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A rare case of subcutaneous phaeohyphomycosis caused by a Rhytidhysteron species: a clinico-therapeutic experience Vikram K. Mahajan1, MD, Vikas Sharma1, MBBS, Neel Prabha1, MBBS, Kamlesh Thakur2, MD, Nand Lal Sharma1, MD, Shivaprakash M. Rudramurthy3, MD, Pushpinder S. Chauhan1, MD, Karaninder S. Mehta1, MD, and C. Abhinav1, MBBS
1 Departments of Dermatology, Venereology and Leprosy, 2Microbiology, Dr Rajendra Prasad Government Medical College, Kangra at Tanda, Himachal Pradesh, and 3 Mycology Division, Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
Correspondence Vikram K. Mahajan, MD Department of Dermatology, Venereology and Leprosy Dr R. P. Government Medical College Kangra at Tanda 176001, Himachal Pradesh, India Tel: + 91 1892 287161 Fax: + 91 1892 267115 E-mail: [email protected]
Abstract Background Subcutaneous phaeohyphomycosis usually results from traumatic inoculation with the fungus and generally occurs in immunosuppressed men. Cladosporium, Exophiala, and Alternaria spp. are commonly implicated pathogens. Objectives We present a case of subcutaneous phaeohyphomycosis caused by Rhytidhysteron sp. that was refractory to conventional antifungal therapy. Case report A 72-year-old man with hypertension and diabetes presented with a multiloculated, large cystic swelling over the right dorsal foot. Laboratory findings and x-rays of the chest and left foot were normal. Results Adequate control of the patient’s diabetes was achieved, and the swelling was excised under itraconazole/terbinafine coverage. Histology showed multiple areas of neutrophilic abscess, epithelioid cells, foreign body giant cells, and multiple septate hyphae and yeast-like cells. Dematiaceous fungus was cultured but failed to produce spores. Sequencing of the isolate showed a match of > 99% with Rhytidhysteron rufulum. The patient demonstrated no response after one year of therapy with itraconazole/terbinafine. Weekly infiltration of the lesion with liposomal amphotericin B resulted in its complete resolution within 15 weeks.
Funding: None. Conflicts of interest: None doi: 10.1111/ijd.12529
Conclusions Lesions of phaeohyphomycosis appear morphologically similar regardless of the organism implicated. Hence, their diagnosis rests entirely on the clinicopathological and microbiological presentation. Molecular studies may be required to identify a fungus if attempts to grow it in artificial culture media fail. Rhytidhysteron spp. are not known as pathogens in humans, and no treatment protocol exists. Intralesional amphotericin was highly effective in our patient and caused no systemic adverse effects. Voriconazole and posaconazole are effective against disseminated/visceral phaeohyphomycotic infections, but their efficacy against Rhytidhysteron spp. remains unstudied.
Introduction The nomenclature phaeohyphomycosis was introduced by Ajello et al.1 in 1974 to describe cutaneous and systemic or disseminated mycoses, including cerebral phaeohyphomycosis, caused by a variety of dematiaceous fungi, which, on clinical, pathological, and mycological grounds, are distinct from chromoblastomycosis. The infection has a wide geographic distribution across all climatic conditions and has been reported in both the extreme south and the extreme north of India, the climatic conditions of which range from tropical to temperate.2 Subcutaneous phaeohyphomycosis usually results from the traumatic inoculation of the fungus or following wound contamination and occurs mostly in adult males ª 2014 The International Society of Dermatology
(aged ≥ 30 years) with some degree of immunosuppression resulting from disease (tuberculosis, diabetes, human immunodeficiency virus [HIV] infection/acquired immunedeficiency syndrome [AIDS], hematological malignancies, or other neoplasia) or iatrogenic causes (organ transplantation, corticosteroids, or immunosuppressive therapies).3 Its manifestation begins with a usually single, slowly enlarging but asymptomatic or mildly painful subcutaneous nodule on the feet, legs, or hands in 60–85% of cases.2–4 These evolve into painless cystic abscesses full of purulent material encapsulated in fibrous connective tissue and have little tendency to rupture. However, papulonodules, verrucous or ulcerated plaques, non-healing ulcers or sinuses, or scaly hyperkerototic lesions resembling chromoblastomycosis are not uncommon, although International Journal of Dermatology 2014
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Subcutaneous phaeohyphomycosis caused by a Rhytidhysteron sp.
