Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 97-103

ISSN: 2319-7706 Volume 4 Number 5 (2015) pp. 97-103 http://www.ijcmas.com

Original Research Article First Reported Human Case of Bilateral Subcutaneous Phaeohyphomycosis by a New Member of () spp.

P.K.Maiti1, A.Naha1, R.Ghosh1*, P.Ghosh1 and G.Chatterjee2

1Department of Microbiology, Institute of Post Graduate Medical Education and Research, Kolkata 700020, West Bengal 2Department of Dermatology, Institute of Post Graduate Medical Education and Research, Kolkata 700020, West Bengal, India *Corresponding author

A B S T R A C T

A 45 years old female from Eastern India presented with painless multi-lobulated subcutaneous swellings on both forearms. She was controlled diabetic. From K e y w o r d s drained pus and biopsy materials dematiaceous fungal hyphae were demonstrated by KOH mount and PAS staining. Coffee brown colonies grew on SDA medium Phaeohyphomycosis, which showed brown branched hyphae with lateral anastomosis but no Dematiaceous, conidiogenesis. By molecular identification it was concluded as a new member of Dothideomycetes, Dothideomycetes (Pleosporales) spp showing 86% sequence similarity with Pleosporales, Microsphaeropsis arundini. By further morphological study bitunicate asci and Microsphaeropsis asexual ascostromal development was noted. Thus it became first reported human case by such plant pathogen fungi. The probable reason for bilateral involvement might be due to her occupational trauma by carrying a type of elephant grass. The patient was managed by surgical excision and itraconazole therapy.

Introduction

Melanized or dematiaceous fungi are human pathogens like Alternaria spp., ubiquitously present saprophytes and also Bipolaris spp., Exophiala spp., enormously present on dead and living Cladosporium spp., Madurella spp., plants. The most common type of clinical Sporothrix spp., various black fungi presentation caused by dematiaceous fungi belonging to Phylum , which is phaeohyphomycosis of skin and are very new to medical mycologists are subcutenous tissue following minor, increasingly causing infections these days unrecognizable trauma by plant or vegetable among immunocompromised as well as material (Revankar and Sutton, 2010). In immunocompetant persons (Revankar and recent time many rare and new members of Sutton, 2010). It imposes difficulties in this group are emerging as important human morphological identification and need for pathogen. Apart from the common known molecular methods in routine mycology

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Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 97-103 laboratory. Here we report the first case of diameters). The reverse appeared ledean bilateral subcutaneous phaeohyphomycosis black to violaceous black (Fig. 2a,b). Lacto by a new member of Dothideomycetes Phenol cotton blue preparation of the (Pleosporales) spp. in an immunocompetant colonies showed thick and thin to dark host. golden brown as well as sub hyaline branching hyphae up to 5µm thick. Case report Characteristic right angle branched hyphae with constriction at the site of origin & A 45 years old female from Howrah district, lateral anastomosis was observed (Fig. 2c). South Bengal, India presented with multi- No conidia or asexual fruiting bodies were lobulated subcutaneous swellings of about 5 evident after prolong incubation and cm sizes on dorsal as well as ventral aspects subculture on cornmeal agar (CMA, of both forearms (Fig. 1a). Lesions were HiMedia). Similar fungi isolated from chronically increasing for last eight months. lesions on both the hands and also present in There was no history of trauma, pain or histopathological examination (Fig. 1d). fever. She was hypertensive and diabetic Based on the morphological characters we though her sugar level (fasting, post- provisionally diagnosed it as Rhizoctonia prandial, HBA1c level) was well controlled spp. though we failed to find sclerotia. Then with medication at the time of presentation. the isolate was subcultured on SDA and sent All the relevant biochemical investigations to National Fungal Culture Collection, India were carried out which includes: Hb%, (NFCCI) at Pune for molecular WBC total count, urea, creatinine, LFT, identification and also deposition of the Lipid profile, RPR, Thyroid profile, serum strain in the culture collection with unique cortisol level (Baseline and after accession number. After sequencing the dexamethasone challenge) as well as USG isolate (NCBI accession JQ759930.1/ isolate whole abdomen, Chest X-Ray, CT Scan of FL0071/ Dothideomycetes sp. Genotype Brain. All reports were within normal limit. 223) showed 86% sequence similarity with She was also non-reactive for HIV, HBsAg, Microsphaeropsis arundinis and diagnosed anti-HCV tests. as a new member of Dothideomycetes (Pleosporales) family (Fig. 3). Then we We took pus and biopsy sample for started further morphological studies after investigation. The KOH mount of aspirated prolong incubation and appreciated few material revealed presence of brown septate structures like bitunicate asci, multi-cellular branching hyphae, which were also PAS fruiting body arising from hyphae (Fig. 4) positive on staining (Fig 1b, 1c). Case was but failed to find any pycnidium. diagnosed as subcutaneous phaeohyphomycosis and itraconazole Following surgical excision itraconazole therapy was initiated empirically. Surgical therapy continued for 3 months, patient excision of lesions was also undertaken. responded and no recurrence reported within one year of follow up period. Material was directly cultured onto Sabauraud s dextrose agar (SDA, HiMedia) Dothideomycetes is a diverse class of and incubated at 25°C and 37°C. After 10 ascomycetes fungi, characterized with days of incubation both the tubes showed bitunicate (thick, double layered) asci floccose to velutinous, dark mouse grey to without any apical pore and ascostromatic or coffee brown colonies (2 3.5 mm in ascolocular development of pseudothecia

