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Subcutaneous Mycosis Produced by Aureobasidium Pullulans Var

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Subcutaneous Mycosis Produced by Aureobasidium Pullulans Var 1 Subcutaneous mycosis produced by Aureobasidium pullulans var. 2 melanogenum in a diabetic old man 3 Bai Shuang2, Li Ping1, Lian Cuihong3, Zhang Zhenying1 4 5 1,HongKong University Shenzhen Hospital 6 2, The Affiliated Hospital of Inner Mongolia Medical University 7 3,Shenzhen Second People’s Hospital 8 9 Correspondence to： 10 Zhenying Zhang, Dermatology Department, HongKong University Shenzhen Hospital. Email: 11 [email protected]. 12 Lian Cuihong, Dermatology Department, Shenzhen Second People’s Hospital . 13 Email:[email protected] 14 15 Aureobasidium pullulans is a saprophytic dematiaceous fungus which widely 16 distribute in soil, decaying wood, leaves, house dust and other environment and it's 17 also can be separated from the hair, skin and nails[1-3].There were four varieties of 18 A.pullulans, namely A.pullulans var, pullulans, A, pullulans var.melanogenum, A, 19 pullulans var.subglaciale and A, pullulans var. Namibiae[4]. 20 The separated A. pullulans was generally considered as a contaminant in the past, 21 it's pathogenicity, especially on immunosuppression patients is gradually be recognized 22 recently[1,2,5]. We reported a case of subcutaneous infection caused by Aureobasidium 23 pullulans var. melanogenum in a patients with diabetes and cases of A. pullulans 24 subcutaneous infection were also reviewed. 25 Case Report 26 A 72-year-old man complained of painful bump with purulent exudation on his 27 left ankle for8 months after local trauma. The antibiotic ointment was apply to the 28 lesion by himself for 2 weeks without any response but aggravation. On physical 29 examination,an infiltrated plaque 5cm in diameter was identified on his left extensor 30 ankle. The plaque with surrounding dark erythema was covered bypurulent exudate 31 and scab. Local skin temperature is high and the tenderness was obvious. Skin biopsy 32 specimens were taken for histopatholigic analysis, microbiological cultures for 33 bacteria, mycobacteria, fungi and PCR analysis. Histological examination disclosed 34 granuloma formation, consisting of epithelial cell clusters, giant cells,lymphocytes 35 and plasma cells in the dermis and the subcutaneous tissue. Tests for bacteria and 36 mycobacteria yield negative results. The cultures on Sabouraud glucose agar grew the 37 velvety, yellow colonies with surrounding inoculation point of dark pigment after 5 38 days and became completely black after 10 days . Microscopic examination of slide 39 culture for 5 days revealed multicellular filamentous hyphal structures of varying 40 accompanied by budding ellipsoidal yeast-like cells. All characteristic features of A. 41 pullulans, hyaline septate hyphae, clusters of hyaline blastoconidia , and thick-walled 42 pigmented arthroconidia were appeared in the slide culture incubated for 10 days. 43 DNA extraction from the tissues was performed following the method we have 44 described previously[6].The nucleotide sequences of the internal transcribed spacer 45 (ITS) regions and 5.8S of rRNA genes of the sample were amplified using the 46 ITS1-ITS4 primer pair. The 581bp fragment of PCR product showed 100% identity to 47 the sequence of Aureobasidium pullulans var. Melanogenum. The sequence has 48 submitted to GenBank and the No. is MK 307635.1. The sequences of A. 49 melanogenum, A,namibiae, A. pullulans, A.subglaciale, A. caulivorum and 50 A.microstictum isolates were obtained from Genbank for phylogenetic analysis using 51 MEGA software[7].(Figure 5 ) . Screening for human immunodeficiency virus, 52 hepatitis B virus, hepatitis C virus and auto antibody were all negative. X-ray and 53 vascular ultrasound of lower limbs were normal.The plaque subsided gradually with a 54 mild atrophic scar after treatment with itraconazole at 400mg/ day for 12 weeks. The 55 patients were followed up for 2 years without relapse. 56 Discussion 57 A.pullulans is classified among dematiaceous fungi because of its capacity to 58 produce melanin-like pigment that may act as virulence factors inhibiting 59 phagocytosis by the host’s immune system[1,2,8]. Despite its limited pathogenicity, 60 peritonitis in patients undergoing peritoneal dialysis, pulmonary mycosis, splenic and 61 mandibular abscess, meningitis, sepsis , keratomycosis, as well as cutaneous and 62 subcutaneous infections have been described[1,3,5,9]. Subcutaneous involvment is rare, 63 and table 1 summarizes the cases of subcutaneous mycosis caused by A. pullulans on 64 the basis of an extensive search of the literature we could find. A. pullulans, like the 65 other dematiaceous fungi, may produce two types of subcutaneous mycosis, 66 chromoblastomycosis and phaeohyphomycosis depending on the configuration