all clinical forms show similar features on histopathology irrespective of the etiological agent. More than 100 different saprophytes of soil, wood and plant detritus, classified in 60 different genera, have been implicated as etiological agents of phaeohyphomycosis. Whereas moulds from the genera Cladosporium and Exophiala are commonly identified pathogens, Alternaria spp. are emerging as a cause of infections in both normal and immunocompromised hosts.5–10 To date, Rhytidhysteron spp. (Ascomycota, Patellariales: Patellariaceae) have not been recognized as infecting humans. However, we describe a case of subcutaneous phaeohyphomycosis caused by a Rhytidhysteron sp. which responded to intralesional amphotericin B and appears to be the first of its kind to be reported. Case report A 72-year-old, HIV-negative man presented with a large, soft, slowly progressive, painless swelling over the right foot of one year’s duration. Over the previous three months, the swelling had developed slight redness and sinuses that occasionally discharged hemorrhagic and purulent material. The patient had no history of prior trauma or manipulation of the swelling. He had been treated for type 2 diabetes mellitus for the past 18 years and for hypertension for 15 years. He had poor diabetic control (his HbA1C value was 7.6). He was taking itraconazole (200 mg twice per day) and terbinafine (250 mg/day)
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which had been prescribed six months previously when fine needle aspiration cytology showed branching septate hyphae within and outside the giant cells in an acute on chronic inflammatory background, suggesting deep fungal infection. Cutaneous examination showed an ill-defined, multiloculated, soft and cystic, non-tender swelling over the dorsum of the right foot. The overlying skin had mild erythema and few sinuses with crusts (Fig. 1a). A small swelling of similar morphology was also present over the sole. The patient’s hair, nails, mucous membranes, and other systemic findings were essentially normal. His blood pressure was within the normal range, and daily fasting blood sugar levels varied between 117 mg/dl and 149 mg/dl. Other laboratory investigations including complete blood counts, hepatorenal function tests, serum electrolytes, urinalysis, and chest x-ray revealed no abnormality. An x-ray of the right foot showed corresponding soft tissue swelling and normal underlying bones. Adequate control of the subject’s diabetic status was achieved, and the swelling was surgically excised under itraconazole/terbinafine coverage. The specimen was subjected to histopathology, culture on Sabouraud's glucose agar (SDA) for fungus and L€ owenstein–Jensen medium for Mycobacterium tuberculosis. Pending investigations, treatment with a saturated solution of potassium iodide (SSKI) was added to the itraconazole/ terbinafine combination but could not be continued as the patient developed a flu-like adverse reaction to it. Histology showed multiple areas of neutrophilic abscess bounded
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Figure 1 (a) A 72-year-old man demonstrates poorly defined, multiloculated, mild erythematous, cystic swellings, sinuses and crusts over the right dorsal foot at presentation (after > 6 months of itraconazole/terbinafine). (b) Persistent swelling and discharging sinuses are evident at 6 months after excision (1 year after itraconazole/terbinafine treatment). (c) The swelling and discharge are remarkably reduced 4 weeks after weekly infiltration with liposomal amphotericin B. (d) The phaeohyphomycotic swelling is completely resolved 6 months after the completion of amphotericin B treatment International Journal of Dermatology 2014
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Subcutaneous phaeohyphomycosis caused by a Rhytidhysteron sp.
by epithelioid cells and foreign body giant cells, and multiple, broad, septate, irregularly branched, dematiaceous hyphae, toruloid hyphae (chains of yeast cells), and yeastlike cells that were periodic acid-Schiff (PAS) positive (Fig. 2). Culture of biopsy tissue on SDA with or without cycloheximide showed the growth of multiple colonies of dematiaceous fungus at 28 °C after two weeks. Initially, colonies were light gray–white and velvety and demonstrated brown pigment on the reverse, but incubation for another two weeks resulted in the development of dark pigmentation. Broad, septate, irregularly branched dematiaceous hyphae and toruloid hyphae were identified on lactophenol cotton blue mounts (Fig. 3). The mould was sent to the Center of Advanced Research and the National Culture Collection of Pathogenic Fungi at the Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India, for identification. The isolate was subjected to morphological identification at the reference center. Despite repeated attempts using various techniques, the isolate failed to produce any conidia. Molecular identification was performed by sequencing various regions such as the 18s rRNA gene (partial), internal transcribed spacer 1 (ITS1), 5.8s, ITS2, and 28s rRNA (partial) using the primer pair ITS1 (5′TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The consensus sequence was prepared using BioNumerics Version 6.6 (Applied Maths, Ghent, Belgium). The consensus sequence was compared against known sequences in the National Center of Biotechnology Information (NCBI) GenBank database using BLAST, and in the Central Bureau of Fungal Cultures (Centraal bureau voor Schimmel cultures, Utrecht, the Nether-
Figure 2 (a) Histology shows multiple areas of neutrophilic abscess bounded by acute on chronic cellular infiltrate, epithelioid cells and foreign body giant cells. Arrows indicate: (b) multiple, broad, septate, irregularly branching dematiaceous hyphae, (c) yeast-like cells, and (d) toruloid hyphae or chains of yeast-like cells. (Hematoxylin and eosin stain; original magnification 940) ª 2014 The International Society of Dermatology
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lands). The sequences of the isolate showed a > 99% and up to 100% similarity with sequences of Rhytidhysteron spp. in both databases. The closest match found refers to Rhytidhysteron rufulum. Treatment outcome
The patient demonstrated no significant improvement after almost one year of therapy with itraconazole and terbinafine. The surgical wound had also developed multiple sinuses discharging purulent material (Fig. 1b). The lesion was infiltrated with 25 ml of liposomal amphotericin B (25 mg/25 ml at a maximum IV dosage of 0.5–1.0 mg/kg/
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Figure 3 (a) Velvety colonies of dematiaceous fungus with brown–black pigmentation appear on prolonged incubation. (b and inset) Lactophenol cotton blue stain staining shows broad, septate, irregularly branching dematiaceous hyphae, and toruloid hyphae (arrows). Note that sporulation is conspicuously absent. (Original magnification 940)
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Subcutaneous phaeohyphomycosis caused by a Rhytidhysteron sp.