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Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 97-103

(perithecia initiated from non-sexual pathogen which are affecting human vegetative hyphal cells (Schoch et al., newly. 2009). Predominantly plant pathogen this class of fungi is divided into several Kingdom: Fungi subclasses and orders though poorly Phylum: Ascomycota understood till date. Amongst the seven orders, Pleosporales comprises the Class: Dothideomycetes commonest causative agents of Order: Pleosporales phaeohyphomycosis. Besides the well- known melanized anamorphic Family/Clade: Didymellaceae hyphomycetes e.g. Alternaria, Curvularia Genera: Microsphaeropsis few unusual coelomycetes e.g. Microsphaeropsis, Phoma, Pleurophoma are Species: sporadically reported as human pathogen Microsphaeropsis arundinis (Guarro et al., 1999; Shah et al., 2001; Microsphaeropsis olivaces Padhye et al., 2004; Stella et al., 2004; Microsphaeropsis callista Galipothu et al., 2015). Microsphaeropsis conielloides A heterogenous group of Ascomycotic fungi Phylogenetic tree of Microsphaeropsis are called as Mitosporic Ascomycota due according to multi-locus DNA sequencing to absence of any sexual stage. (Zhang et al., 2009). Microsphaeropsis is a member of this group and also comes under coelomycetes group as Our case was apparently healthy patient conidia bourn inside closed sac which forms without any systemic disease presented with only asexual fruiting body, called Pycnidia bilateral lesions and there was no organ with small aseptate conidia (Sutton, 1980). dissemination found after radiological Pycnidium is an asexual version of investigations. Meticulous history taking perithecium, as the first one produces uncover her habit of carrying bundles of conidia and the other produces sexual hogla leaves on her forearm leading to ascospores. minor trauma. Hogla plants (Typha elephantina) or elephant grass, a gigantic Different species of Microsphaeropsis are marsh plant luxuriously grown in canals, identified on the basis of structural roadside shallow water of southern Bengal is difference of pycnidia and conidia. But used for thaching. Literature study revealed production of pycnidium is very difficult Microsphaeropsis arundinis was first and slow, needs various special sporulating isolated from Arundo donax, similar type of culture media like carnation leaf agar and grass growing in wetlands (Ahmad, 1971) long term incubation. and thereafter many marine plants (Wanderley Costa et al., 2012). Further In our routine laboratory with only two research may explore relation of this fungus simple fungal media in hand, it was not with hogla plant. In present case bilateral possible to sporulate the isolate. Previous traumatic implantation might be possible reports expressed such difficulties and took reason for bilateral disease. In subcutaneous help of reference laboratory of morpho- mycoses among immune-competent bilateral identification of such isolates (Stella et al., incidence is exception rather than rule (Maiti 2004). Therefore molecular techniques are et al., 2000). the new tool to solve the mystery of plant 99

Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 97-103

Fig.1(a) Multi-lobulated subcutaneous lesions before treatment. (b) KOH mount of pus showed brown septate branching hyphae (400x). (c) Pas positive branching fungal hyphae (1000x). (d) Fungal elements present in histopathological slide (1000x).

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Fig.2 (a) and (b) Obverse and reverse side of dematiaceous fungal colony on Sabauraud s dextrose agar. (c) Lacto Phenol cotton blue preparation of the colonies showed brown right angle branching hyphae with lateral anastomosis (400x)

Fig.3 Sequence analysis of ~500bp rDNA fragment showed 86% sequence similarity with Microsphaeropsis arundinis. Possibly a new member of Dothideomycetes (Pleosporales) spp

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Fig.4(a) Bitunicate asci, (b) multi-cellular fruiting body arising from hyphae were observed under microscope (400x)

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2004. Phaeohyphomycotic soft tissue infections caused by the coelomycetous fungus Microsphaeropsis arundinis. J. Clin. Microbiol., 42: 5315 9. Sutton, B.C. 1980. The coelomycetes: fungi imperfecti with pycnidia acervuli and stromata. Commonwealth Mycological Institute, Kew, England. Wanderley Costa, I.P., Maia, L.C., Cavalcanti, M.A. 2012. Diversity of leaf endophytic fungi in mangrove plants of northeast Brazil. Braz. J. Microbiol., 43(3):1165 73. Zhang, Y., Schoch, CL., Fournier, J., Crous, PW., de Gruyter, J., Woudenberg, J.H., Hirayama, K., Tanaka, K., Pointing, S.B., Spatafora, J.W., Hyde, K.D. 2009. Multi-locus phylogeny of Pleosporales: a taxonomic, ecological and evolutionary re-evaluation. Stud. Mycol., 64: 85 102,S5.

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