Post-traumatic Corneal Mucormycosis Caused by Absidia corymbifera

Post--traumatic Corneal Mucormycosis Caused by Absidia corymbifera David H. Marshall, MD,I.2 Seymour Brownstein, MD/,2 W. Bruce Jackson, MD,1 George Mintsioulis, MD,1 Steven M. Gilberg, MD,1 Basil F. Al~Zeerah, MD1 Objective: The purpose of the study was to report a case of mycotic keratitis caused by the organism Absidia corymbifera (class Zygomycetes, order Mucorales, family Mucoraceae). Design: Case report. Participant: A healthy 37-year-old farmer scratched his left cornea on a galvanized nail while working in his barn. Within 24 hours, an infiltrate in the inferior cornea developed that advanced superiorly, reducing the vision to hand motion by the following day. He was treated with topical and systemic antibiotics and antifungal medications, but the infiltrate spread to the adjacent nasal limbus. Intervention: An 11-mm penetrating keratoplasty was performed with an adjacent nasal 7-mm superficial lamellar sclerectomy. Main Outcome Measures: Pathologic examination of the keratoplasty specimen. Results: Corneal cultures grew A. corymbifera. The organisms were identified in tissue sections by light, fluorescent, electron, and immunoelectron microscopy. Conclusions: The authors believe that this is the first reported case of keratitis caused by an Absidia species and, as such, represents an unusual form of mucormycosis in an otherwise healthy individual. Ophthalmology 1997; 104:1107-1111

Fungal keratitis caused by an agent of mucormycosis is unusual. Mucormycosis encompasses a spectrum of diseases caused by the nonseptate fungi of the class Zygomycetes, order Mucorales, and usually occurs in patients with an underlying systemic illness such as diabetic ketoacidosis, a hematologic malignancy, or who are otherwise immunocompromised. Well-recognized clinical forms of mucormycosis include rhino-orbitocerebral, pulmonary, cutaneous, central nervous system, gastrointestinal, and disseminated. I ,2 Ocular involvement with these organisms usually occurs in association with sinusitis or rhinitis.2,3 One case of post-traumatic keratitis due to mucormycosis (genus Rhizopus) has been reported in an individual who initially had been treated with corticosteroids. 4 Originally received: July 30, 1996. Revision accepted: March 6, 1997. 1 Department of Ophthalmology, University of Ottawa Eye Institute, Ottawa General Hospital, Ottawa, Ontario, Canada. 2 Department of Pathology, University of Ottawa, Ottawa General Hospital, Ottawa, Ontario, Canada. Reprint requests to Seymour Brownstein, MD, University of Ottawa Eye Institute, 501 Smyth Rd, Room 3818, Ottawa, ON KIH 8L6, Canada.

We report a case of fulminant keratitis caused by the organism Absidia corymbifera (order Mucorales, family Mucoraceae) in an otherwise healthy 37-year-old farmer. The infection was documented by culturing the organism and by observing it in corneal sections by light, fluorescent, and electron microscopy. We believe that this is the first reported case of keratitis caused by this organism and, as such, represents a unique form of mucormycosis in an immunocompetent individual without evidence of systemic illness. Furthermore, our case displays that fungal infections should be included in the differential diagnosis of acute, rapidly progressive keratitis, although most cases of keratomycosis have a more insidious presentation.

Case Report A 37-year-old farmer scratched his left cornea on a galvanized nail while working in his bam. The following day, he had pain and photophobia and noted an infiltrate in the inferior cornea that became totally opacified 1 day later with marked deteriora-

1107

Ophthalmology

Volume 104, Number 7, luly 1997

I

Figure 1. A fluffy, white, irregular left corneal infiltrate is seen in a ring configuration 5 days after the injury. The conjunctiva is chemotic and injected.

.

Figure 4. The organisms (arrows) are seen more readily with the GrocortGomori methenamine-silver stain (original magnification, X1250).