day) once per week under aseptic conditions. The swelling and discharge reduced remarkably during the initial four weeks (Fig. 1c). In view of this encouraging response, intralesional liposomal amphotericin 10 ml/week (10 mg/ 10 ml) was infiltrated for the next seven weeks, followed by two injections of 10 mg administered every fortnight. The patient achieved complete resolution of the lesion but developed mild abnormal renal functions (blood urea/creatinine 91/1.49 mg/dl), which improved over the next eight weeks subsequent to his decision to stop the itraconazole and terbinafine. The patient was advised to continue intralesional liposomal amphotericin B at 5 mg/5 ml once per month for another 16 weeks and to attend for regular follow-up. No recurrence has been noted during post-treatment follow-up of > 1 year (Fig. 1d). Discussion Cystic lesions of subcutaneous phaeohyphomycosis are usually well circumscribed and encapsulated granulomas, the walls of which show epithelioid histiocytic inflammatory reaction and giant cells surrounded by connective tissue, the centers of which show necrotic debris, fibrin, and degenerated polymorphonuclear cells. However, the presence of brown-colored fungal hyphae and yeast-like elements with the giant cells or in the background of a granulomatous inflammatory reaction containing central neutrophilic abscess in a hematoxylin and eosin-stained section is pathognomonic.3,11 Planate septa and thickwalled cells are observed occasionally, but the typical copper penny cell, a more consistent feature of chromoblastomycosis, is not seen. Our patient showed all the classic clinicopathological features of phaeohyphomycosis. The various species belonging to the genera Cladosporium (C. cladosporoides), Phialophora (P. richardsiae), Curvularia (C. geniculata, C. lunata, C. pallescens), Exophiala (E. jeanselmei, E. mansonii, E. castellani), Fonsecaea (F. pedrosoi), Wangiella (W. dermatitidis), Cladophialophora (C. banitiana, C. modesta, C. devriesii), and Alternaria (A. infectoria, A. alternata, A. chlamydospora) constitute major pathogens for the disease.2,4,12 The lesions of phaeohyphomycosis appear morphologically similar regardless of the organism implicated. Hence, the diagnosis of phaeohyphomycosis rests entirely on the clinicopathological and microbiological presentation in the absence of commercially available diagnostic serological tests. Most isolates obtained from culture on SDA are identified from colony morphology and microscopic morphology of conidia, their arrangement on the conidiogenous cell, and the morphology of the conidiogenous cell. Additionally, nitrate and carbohydrate assimilation tests, exoantigen tests, and proteolytic activity or growth temperature response are sometimes required in order to identify an isolate, despite their limited International Journal of Dermatology 2014
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application in the identification of dematiaceous fungi.3 Molecular studies may sometimes be necessary to identify a causative fungus if attempts to grow it in artificial culture media fail. The isolates of dematiaceous fungus obtained from the present patient on SDA did not produce spores despite repeated efforts, and Rhytidhysteron sp. was identified by sequencing the ITS1–5.8s and ITS2 regions of the rDNA gene. The genus Rhytidhysteron includes two species: R. rufulum and R. hysterinum. The fungus has a worldwide distribution and occurs particularly in the tropics and subtropics. This poorly known family includes fungi with discoidal ascomata and butinicate asci with darkly pigmented reniform, elliptical, or fusiform and septate ascospores. Rhytidhysteron rufulum is a lignicolus species and produces its spores exclusively in natural substrates, mainly in the wood of living or dead dicotyledonous plants, and develops coelomycete anamorphs in artificial culture media.13 Rhytidhysteron spp. are not known as pathogens in humans and have been implicated only once, by Chowdhary et al.,14 in a case of chromoblastomycosis in a renal transplant patient, in which attempts to induce sporulation also failed. Surgical excision, drainage, debridement, and cryotherapy, maximum doses of antifungal drugs (amphotericin B, 5-flucytosine, ketoconazole, fluconazole, itraconazole), superficial x-ray therapy, and thiabendazole have been used in various permutations and combinations to treat phaeohyphomycosis, with variable degrees of success.4,15,16 Oral itraconazole at 200–400 mg/day or at a dose as high as 800 mg/day, for a period of 4–6 weeks, appears to be effective in phaeohyphomycosis.16 Sharkey et al.17 observed clinical improvement or remission using itraconazole (50–500 mg/day) for 1–48 months in the 50% of their patients in whom earlier treatment with amphotericin B, ketoconazole, or miconazole had failed. The effectiveness of itraconazole against Rhytidhysteron spp. remains unknown as the only patient reported died of multi-organ failure two weeks after therapy with itraconazole (100 mg twice per day) was initiated.14 In the present patient, a combination of itraconazole and terbinafine (for six months prior to surgery and for 19 weeks after surgery in combination with amphotericin B) did not prove useful, and surgical excision helped only to reduce the mass. Although Vieira et al.10 treated a patient with cutaneous phaeohyphomycosis caused by A. alternata with intralesional amphotericin B (1 mL at 1 mg/ml) twice per week for five weeks, there exists no treatment protocol. Intralesional amphotericin was highly effective in our patient and was administered for 35 weeks without causing any systemic adverse effects. Mild renal function test impairment can be attributed to terbinafine (an uncommon ª 2014 The International Society of Dermatology
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Subcutaneous phaeohyphomycosis caused by a Rhytidhysteron sp.
