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Fungal Keratitis Caused by Macrophomina Phaseolina – a Case Report

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Fungal Keratitis Caused by Macrophomina Phaseolina – a Case Report Medical Mycology Case Reports 1 (2012) 123–126 Contents lists available at SciVerse ScienceDirect Medical Mycology Case Reports journal homepage: www.elsevier.com/locate/mmcr Fungal keratitis caused by Macrophomina phaseolina – A case report T. Premamalini a,n, B.T. Ambujavalli a, R. Vijayakumar a, S.V. Rajyoganandh a, S. Kalpana b, Anupma J Kindo a a Department of Microbiology, Sri Ramachandra Medical College & Research Institute, SRU, Chennai 600116, India b Department of Ophthalmology, Sri Ramachandra Medical College & Research Institute, SRU, Chennai 600116, India article info abstract Article history: A 70 year old female patient presented with complaints of pain, watering and swelling in the right eye. Received 3 September 2012 She gave a history of fall, as she was walking in the paddy field of her farm. Ophthalmological and Received in revised form Microbiological investigation revealed a fungal keratitis with an unusual fungus Macrophomina 11 October 2012 phaseolina which is primarily a plant pathogen, with a potential to cause human infections especially Accepted 15 October 2012 in immuno-compromised patients. The patient responded well to the antifungal treatment with Oral Voriconazole with absence of recurrence and dissemination. Keywords: & 2012 International Society for Human and Animal Mycology. Published by Elsevier B.V. All rights Fungal keratitis reserved. Macrophomina phaseolina DNA sequencing 1. Introduction watering and swelling in the right eye. She gave a history of fall when suddenly a cow came on her way as she was walking in the Macrophomina phaseolina is a soil borne fungus. This fungus is paddy field of her farm (day 14). primarily a plant pathogen infecting the root and lower stem of Following the injury she went to a local doctor in her area over 500 plant species [1]. M. phaseolina commonly causes disease where she is living. She was prescribed hourly dose of Ciproflox- on soybean (charcoal rot), peanut, and corn. It survives as acin eye drops with steroid combination (day 3). Then she microsclerotia in the soil and on infected plant debris [2]. The developed severe pain and swelling in the injured eye (day 1). microsclerotia forms the main source of infection and can survive On examination of the right eye, the visual acquity was reduced to in soil for up to three years [3]. The microsclerotia are black, hand movements. Patient had periorbital edema along with edema of multi-celled structures that are produced in the plant which is the the eyelid (Fig. 1a). There was matting of the eye lashes, discharge host for this fungus, and gets passed on from the decayed plant to and conjunctival congestion with blepharitis. The cornea showed the soil. These multi-celled structures like the bacterial spores can central epithelial defect with corneal infiltrates measuring around survive under adverse conditions even at temperature above 8 Â 5mm2 with satellite lesions (Fig. 1a). There was stromal edema 30 1C. Their survival is greatly reduced in wet soil where, micro- and descemet membrane folding. Anterior chamber was irregular sclerotia survives not more than 7–8 weeks and mycelium with a 2 mm hypopyon. The pupil was 3 mm reacting to light. The survives not more than 7 days. This fungus rarely causes disease duct showed clear regurgitation. The left eye had a visual acquity of in humans. There have been only two reports of this fungus 3/60 with no clinical features of injury. causing infection in humans one from a renal transplant recipient Her investigation parameters were within normal limits. and one in a child with acute myeloid leukemia [4,5].To the best Hemoglobin 80 g/L, Total White Blood cell count 7.8 Â 109 cells/L, of our knowledge there has been no report of this plant pathogen Liver function test showed SGOT – 13 U/L, SGPT – 21 U/L, Alkaline causing keratitis in humans. phosphatase 102 U/L, Total protein 65 g/L, Albumin 30 g/L and Globulin 35 g/L. Among the renal parameters, Creatinine was raised to 282.88 umol/L. She was not a known case of diabetes or 2. Case hypertension. In lieu of the high Creatinine level medicine opinion was sought, and a diagnosis of anemia with secondary chronic A 70 year old female patient came to the outpatient depart- kidney disease was made. Patient wasadvisedforECG,2Dechoand ment of Ophthalmology (day 0) with the complaints of pain, Ultra sonogram abdomen (for kidney size). Due to monetary reasons these investigations were not done. n Corresponding author. Tel.: þ91 44 24768403x139; fax: þ91 44 24765993. A corneal scraping was done by the ophthalmologist and E-mail address: [email protected] (T. Premamalini). specimen was subjected to 10% KOH mount and inoculated 2211-7539/$ - see front matter & 2012 International Society for Human and Animal Mycology. