The genus Alternaria is one of the most common black moulds and appears to be increasing as a causative agent of subcutaneous phaeohyphomycosis, particularly among immunosuppressed patients. A 53-year-old patient who had received a kidney transplant presented with multiple verrucous lesions on the distal extremities. Positive histopathology and cultures, in addition to rDNA ITS region sequencing, identified the fungal isolate as Alternaria infectoria. Oral itraconazole was administered for 10 months. A follow-up at 15 months demonstrated no signs of infection. Clinical manifestations of cutaneous alternariosis vary significantly and only a few cases have been described in the literature. Although optimal treatment options remain controversial, this case of phaeohyphomycosis was successfully treated with itraconazole monotherapy

Afonso, A

Amaro, C

Cardoso, J

Cunha, D

Inácio, J

Maduro, AP

Marques Pinto, G

Martins, ML

Vieira, MR

Repository of the Hospital Center of Central Lisbon

Feohifomicosis Producida por Alternaria Infectoria con Presentación Clínica de Múltiples Lesiones Vegetantes en un Paciente Sometido a un Trasplante Renal

Repositório do Centro Hospitalar de Lisboa Central, EPE

NPvDJabcdaARAAKMKIIPMTIPab1dRev Iberoam Micol. 2012;29(1):44–46Revista  Iberoamericanade  Micologíawww.elsev ier .es / rev iberoammicolotehaeohyphomycosis  caused  by  Alternaria  infectoria  presenting  as  multipleegetating  lesions  in  a  renal  transplant  patientaniela  Cunhaa,∗,  Cristina  Amaroa, María  Raquel  Vieiraa, María  da  Luz  Martinsb,  Ana  Paula  Madurob,oão Inácioc,  Ana  Afonsod,  Gabriela  Marques  Pintoa,  Jorge  CardosoaDermatology Department, Hospital de Curry Cabral, Lisbon, PortugalMycology Laboratory, Instituto de Higiene e Medicina Tropical/CREM, Universidade Nova de Lisboa, Lisbon, PortugalLaboratório Nacional de Investigac¸ ão Veterinária, Instituto Nacional de Recursos Biológicos, Lisbon, PortugalHistopathology Department, Hospital de Curry Cabral, Lisbon, Portugal r  t  i  c  l  e  i  n  f  orticle history:eceived 1 December 2010ccepted 22 June 2011vailable online 7 July 2011eywords:ycosisidney transplantationtraconazolemmunocompromised patienta  b  s  t  r  a  c  tThe  genus  Alternaria  is  one  of the  most  common  black  moulds  and  appears  to  be  increasing  as  a  causativeagent of  subcutaneous  phaeohyphomycosis,  particularly  among  immunosuppressed  patients.A 53-year-old  patient  who  had  received  a kidney  transplant  presented  with  multiple  verrucous  lesionson the  distal  extremities.  Positive  histopathology  and  cultures,  in addition  to  rDNA  ITS region  sequencing,identiﬁed  the  fungal  isolate  as Alternaria  infectoria.  Oral  itraconazole  was  administered  for  10  months.  Afollow-up  at 15 months  demonstrated  no signs  of  infection.Clinical  manifestations  of  cutaneous  alternariosis  vary  signiﬁcantly  and  only  a  few  cases  have  beendescribed  in the  literature.  Although  optimal  treatment  options  remain  controversial,  this  case  of phaeo-hyphomycosis  was  successfully  treated  with  itraconazole  monotherapy.© 2010  Revista  Iberoamericana  de Micología.  Published  by  Elsevier  España,  S.L.  All rights  reserved.Feohifomicosis  producida  por  Alternaria  infectoria  con  presentación  clínica  demúltiples  lesiones  vegetantes  en  un  paciente  sometido  a  un  trasplante  renalalabras clave:icosisrasplante renaltraconazolaciente inmunocomprometidor  e  s  u  m  e  nLas  especies  del  género  Alternaria  son  mohos  ubicuos  que  se  caracterizan  por presentar  colonias  de  colorgris;  parecen  ser  cada  vez  más  frecuentes  como  agente  causal  de  las feohifomicosis  subcutáneas,  enespecial  entre  pacientes  inmunocomprometidos.Un paciente  de  53 an˜os  de  edad,  que  se había  sometido  a  un  trasplante  renal,  consultó  por  presentartumores  verrugosos  en  el tercio  distal  de  las  extremidades.  La  histopatología  y  cultivo  positivos,  ademásde la  secuenciación  de  la  región  de transcripción  interna  ITS  del rDNA,  permitieron  la  identiﬁcacióndel  aislamiento  fúngico  como  Alternaria  infectoria.  