but known side effect) as this improved after the patient discontinued terbinafine, despite the continuation of amphotericin B. We used an initially high dose in order to achieve an early clinical response and a low dose to maintain remission. Voriconazole and posaconazole, in combination with other drugs, have been found to be effective for disseminated/visceral phaeohyphomycotic infections in both humans and experimental animals,18–25 but their efficacy against Rhytidhysteron spp. remains unstudied. Acknowledgment The erudite help and support extended by Dr. Arunaloke Chakrabarti, Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India, in the molecular identification of the isolate is gratefully acknowledged. References 1 Ajello L, Georg LK, Steigbigel RT, et al. A case of phaeohyphomycosis caused by a new species of Phialophora. Mycologia 1974; 66: 490–498. 2 Revankar SG, Patterson JE. Disseminated phaeohyphomycosis: review of an emerging mycosis. Clin Infect Dis 2002; 34: 467–476. 3 Kwon-Chung KJ, Bennett JE. Phaeohyphomycosis. In: Kwon-Chung KJ, Bennett JE, eds. Medical Mycology. Philadelphia, PA: Lea & Febiger, 1992: 620. 4 Sharma NL, Mahajan V, Sharma RC, et al. Subcutaneous phaeohyphomycosis in India – a case report and review. Int J Dermatol 2002; 41: 16–20. 5 Gugnani HC, Sood N, Singh B, et al. Case report: subcutaneous phaeohyphomycosis due to Cladosporium cladosporioides. Mycoses 2000; 43: 85–87. 6 Gonzalez MS, Alfonso B, Seckinger D, et al. Subcutaneous phaeohyphomycosis caused by Cladosporium devriesii, sp. nov. Sabouraudia 1984; 22: 427–432. 7 Qiu-Xia C, Chang-Xing L, Wen-Ming H, et al. Subcutaneous phaeohyphomycosis caused by Cladosporium sphaerospermum. Mycoses 2007; 51: 79–80. 8 Murayama N, Takimoto R, Kawai M, et al. A case of subcutaneous phaeohyphomycotic cyst due to Exophiala jeanselmei complicated with systemic lupus erythematous. Mycoses 2003; 46: 145–148. 9 Silva MRR, Fernandes OFL, Costa CR, et al. Subcutaneous phaeohyphomycosis by Exophiala jeanselmei in a cardiac transplant recipient. Rev Inst Med Trop Sao Paulo 2005; 47: 55–57. 10 Vieira MR, Martins ML, Afonso1 A, et al. Cutaneous alternariosis. Rev Iberoam Micol 1998; 15: 97–99. 11 Ben-Ami R, Lewis RE, Raad II, et al. Phaeohyphomycosis in a tertiary care cancer center. Clin Infect Dis 2009; 48: 1033–1041.
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12 Cooper CR Jr. Deep phaeohyphomycosis. In: Merz WG, Hay RJ, eds. Topley & Wilson’s Microbiology & Microbial Infections: Medical Mycology, 10th edn. Washington, DC: ASM Press, 2005: 739–748. 13 Samuels GJ, Muller E. Life-history studies of Brazilian ascomycetes. Rhytidhysteron rufulum and the genus Eutryblidiella. Sydowia 1979; 32: 277–292. 14 Chowdhary A, Guarro J, Randhawa HS, et al. A rare case of chromoblastomycosis in a renal transplant recipient caused by a non-sporulating species of Rhytidhysteron. Med Mycol 2008; 46: 163–166. 15 Suh MK. Phaeohyphomycosis in Korea. Jpn J Med Mycol 2005; 46: 67–70. 16 Ogawa MM, Galante NZ, Godoy P, et al. Treatment of subcutaneous phaeohyphomycosis and prospective follow-up of 17 kidney transplant recipients. J Am Acad Dermatol 2009; 61: 977–985. 17 Sharkey PK, Graybill JR, Rinaldi MG, et al. Itraconazole treatment of phaeohyphomycosis. J Am Acad Dermatol 1990; 23: 577–586. 18 Larsen CG, Arendrup MC, Krarup E, et al. Subcutaneous phaeohyphomycosis in a renal transplant recipient successfully treated with voriconazole. Acta Derm Venereol 2009; 89: 657–658. 19 Graybill JR, Najvar LK, Johnson E, et al. Posaconazole therapy of disseminated phaeohyphomycosis in a murine model. Antimicrob Agents Chemother 2004; 48: 2288– 2291. 20 Negroni R, Helou SH, Petri N, et al. Case study: posaconazole treatment of disseminated phaeohyphomycosis due to Exophiala spinifera. Clin Infect Dis 2004; 38: e15–e20. 21 Nesky MA, McDougal EC, Peacock JE Jr. Pseudallscheria boydii brain abscess successfully treated with voriconazole and surgical drainage: case report and review of central nervous system pseudallescheriasis. Clin Infect Dis 2000; 31: 673–677. 22 Poza G, Montoya J, Redondo C, et al. Meningitis caused by Pseudallscheria boydii treated with voriconazole. Clin Infect Dis 2000; 30: 981–982. 23 Mellinghoff IK, Winston DJ, Mukwaya G, et al. Treatment of Scedosporium apiospermum brain abscesses with posaconazole. Clin Infect Dis 2002; 34: 1648–1650. 24 Howden BP, Slavin MA, Schwarer AP, et al. Successful control of disseminated Scedosporium prolificans infection with combination of voriconazole and terbinafine. Eur J Clin Microbiol Infect Dis 2003; 22: 111–113. 25 Barbaric D, Shaw PJ. Scedosporium infection in immunocompromised patients: successful use of liposomal amphotericin B and itraconazole. Med Pediatr Oncol 2001; 37: 122–125.