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mmcr.2012.10.007 124 T. Premamalini et al. / Medical Mycology Case Reports 1 (2012) 123–126 Fig. 3. Black color colony seen on water culture. Fig. 1. Periorbital edema along with edema of the eyelid with corneal infilterate. 15 min, inoculated with the culture and incubated at room temperature for one week. Conidiation and formation of pycnidia was observed in banana peel culture which made the identification little complicated (Fig. 4aandb). The identification of the isolate based only on morphological criteria did not give a confirmed identification due to variations in the phenotype. Hence, the confirmation had to be made by molecular techniques [7]. The culture grown on Sabouraud’s Dextrose Agar was used for DNA extraction using the Qiagen kit as per manufacturer instruc- tions. The internal Transcribed Spacer (ITS) regions (ITS1-5.8S- ITS2) were amplified using ITS1 (50-TCCGTAGGTGAACCTGCGG-3) and ITS4 (50-TCCTCCGCTTATTGATATGC-30) as described pre- viously [5]. The thermocycler used was Eppendorf Mastercycler gradient (Germany). The program was as follows; 30 cycles of denaturation at 95 1C for 30 s, annealing at 52 1C for 30 s and extension at 72 1C for 45 s. The amplicon was electrophoresed on 1.5% agarose gel, stained with Ethidium bromide and visualized on Biorad XR (California, USA) gel documentation unit. The cleanup was done using a Qiaquick PCR purification kit (Qiagen, Fig. 2. The KOH mount showed plenty of septate hyphae (40 Â magnification). Inc., Valencia, CA). The amplicon was sent for DNA sequencing, to the Department of Genetics (a unit of Sankara Nethralaya), Chennai, India. The PCR (C shape inoculation) on Sabouraud’s dextrose agar with and amplified products were sequenced by the dideoxynucleotide without cycloheximide and incubated at 37 1Cinthemycology chain termination method using an ABI PRISM 3100 Genetic laboratory. Analyzer (Applied Biosystems, USA). The sequence was then used The KOH mount showed plenty of septate hyphae (Fig. 2). The for a nucleotide - nucleotide search using the BLAST algorithm at ophthalmology consultant was informed about it, and patient was the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST/). BLAST started on 1% Atropine eye drops thrice daily, 5% Natamycin eye hits more than 98% were considered. The identity was with drops hourly, Oral ketoconazole 200 mg twice daily, Oral Haema- M. phaseolina. Hence, the isolate was genotypically confirmed to tinics, Oral Ibuprofen and Oral Ranitidine for 10 days. be M. phaseolina. The culture of the corneal scraping grew a black color colony The clinician was informed and Oral Voriconazle 200 mg twice on Sabouraud’s dextrose agar which was floccose. The reverse daily was added along with 5% Natamycin eye drops two hourly was also black. There was no growth on plate containing cyclo- for 10 days (day 14). Patient’s vision improved within two days. heximide. The culture grew at 37 1C as well as 40 1C. Slide culture The patient was asked to continue Oral Voriconazole 200 mg was done on potato flakes agar to enhance the production of twice daily for 6 weeks (day 24). The patient came back for follow morphological structures required for identification [6]. Micro- up and the lesion had subsided and she was relieved of the scopically it showed smooth, subhyaline, septate hyphae and clinical symptoms. some clumps of multicelled structures later maturing to sclerotia. Since there was no specific sporulation seen on Sabouraud’s dextrose agar and potato flakes agar, water culture (Fig. 3) was 3. Discussion done to induce sporulation. In addition, Banana peel culture was also done as an indigenous method, to simulate the natural The fungus M. phaseolina belongs to the phylum Ascomycota and habitat of this fungus and to induce sporulation. About class Dothideomycetes of the Botryosphaeriaceae family [5].Syno- 1 Â 1cm2 sized Banana peels were sterilized by autoclaving in nyms historically are: M. phaseolina Tassi (1901), M. phaseoli Maubl. glass petri dishes with 5 ml of distilled water at 121 1C, 15 lbs for (1905), Sclerotium bataticola Taub (1913), Rhizoctonia bataticola (Taub) T. Premamalini et al. / Medical Mycology Case Reports 1 (2012) 123–126 125 Fig. 4. (a and b) Pycnidia and conidiation in sterilized banana peel culture.(40 Â magnification). M. phaseolina is primarily a soil borne plant pathogen. The accidental cause of human infection could be due to traumatic implantation as would have happened in this patient. The first case reported in humans, was a disseminated infec- tion in an immunocompromised adult male following renal transplantation [4]. The patient subsequently succumbed to invasive infection with Scytalidium dimidiatum, which is a related species within the ascomycete family Botryosphaeriaceae. The patient responded well to treatment with Voriconazole alone [4]. Another case of cutaneous infection caused by M. phaseolina was reported in a child with acute myeloid leukemia. The child responded well to treatment with Posaconazole without recur- rence, after Haematopoietic Stem Cell Transplantation, and also the patient had no signs of systemic fungal involvement [5].
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