies sub-globuleusesàparoi lisse(?1000).

Trichoderma atroviride in a liver biopsy235

contamination of the patient’s sample is unlikely because Trichoderma spp.are seldom isolated from our hospital’s environment and T.atroviride has not been previously cultu-red in our laboratory.There was no de?nitive evidence in favor of a disseminated infection in this patient,and the overall pathological importance of this mold infection can only be conjectured with respect to the complex clinical presentation of this patient who ultimately died.A retro-spective analysis of the patient’s?le revealed no particular expositional risk to Trichoderma spp.or any other mold other than immunosuppression.

In a comprehensive review of the literature,Chouaki et al.found22human infections caused by Trichoderma spp[1].Recently,De Miguel at al.reported another case of Trichoderma viride infection[2].Including one personal observation of a fatal Trichoderma longibrachiatum(NRCMA No200300360)cholecystitis and peritonitis treated with voriconazole and caspofungin in a70-year-old man who underwent splenectomy to diagnose a Castelman’s disease and the present T.atroviride infection,brings to24the reports of Trichoderma spp.infections in humans to date.Six species:Trichoderma harzianum,Trichoderma koningii,Tri-choderma longibrachiatum,Trichoderma pseudokoningii, Trichoderma citrinoviride,and T.viride have been identi?ed as etiologic agents of infections in immunocompromised hosts or in continuous ambulatory peritoneal dialysis associated peritonitis[1].In this report,a seventh species, T.atroviride,is implicated for the?rst time.We may anti-cipate that emergent Trichoderma spp.infections will conti-nue to develop in the settings of permissive environmental conditions,selective antifungal pressure and an expanding population of immunocompromised hosts.

References

[1]Chouaki T,Lavarde V,Lachaud L,Raccurt CP,Hennequin C.

Invasive infections due to Trichoderma species:report of2 cases,?ndings of in vitro susceptibility testing,and review of the literature.Clin Infect Dis2002;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 of one case and review of the literature.Diagn Microbiol Infect Dis 2005;53:33—7.

[3]Gams W,Bissett J.Morphology and identi?cation of

Trichoderma.In:Kubicek CP,Harman GE,editors.Trichoderma and 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.Trichoderma

species-opportunistic,avirulent plant symbionts.Nat Rev Micro-biol2004;2:43—56.

236S.Ranque et al.