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A rare case of subcutaneous phaeohyphomycosis caused by a Rhytidhysteron species: a clinico-therapeutic experience Vikram K. Mahajan1, MD, Vikas Sharma1, MBBS, Neel Prabha1, MBBS, Kamlesh Thakur2, MD, Nand Lal Sharma1, MD, Shivaprakash M. Rudramurthy3, MD, Pushpinder S. Chauhan1, MD, Karaninder S. Mehta1, MD, and C. Abhinav1, MBBS
1 Departments of Dermatology, Venereology and Leprosy, 2Microbiology, Dr Rajendra Prasad Government Medical College, Kangra at Tanda, Himachal Pradesh, and 3 Mycology Division, Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
Correspondence Vikram K. Mahajan, MD Department of Dermatology, Venereology and Leprosy Dr R. P. Government Medical College Kangra at Tanda 176001, Himachal Pradesh, India Tel: + 91 1892 287161 Fax: + 91 1892 267115 E-mail: [email protected]
Abstract Background Subcutaneous phaeohyphomycosis usually results from traumatic inoculation with the fungus and generally occurs in immunosuppressed men. Cladosporium, Exophiala, and Alternaria spp. are commonly implicated pathogens. Objectives We present a case of subcutaneous phaeohyphomycosis caused by Rhytidhysteron sp. that was refractory to conventional antifungal therapy. Case report A 72-year-old man with hypertension and diabetes presented with a multiloculated, large cystic swelling over the right dorsal foot. Laboratory findings and x-rays of the chest and left foot were normal. Results Adequate control of the patient’s diabetes was achieved, and the swelling was excised under itraconazole/terbinafine coverage. Histology showed multiple areas of neutrophilic abscess, epithelioid cells, foreign body giant cells, and multiple septate hyphae and yeast-like cells. Dematiaceous fungus was cultured but failed to produce spores. Sequencing of the isolate showed a match of > 99% with Rhytidhysteron rufulum. The patient demonstrated no response after one year of therapy with itraconazole/terbinafine. Weekly infiltration of the lesion with liposomal amphotericin B resulted in its complete resolution within 15 weeks.
Funding: None. Conflicts of interest: None doi: 10.1111/ijd.12529
Conclusions Lesions of phaeohyphomycosis appear morphologically similar regardless of the organism implicated. Hence, their diagnosis rests entirely on the clinicopathological and microbiological presentation. Molecular studies may be required to identify a fungus if attempts to grow it in artificial culture media fail. Rhytidhysteron spp. are not known as pathogens in humans, and no treatment protocol exists. Intralesional amphotericin was highly effective in our patient and caused no systemic adverse effects. Voriconazole and posaconazole are effective against disseminated/visceral phaeohyphomycotic infections, but their efficacy against Rhytidhysteron spp. remains unstudied.
Introduction The nomenclature phaeohyphomycosis was introduced by Ajello et al.1 in 1974 to describe cutaneous and systemic or disseminated mycoses, including cerebral phaeohyphomycosis, caused by a variety of dematiaceous fungi, which, on clinical, pathological, and mycological grounds, are distinct from chromoblastomycosis. The infection has a wide geographic distribution across all climatic conditions and has been reported in both the extreme south and the extreme north of India, the climatic conditions of which range from tropical to temperate.2 Subcutaneous phaeohyphomycosis usually results from the traumatic inoculation of the fungus or following wound contamination and occurs mostly in adult males ª 2014 The International Society of Dermatology
(aged ≥ 30 years) with some degree of immunosuppression resulting from disease (tuberculosis, diabetes, human immunodeficiency virus [HIV] infection/acquired immunedeficiency syndrome [AIDS], hematological malignancies, or other neoplasia) or iatrogenic causes (organ transplantation, corticosteroids, or immunosuppressive therapies).3 Its manifestation begins with a usually single, slowly enlarging but asymptomatic or mildly painful subcutaneous nodule on the feet, legs, or hands in 60–85% of cases.2–4 These evolve into painless cystic abscesses full of purulent material encapsulated in fibrous connective tissue and have little tendency to rupture. However, papulonodules, verrucous or ulcerated plaques, non-healing ulcers or sinuses, or scaly hyperkerototic lesions resembling chromoblastomycosis are not uncommon, although International Journal of Dermatology 2014
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Subcutaneous phaeohyphomycosis caused by a Rhytidhysteron sp.