Se instauró  tratamiento  con  itraconazol  oral  durante10 meses  y,  a  los 15  meses  de  seguimiento,  se  demostró  la  ausencia  de  signos  de  infección.Las  manifestaciones  clínicas  de la  alternariosis  cutánea  son  muy  variables  y  tan  sólo  se  han  descritounos  pocos  casos  en los  que se  presenta  en  forma  de  tumoraciones  verrugosas.  Aunque  el  tratamientode  elección  sigue  siendo  motivo  de controversia,  el paciente  presentado  en este  estudio  fue tratadosatisfactoriamente  con  itraconazol.© 2010  Revista  Iberoamericana  de  Micología.  Publicado  por  Elsevier  España,  S.L.  Todos  los  derechosSubcutaneous phaeohyphomycosis is an infection caused byn heterogeneous group of dematiaceous fungi.15 Infectionsy species of Alternaria accounted for a signiﬁcant number of cases∗ Corresponding author.E-mail address: danielaccunha@gmail.com (D. Cunha).130-1406/$ – see front matter © 2010 Revista Iberoamericana de Micología. Published boi:10.1016/j.riam.2011.06.010reservados.reported in recent years.2,3,5,6,9,11,12,14,16,18 Members of this genusare generally found in aerosols, in soil, or on plants, and are typ-ically pathogenic only to immunocompromised hosts.2,3,5,6,8–16,18Nevertheless, a few reports describing cutaneous infections amongimmunocompetent individuals have also been described.17The development and reﬁnement of modern molecularbiologic techniques have lead to a better genotypic characterizationy Elsevier España, S.L. All rights reserved.D. Cunha et al. / Rev Iberoam Micol. 2012;29(1):44–46 45ordCtenuetlbanvwm1pmwrsocbsbiawfwhdwf(ap(cers have also been reported. Among the Alternaria speciesresponsible for opportunistic infections in organ recipient patients,A. infectoria accounts for only a minority of cases reported over thepast decade.2,3,5,6,9,11,12,14,16,18Figure 1. Widespread verrucous hyperkeratotic tumours.f dematiaceous species associated with phaeohyphomycosis,esulting in a better understanding of these infections over the pastecade.ase reportA 53-year-old male gardener presented with multiple asymp-omatic vegetating hyperkeratotic lesions and nodules on the distalxtremities that had evolved over the past two months. There waso previous history of skin lesions or trauma. The patient hadndergone kidney transplantation 16 months previously, due tond-stage renal disease of unknown etiology. Since transplantation,he patient was treated with tacrolimus plus mofetil mycopheno-ate and prednisolone and, at presentation, the dosages were 6 mgid, 750 mg  bid and 10 mg  o.d., respectively.On examination, there were widespread, multiple keratoticnd vegetating tumours, as well as several purplish non-painfulodules on the forearms, dorsum of the hands and shins. Lesionsaried in size but a few reached 3.0 cm in diameter (Fig. 1). Thereas no edema or lymphadenopathy and vital signs were nor-al. The patient was treated with itraconazole 400 mg  o.d. for0 months and the tacrolimus dose was adjusted accordingly. Com-lete resolution of skin lesions was achieved 9 months after treat-ent and itraconazole was administered for 4 weeks more. Thereas no recurrence 15 months after completion of the treatment.Routine laboratory tests, including renal function and chestadiography, were unremarkable. Histopathology of the skin biopsyhowed a granulomatous inﬁltrate with septate hyphae andval-shaped structures following periodic acid-Schiff and Gro-ott’s staining (Fig. 2). Direct examination of portions of theiopsy performed with 20% potassium hydroxide solution revealedeveral hyaline septate hyphae. For isolation, the remainder of theiopsy was inoculated onto Sabouraud dextrose agar, Brain–heart-nfusion agar and Malt agar (DIFCO®, Detroit, USA) and incubatedt 24 ◦C and 37 ◦C. After seven days incubation, fungal coloniesere discernable on the respective media. By day 21, the sur-ace of the colonies was  gray-white and cottony and the reverseas dark-brown. Microscopic examination revealed septate, brownyphae with simple or branched conidiophores and multiple coni-ia. Conidia were mainly