of 67 fungi in the skin tissue[12]. Phaeohyphomycosis contains dematiaceous yeast-like cells 68 and hyphae,while chromoblastomycosis is characterized by the presence of 69 thick-walled septate round sclerotic bodies[12]. In our case, morphological distinction 70 is difficult because neither sclerotic bodies nor hyphae has been confirmed 71 microscopically. However, the macroscopic and microscopic analysis of colonies, in 72 combination with molecular identification support the diagnosis of subcutaneous 73 mycosis infected by Aureobasidium pullulans var. Melanogenum. Most clinical 74 isolates was identified as A.pullulans without detailed species variety. So far, only 75 seven clinical A. pullulans var. melanogenum isolates including the present isolate 76 and one A.pullulans var.pullulans clinical isolate were sequenced and available in 77 GenBank, which indicated A. pullulans var. melanogenum might has a higher 78 pathogenic potency. 79 Several conditions can predispose patient to infection by fungal saprophytes, 80 including immunosuppression,which can be due to cancer chemotherapy or organ 81 transplantation, the broad and extensive spectrum of antimicrobial therapy, and the 82 distribution of natural barriers, which occurs during major surgery or trauma[1]. The 83 traumatic injuries experienced by this patient might led to accidental inoculation of 84 the fungus in to the ankle of the host. Hyperglycemia-related impairment of immune 85 response and vascular insufficiency might contribute to the progressive growth of the 86 lesion.To our knowledge,this is the first case of subcutaneous infection caused by A. 87 pullulansin diabetic patient. 88 Antifungal treatment for A. pullulans include amphotericillin B, itraconazole and 89 fluconazole,etc[1]. The protocols of oral fluconazole and itraconazole for 4-8 weeks 90 were widely used in the patient with cutaneous or subcutaneous infection on account 91 of less side effects of drugs and weak virulence of A. pullulans[1]. The prognosis of A. 92 pullulans infections vary from complete recovery to death depending on the extent of 93 infections and host conditions. Considering the deep lesions, long duration and 94 concomitant diabtetes, our patient was prescribed to oral itraconazole per day for 12 95 weeks. No recurrence occurred during the 2-years follow up. 96 Clinicians must be aware of these weak, opportunistic pathogens as a cause of chronic 97 infections even in the immunocompetents. Molecular identification of pathogen is 98 vital for these rare opportunistic skin infection. 99 100 Acknowledgements 101 This manuscript was supported by the National Natural Science Foundation of China 102 (Grant no. 81472891). 103 References: 104 1. Oliveira LR,Moraes-Souza H,Maltos AL,Santos KC,Molina RJ,Barata CH. 105 Aureobasidium pullulans infection in a patient with chronic lymphocytic leukemia. 106 Revista da Sociedade Brasileira de Medicina Tropical.2013,46(5)：660-662. 107 2. Bolignano G; Criseo G. Disseminated nosocomial fungal infection by 108 Aureobasidium pullulans var. melanigenum: a case report. Journal of Clinical 109 Microbiology.2003,41(9)：4483-4485. 110 3.Mahesh E , Vijay V P , Rakesh M , et al. Unusual Fungal Infections in Renal 111 Transplant Recipients. Case Reports in Transplantation, 2015, 2015:1-4. 112 4. Chen W T , Tu M E , Sun P L . Superficial Phaeohyphomycosis Caused by 113 Aureobasidium melanogenum Mimicking Tinea Nigra in an Immunocompetent 114 Patient and Review of Published Reports. Mycopathologia, 2016, 181(7-8):555-560. 115 5. Joshi, A Singh, R Shah, M S Umesh, S Khattry, N. Subcutaneous mycosis 116 and fungemia by Aureobasidium pullulans: a rare pathogenic fungus in a post 117 allogeneic BM transplant patient. Subcutaneous mycosis and fungemia by 118 Aureobasidium pullulans: a rare pathogenic fungus in a post allogeneic BM transplant 119 patient.Bone marrow transplantation.2010, 45(1). 120 6. Liu Xiaoming,Zhang Zhenying, Hou Binbin. Rapid identification of Sporothrix 121 schenckii in biopsy tissue by PCR. J Eur Acad Dermatol Venereol. 122 2013,27(12):1491-7 123 7. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S MEGA6: molecular 124 evolutionary genetics analysis, version 6.0.; 2013. 125 8. Nalcacioglu H , Yakupoglu Y K , Genc G , et al. Disseminated fungal infection by 126 Aureobasidium pullulans in a renal transplant recipient. Pediatric Transplantation, 127 2018, 22(3):e13152. 128 9. Huang Y T , Liaw S J , Liao C H , et al. Catheter-related septicemia due to 129 Aureobasidium pullulans. International Journal of Infectious Diseases, 2008, 130 12(6):e137-e139. 131 10. RedondoBellon P;Idoate M;Rubio M;IgnacioHerrero J.Chromoblastomycosis 132 produced by Aureobasidium pullulans in an immunosuppressed patient. Archives of 133 Dermatology.1997,133(5)：663-664. 134 11. Fernandes H , Pinto A C , Dias M , et al. Fungal foot abscess
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