all clinical forms show similar features on histopathology irrespective of the etiological agent. More than 100 different saprophytes of soil, wood and plant detritus, classified in 60 different genera, have been implicated as etiological agents of phaeohyphomycosis. Whereas moulds from the genera Cladosporium and Exophiala are commonly identified pathogens, Alternaria spp. are emerging as a cause of infections in both normal and immunocompromised hosts.5–10 To date, Rhytidhysteron spp. (Ascomycota, Patellariales: Patellariaceae) have not been recognized as infecting humans. However, we describe a case of subcutaneous phaeohyphomycosis caused by a Rhytidhysteron sp. which responded to intralesional amphotericin B and appears to be the first of its kind to be reported. Case report A 72-year-old, HIV-negative man presented with a large, soft, slowly progressive, painless swelling over the right foot of one year’s duration. Over the previous three months, the swelling had developed slight redness and sinuses that occasionally discharged hemorrhagic and purulent material. The patient had no history of prior trauma or manipulation of the swelling. He had been treated for type 2 diabetes mellitus for the past 18 years and for hypertension for 15 years. He had poor diabetic control (his HbA1C value was 7.6). He was taking itraconazole (200 mg twice per day) and terbinafine (250 mg/day)
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which had been prescribed six months previously when fine needle aspiration cytology showed branching septate hyphae within and outside the giant cells in an acute on chronic inflammatory background, suggesting deep fungal infection. Cutaneous examination showed an ill-defined, multiloculated, soft and cystic, non-tender swelling over the dorsum of the right foot. The overlying skin had mild erythema and few sinuses with crusts (Fig. 1a). A small swelling of similar morphology was also present over the sole. The patient’s hair, nails, mucous membranes, and other systemic findings were essentially normal. His blood pressure was within the normal range, and daily fasting blood sugar levels varied between 117 mg/dl and 149 mg/dl. Other laboratory investigations including complete blood counts, hepatorenal function tests, serum electrolytes, urinalysis, and chest x-ray revealed no abnormality. An x-ray of the right foot showed corresponding soft tissue swelling and normal underlying bones. Adequate control of the subject’s diabetic status was achieved, and the swelling was surgically excised under itraconazole/terbinafine coverage. The specimen was subjected to histopathology, culture on Sabouraud's glucose agar (SDA) for fungus and L€ owenstein–Jensen medium for Mycobacterium tuberculosis. Pending investigations, treatment with a saturated solution of potassium iodide (SSKI) was added to the itraconazole/ terbinafine combination but could not be continued as the patient developed a flu-like adverse reaction to it. Histology showed multiple areas of neutrophilic abscess bounded
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Figure 1 (a) A 72-year-old man demonstrates poorly defined, multiloculated, mild erythematous, cystic swellings, sinuses and crusts over the right dorsal foot at presentation (after > 6 months of itraconazole/terbinafine). (b) Persistent swelling and discharging sinuses are evident at 6 months after excision (1 year after itraconazole/terbinafine treatment). (c) The swelling and discharge are remarkably reduced 4 weeks after weekly infiltration with liposomal amphotericin B. (d) The phaeohyphomycotic swelling is completely resolved 6 months after the completion of amphotericin B treatment International Journal of Dermatology 2014
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Subcutaneous phaeohyphomycosis caused by a Rhytidhysteron sp.
by epithelioid cells and foreign body giant cells, and multiple, broad, septate, irregularly branched, dematiaceous hyphae, toruloid hyphae (chains of yeast cells), and yeastlike cells that were periodic acid-Schiff (PAS) positive (Fig. 2). Culture of biopsy tissue on SDA with or without cycloheximide showed the growth of multiple colonies of dematiaceous fungus at 28 °C after two weeks. Initially, colonies were light gray–white and velvety and demonstrated brown pigment on the reverse, but incubation for another two weeks resulted in the development of dark pigmentation. Broad, septate, irregularly branched dematiaceous hyphae and toruloid hyphae were identified on lactophenol cotton blue mounts (Fig. 3). The mould was sent to the Center of Advanced Research and the National Culture Collection of Pathogenic Fungi at the Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India, for identification. The isolate was subjected to morphological identification at the reference center. Despite repeated attempts using various techniques, the isolate failed to produce any conidia. Molecular identification was performed by sequencing various regions such as the 18s rRNA gene (partial), internal transcribed spacer 1 (ITS1), 5.8s, ITS2, and 28s rRNA (partial) using the primer pair ITS1 (5′TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The consensus sequence was prepared using BioNumerics Version 6.6 (Applied Maths, Ghent, Belgium). The consensus sequence was compared against known sequences in the National Center of Biotechnology Information (NCBI) GenBank database using BLAST, and in the Central Bureau of Fungal Cultures (Centraal bureau voor Schimmel cultures, Utrecht, the Nether-
Figure 2 (a) Histology shows multiple areas of neutrophilic abscess bounded by acute on chronic cellular infiltrate, epithelioid cells and foreign body giant cells. Arrows indicate: (b) multiple, broad, septate, irregularly branching dematiaceous hyphae, (c) yeast-like cells, and (d) toruloid hyphae or chains of yeast-like cells. (Hematoxylin and eosin stain; original magnification 940) ª 2014 The International Society of Dermatology