ovoid but some were muriform, smoothalled or verrucous, and a few ended with apical beaks, mostorming acropetal chains (Fig. 3). After DNA extraction, the ITSinternal transcribed spacer) region of rDNA was PCR-ampliﬁednd sequencing of the ITS region was carried out using forwardrimer ITS1 and reverse primer ITS4 following standard protocolsSTAB Vida Lda, Caparica, Portugal). The nucleotide sequence wasFigure 2. Histopathological examination with Grocott’s stain (magniﬁcation ×400).deposited in GenBank with the accession number HM467255. Stan-dard BLAST searches at GenBank database revealed a high similaritybetween this sequence and others representing Alternaria species(which are assigned in GenBank as Lewia, the designation of therespective teleomorphs). A phylogenetic tree was  computed usingPAUP software (Sinauer Associates, Inc., Sunderland, MA), includingITS sequences belonging to reference strains of Alternaria species,and to other closely related fungi, retrieved from GenBank (datanot shown). This phylogenetic analysis placed our fungal isolate inthe Alternaria infectoria species-group1 and the 99.6% nucleotideidentity with the type strain of A. infectoria, CBS 112250T, furtherclassiﬁed the isolate as this species.DiscussionCutaneous infections caused by Alternaria spp. were rarelyreported prior to the use of highly potent immunosuppressivedrugs in solid organ transplantation. These types of infections, how-ever, are likely to become more frequent as the number of patientson these drug therapies and their survival time increase. Infec-tions in patients with underlying immunosuppressive disease, suchas diabetes mellitus, Cushing’s syndrome, or haematological can-8,10,13Figure 3. Microscopic examination following lactophenol blue staining (magniﬁca-tion ×1500).4 oam MaialbtssrcofsTmFiawcoiginssrrcslcaszB2ttit6 D. Cunha et al. / Rev IberThe spectrum of cutaneous manifestations resulting fromlternariosis is broad and includes papules, tumours, and ulcerat-ng lesions. There is no correlation between any given presentationnd a speciﬁc causal agent among Alternaria species. Neverthe-ess, the great majority of the reported cutaneous infections causedy A. infectoria presented as asymptomatic nodules distributed athe extremities.2,3,5,6,12,18 The anatomic distribution of the lesionseems to result from direct inoculation of the fungus into damagedkin following contact with a contaminated fomite or plant.4As A. infectoria is a common saprophyte prevalent in the envi-onment inhabiting primarily leaves and kernels of grasses andereals,14 fungi may  be present at the superﬁcial epidermal layersf human skin, representing environmental contaminants. There-ore, when phaeohyphomycosis is suspected, a deep tissue samplehould be obtained for isolation and identiﬁcation of the fungus.4he morphological ﬁndings of cutaneous alternariosis include der-al  granulomatous inﬁltrates with variable epidermal changes.ungal elements are usually seen within these inﬁltrates, present-ng as spheroidal chlamydospore-like structures and hyphae, withnd without pigmentation.6,7 Fungal culture on Sabouraud agarithout cycloheximide yields “greyish”, “green, powdery” or “felty”olonies. By lactophenol cotton blue staining, the microscopicbservation of pigmented conidiophores giving rise to dictyosporess highly suggestive of A. infectoria. The apical growth of longeniculate secondary conidiophores is a typical feature of the A.nfectoria species-group.Molecular studies may  provide additional time-efﬁcient diag-ostic information. Comparative analysis of rDNA ITS nucleotideequences is commonly used for the identiﬁcation of fungalpecies,4 including Alternaria spp.12,14,16 This approach led to aapid and accurate identiﬁcation of the isolate described in thiseport that was  otherwise difﬁcult to identify using traditionalulture-based mycologic methods.Different treatment options for this infection are availableince no standard treatment has been deﬁned. Small, isolatedesions may  be successfully treated by simple surgical excision14 orryosurgery.2 Multiple and larger lesions usually require systemicntimycotic