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lands). The sequences of the isolate showed a > 99% and up to 100% similarity with sequences of Rhytidhysteron spp. in both databases. The closest match found refers to Rhytidhysteron rufulum. Treatment outcome
The patient demonstrated no significant improvement after almost one year of therapy with itraconazole and terbinafine. The surgical wound had also developed multiple sinuses discharging purulent material (Fig. 1b). The lesion was infiltrated with 25 ml of liposomal amphotericin B (25 mg/25 ml at a maximum IV dosage of 0.5–1.0 mg/kg/
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Figure 3 (a) Velvety colonies of dematiaceous fungus with brown–black pigmentation appear on prolonged incubation. (b and inset) Lactophenol cotton blue stain staining shows broad, septate, irregularly branching dematiaceous hyphae, and toruloid hyphae (arrows). Note that sporulation is conspicuously absent. (Original magnification 940)
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day) once per week under aseptic conditions. The swelling and discharge reduced remarkably during the initial four weeks (Fig. 1c). In view of this encouraging response, intralesional liposomal amphotericin 10 ml/week (10 mg/ 10 ml) was infiltrated for the next seven weeks, followed by two injections of 10 mg administered every fortnight. The patient achieved complete resolution of the lesion but developed mild abnormal renal functions (blood urea/creatinine 91/1.49 mg/dl), which improved over the next eight weeks subsequent to his decision to stop the itraconazole and terbinafine. The patient was advised to continue intralesional liposomal amphotericin B at 5 mg/5 ml once per month for another 16 weeks and to attend for regular follow-up. No recurrence has been noted during post-treatment follow-up of > 1 year (Fig. 1d). Discussion Cystic lesions of subcutaneous phaeohyphomycosis are usually well circumscribed and encapsulated granulomas, the walls of which show epithelioid histiocytic inflammatory reaction and giant cells surrounded by connective tissue, the centers of which show necrotic debris, fibrin, and degenerated polymorphonuclear cells. However, the presence of brown-colored fungal hyphae and yeast-like elements with the giant cells or in the background of a granulomatous inflammatory reaction containing central neutrophilic abscess in a hematoxylin and eosin-stained section is pathognomonic.3,11 Planate septa and thickwalled cells are observed occasionally, but the typical copper penny cell, a more consistent feature of chromoblastomycosis, is not seen. Our patient showed all the classic clinicopathological features of phaeohyphomycosis. The various species belonging to the genera Cladosporium (C. cladosporoides), Phialophora (P. richardsiae), Curvularia (C. geniculata, C. lunata, C. pallescens), Exophiala (E. jeanselmei, E. mansonii, E. castellani), Fonsecaea (F. pedrosoi), Wangiella (W. dermatitidis), Cladophialophora (C. banitiana, C. modesta, C. devriesii), and Alternaria (A. infectoria, A. alternata, A. chlamydospora) constitute major pathogens for the disease.2,4,12 The lesions of phaeohyphomycosis appear morphologically similar regardless of the organism implicated. Hence, the diagnosis of phaeohyphomycosis rests entirely on the clinicopathological and microbiological presentation in the absence of commercially available diagnostic serological tests. Most isolates obtained from culture on SDA are identified from colony morphology and microscopic morphology of conidia, their arrangement on the conidiogenous cell, and the morphology of the conidiogenous cell. Additionally, nitrate and carbohydrate assimilation tests, exoantigen tests, and proteolytic activity or growth temperature response are sometimes required in order to identify an isolate, despite their limited International Journal of Dermatology 2014
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application in the identification of dematiaceous fungi.3 Molecular studies may sometimes be necessary to identify a causative fungus if attempts to grow it in artificial culture media fail. The isolates of dematiaceous fungus obtained from the present patient on SDA did not produce spores despite repeated efforts, and Rhytidhysteron sp. was identified by sequencing the ITS1–5.8s and ITS2 regions of the rDNA gene. The genus Rhytidhysteron includes two species: R. rufulum and R. hysterinum. The fungus has a worldwide distribution and occurs particularly in the tropics and subtropics. This poorly known family includes fungi with discoidal ascomata and butinicate asci with darkly pigmented reniform, elliptical, or fusiform and septate ascospores. Rhytidhysteron rufulum is a lignicolus species and produces its spores exclusively in natural substrates, mainly in the wood of living or dead dicotyledonous plants, and develops coelomycete anamorphs in artificial culture media.13 Rhytidhysteron spp. are not known as pathogens in humans and have been implicated only once, by Chowdhary et al.,14 in a case of chromoblastomycosis in a renal transplant patient, in which attempts to induce sporulation also failed. Surgical excision, drainage, debridement, and cryotherapy, maximum doses of antifungal drugs (amphotericin B, 5-flucytosine, ketoconazole, fluconazole, itraconazole), superficial x-ray therapy, and thiabendazole have been used in various permutations and combinations to treat phaeohyphomycosis, with variable degrees of success.4,15,16 Oral itraconazole at 200–400 mg/day or at a dose as high as 800 mg/day, for a period of 4–6 weeks, appears to be effective in phaeohyphomycosis.16 Sharkey et al.17 observed clinical improvement or remission using itraconazole (50–500 mg/day) for 1–48 months in the 50% of their patients in whom