therapy. On occasion, a combination of surgical andystemic antinfungal agents has proved efﬁcacious.7,16 Itracona-ole is the most commonly used drug, followed by amphotericin.3–5,9 Itraconazole doses used range between 100 and 400 mg  for–8 months in monotherapy regimens.3,6,7This case report is remarkable due to its clinical presenta-ion with multiple nodules and vegetating tumours as well ashe slow but successful response to monotherapy with high-dosetraconazole. Furthermore, it illustrated the efﬁcacy of molecularechniques in Alternaria species identiﬁcation.icol. 2012;29(1):44–46Conﬂict of interestThe authors have nothing to declare.References1. Andersen B, Sørensen JL, Nielsen KF, Gerrits van den Ende B, de Hoog S. Apolyphasic approach to the taxonomy of the Alternaria infectoria species–group.Fungal Genet Biol. 2009;46:642–56.2. Ara M,  Aspiroz C, Zaballos P, Alvarez R, Rezusta A, Giménez JA. Relapse of cuta-neous Alternaria infectoria in a renal transplant recipient after 2 years. ActaDerm Venereol. 2006;86:154–5.3. Brasch J, Busch JO, de Hoog GS. Cutaneous phaeohyphomycosis caused byAlternaria infectoria. Acta Derm Venereol. 2008;88:160–1.4.  Dubois D, Pihet M,  Clec’h CL, Croué A, Beguin H, Bouchara JP, et al.Cutaneous phaeohyphomycosis due to Alternaria infectoria. Mycopathologia.2005;160:117–23.5. Gallelli B, Viviani M,  Nebuloni M,  Marzano AV, Pozzi C, Messa P, et al.Skin infection due to Alternaria species in kidney allograft recipients:report of a new case and review of the literature. J Nephrol. 2006;19:668–72.6.  Gerdsen R, Uerlich M,  De Hoog GS, Bieber T, Horré R. Sporotrichoidphaeohyphomycosis due to Alternaria infectoria. Br J Dermatol. 2001;145:484–6.7. Gilaberte M,  Bartralot R, Torres JM,  Reus FS, Rodríguez V, Alomar A, et al. Cuta-neous alternariosis in transplant recipients: clinicopathologic review of 9 cases.J  Am Acad Dermatol. 2005;52:653–9.8. Guerin V, Barbaud A, Duquenne M,  Contet-Audonneau N, Amiot F,Ortega F, et al. Cushing’s disease and cutaneous alternariosis. Arch Intern Med.1991;151:1865–8.9.  Halaby T, Boots H, Vermeulen A, van der Ven A, Beguin H, van Hooff H, et al.Phaeohyphomycosis caused by Alternaria infectoria in a renal transplant recip-ient. J Clin Microbiol. 2001;39:1952–5.10. Ioannidou D, Maraki S, Krüger Krasagakis S, Stefanidou M,  Krasagakis K,Alexandrakis M,  et al. Cutaneous alternariosis revealing acute myeloidleukaemia in an adult patient. Mycoses. 2004;47:227–30.11. Laumaillé C, Le Gall F, Degeilh B, Guého E, Huerre M.  CutaneousAlternaria infectoria infection after liver transplantation. Ann Pathol. 1998;18:192–4.12. Lo Cascio G, Ligozzi M,  Maccacaro L, Fontana R. Utility of molecular identi-ﬁcation in opportunistic mycotic infections: a case of cutaneous Alternariainfectoria infection in a cardiac transplant recipient. J Clin Microbiol.2004;42:5334–6.13. Lyke KE, Miller NS, Towne L, Merz WG.  A case of cutaneous ulcerative alternar-iosis: rare association with diabetes mellitus and unusual failure of itraconazoletreatment. Clin Infect Dis. 2001;32:1178–87.14. Nulens E, De Laere E, Vandevelde H, Hilbrands LB, Rijs AJ, Melchers WJ,  et al.Alternaria infectoria phaeohyphomycosis in a renal transplant patient. MedMycol. 2006;44:379–82.15. Porto E, Martins JE, Heins-Vaccari E. Feo-Hifomicoses. In: Porto E, Martins JE,Heins-Vaccari E, et al., editors. Tratado de Micologia Médica. São Paulo: Sarvier;2002. p. 519–61.16. Segner S, Jouret F, Durant JF, Marot L, Kanaan N. Cutaneous infectionby  Alternaria infectoria in a renal transplant patient. Transpl Infect Dis.2009;11:330–2.17.  Sood N, Gugnani HC, Guarro J, Paliwal-Joshi A, Vijayan VK. Subcutaneous phaeo-hyphomycosis caused by Alternaria alternata in an immunocompetent patient.Int J Dermatol. 2007;46:412–3.18. Vieira R, Veloso J, Afonso A, Rodrigues A. Cutaneous alternariosis in a livertransplant recipient. 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Feohifomicosis Producida por Alternaria Infectoria con Presentación Clínica de Múltiples Lesiones Vegetantes en un Paciente Sometido a un Trasplante Renal

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