earlier treatment with amphotericin B, ketoconazole, or miconazole had failed. The effectiveness of itraconazole against Rhytidhysteron spp. remains unknown as the only patient reported died of multi-organ failure two weeks after therapy with itraconazole (100 mg twice per day) was initiated.14 In the present patient, a combination of itraconazole and terbinafine (for six months prior to surgery and for 19 weeks after surgery in combination with amphotericin B) did not prove useful, and surgical excision helped only to reduce the mass. Although Vieira et al.10 treated a patient with cutaneous phaeohyphomycosis caused by A. alternata with intralesional amphotericin B (1 mL at 1 mg/ml) twice per week for five weeks, there exists no treatment protocol. Intralesional amphotericin was highly effective in our patient and was administered for 35 weeks without causing any systemic adverse effects. Mild renal function test impairment can be attributed to terbinafine (an uncommon ª 2014 The International Society of Dermatology
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but known side effect) as this improved after the patient discontinued terbinafine, despite the continuation of amphotericin B. We used an initially high dose in order to achieve an early clinical response and a low dose to maintain remission. Voriconazole and posaconazole, in combination with other drugs, have been found to be effective for disseminated/visceral phaeohyphomycotic infections in both humans and experimental animals,18–25 but their efficacy against Rhytidhysteron spp. remains unstudied. Acknowledgment The erudite help and support extended by Dr. Arunaloke Chakrabarti, Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India, in the molecular identification of the isolate is gratefully acknowledged. References 1 Ajello L, Georg LK, Steigbigel RT, et al. A case of phaeohyphomycosis caused by a new species of Phialophora. Mycologia 1974; 66: 490–498. 2 Revankar SG, Patterson JE. Disseminated phaeohyphomycosis: review of an emerging mycosis. Clin Infect Dis 2002; 34: 467–476. 3 Kwon-Chung KJ, Bennett JE. Phaeohyphomycosis. In: Kwon-Chung KJ, Bennett JE, eds. Medical Mycology. Philadelphia, PA: Lea & Febiger, 1992: 620. 4 Sharma NL, Mahajan V, Sharma RC, et al. Subcutaneous phaeohyphomycosis in India – a case report and review. Int J Dermatol 2002; 41: 16–20. 5 Gugnani HC, Sood N, Singh B, et al. Case report: subcutaneous phaeohyphomycosis due to Cladosporium cladosporioides. Mycoses 2000; 43: 85–87. 6 Gonzalez MS, Alfonso B, Seckinger D, et al. Subcutaneous phaeohyphomycosis caused by Cladosporium devriesii, sp. nov. Sabouraudia 1984; 22: 427–432. 7 Qiu-Xia C, Chang-Xing L, Wen-Ming H, et al. Subcutaneous phaeohyphomycosis caused by Cladosporium sphaerospermum. Mycoses 2007; 51: 79–80. 8 Murayama N, Takimoto R, Kawai M, et al. A case of subcutaneous phaeohyphomycotic cyst due to Exophiala jeanselmei complicated with systemic lupus erythematous. Mycoses 2003; 46: 145–148. 9 Silva MRR, Fernandes OFL, Costa CR, et al. Subcutaneous phaeohyphomycosis by Exophiala jeanselmei in a cardiac transplant recipient. Rev Inst Med Trop Sao Paulo 2005; 47: 55–57. 10 Vieira MR, Martins ML, Afonso1 A, et al. Cutaneous alternariosis. Rev Iberoam Micol 1998; 15: 97–99. 11 Ben-Ami R, Lewis RE, Raad II, et al. Phaeohyphomycosis in a tertiary care cancer center. Clin Infect Dis 2009; 48: 1033–1041.
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12 Cooper CR Jr. Deep phaeohyphomycosis. In: Merz WG, Hay RJ, eds. Topley & Wilson’s Microbiology & Microbial Infections: Medical Mycology, 10th edn. Washington, DC: ASM Press, 2005: 739–748. 13 Samuels GJ, Muller E. Life-history studies of Brazilian ascomycetes. Rhytidhysteron rufulum and the genus Eutryblidiella. Sydowia 1979; 32: 277–292. 14 Chowdhary A, Guarro J, Randhawa HS, et al. A rare case of chromoblastomycosis in a renal transplant recipient caused by a non-sporulating species of Rhytidhysteron. Med Mycol 2008; 46: 163–166. 15 Suh MK. Phaeohyphomycosis in Korea. Jpn J Med Mycol 2005; 46: 67–70. 16 Ogawa MM, Galante NZ, Godoy P, et al. Treatment of subcutaneous phaeohyphomycosis and prospective follow-up of 17 kidney transplant recipients. J Am Acad Dermatol 2009; 61: 977–985. 17 Sharkey PK, Graybill JR, Rinaldi MG, et al. Itraconazole treatment of phaeohyphomycosis. J Am Acad Dermatol 1990; 23: 577–586. 18 Larsen CG, Arendrup MC, Krarup E, et al. Subcutaneous phaeohyphomycosis in a renal transplant recipient successfully treated with voriconazole. Acta Derm Venereol 2009; 89: 657–658. 19 Graybill JR, Najvar LK, Johnson E, et al. Posaconazole therapy of disseminated phaeohyphomycosis in a murine model. Antimicrob Agents Chemother 2004; 48: 2288– 2291. 20 Negroni R, Helou SH, Petri N, et al. Case study: posaconazole treatment of disseminated phaeohyphomycosis due to Exophiala spinifera. Clin Infect Dis 2004; 38: e15–e20. 21 Nesky MA, McDougal EC, Peacock JE Jr. Pseudallscheria boydii brain abscess successfully treated with voriconazole and surgical drainage: case report and review of central nervous system pseudallescheriasis. Clin Infect Dis 2000; 31: 673–677. 22 Poza G, Montoya J, Redondo C, et al. Meningitis caused by Pseudallscheria boydii treated with voriconazole. Clin Infect Dis 2000; 30: 981–982. 23 Mellinghoff IK, Winston DJ, Mukwaya G, et al. Treatment of Scedosporium apiospermum brain abscesses with posaconazole. Clin Infect Dis 2002; 34: 1648–1650. 24 Howden BP, Slavin MA, Schwarer AP, et al. Successful control of disseminated Scedosporium prolificans infection with combination of voriconazole and terbinafine. Eur J Clin Microbiol Infect Dis 2003; 22: 111–113. 25 Barbaric D, Shaw PJ. Scedosporium infection in immunocompromised patients: successful use of liposomal amphotericin B and itraconazole. Med Pediatr Oncol 2001; 37: 122–125.
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