.

Figure 2. Cornea displays a paracentral ring-like ulceration (arrows) joined by an intense stromal infiltrate (stain, hematoxylin and eosin; original magnification, XIS).

Figure 5. Fluorescence of fungal cell walls in the superficial corneal stroma just deep to Bowman layer (stain, calcofluor white; original magnification,

x500).

Figure 3. Irregular tubulocystic nonseptate hyphae (arrows), including one cut along its longitudinal axis, are evident in the superficial corneal stroma just deep to Bowman layer (stain, hematoxylin and eosin; original magnification, X 1250).

Figure 6. Fluorescence of organisms mainly superficially but also in the mid corneal stroma (arrows) (stain, calcofluor white; original magnification, x500).

1108

Marshall et al . Post-traumatic Corneal Mucormycosis

Figure 7. The tubular structure (arrow) represents a fungal hypha cut in cross section. The organism is almost completely engulfed by a macrophage situated among the collagen lamellae. Abundant granular debris (D) is noted within and between the stromal lamellae (original magnification,

The corneal graft showed signs of rejection by 21 days after the operative procedure. Despite oral and topical corticosteroid therapy, the cornea became progressively opacified and vascularized. An 8-mm penetrating keratoplasty performed 1 year later resulted in a clear graft and a visual acuity of 20/400 for 4 months after surgery, after which the graft became edematous and opacified. The results of laboratory investigations after initial hospitalization including a complete blood count (with erythrocyte indices and leukocyte differential), serum electrolytes, coagulation studies (partial thromboplastin time and prothrombin time), and a urinalysis were within the normal range. The blood glucose initially was elevated slightly but subsequently normalized. Radiographic studies included a chest x-ray and computerized tomography of the orbits and sinuses, which were negative. Testing for human immunodeficiency virus by the enzyme-linked immunosorbent assay test showed that the patient was human immunodeficiency virus negative. His leukocyte count and Thelper-to-T -suppression ratio were normal.

x8550).

Pathologic Findings tion of his vision. He was referred to our institution approximately 48 hours after his injury. Historic inquiry showed that the patient was otherwise healthy, had no systemic illnesses, was not receiving any medications, and had no risk factors for human immunodeficiency virus infection. Abnormal physical findings were confined to the left eye, which had hand motion vision and a normal intraocular pressure. The cornea displayed a fluffy, white, irregular ringlike infiltrate (Fig 1). A mucous plaque was adherent to the epithelium centrally. The peripheral cornea was hazy, obscuring the anterior chamber details, but it was thought that no hypopyon was present. The bulbar conjunctiva was markedly chemotic with dilated blood vessels. The fundus could not be visualized. The patient was hospitalized with a diagnosis of keratitis of either bacterial or fungal cause. Corneal scrapings were obtained for culture, and he was treated with intravenous antibiotics, fortified topical antibiotics, and topical antifungal medications. Thirty hours later, the cultures showed a heavy growth of fungus belonging to the family Mucoraceae. The intravenous therapy was changed to amphotericin B (30 to 50 mg daily; BristolMyers Squibb, Montreal, Canada). Topical antifungal medications included natamycin (5% every 30 minutes) and amphotericin B (2.5 mg/ml every 30 minutes; Bristol-Myers Squibb). Further examination of the fungus by the provincial laboratory identified it as belonging to the genus Absidia, species corymbifera. The corneal infiltrate progressively extended to the nasal limbus with an area of adjacent scleritis. Because of concern over possible scleral extension of this potentially invasive organism, a lO-mm penetrating keratoplasty in continuity with an adjacent nasal 7- X 7-mm lamellar sclerectomy was performed 8 days after the injury. A scleral biopsy also was taken deep to the excised sclera to determine whether organisms had extended int~rior to the deep surgical margin. An 11-mm corneal graft and a 7 -mm scleral patch graft then were sutured into position. The antifungal therapy was continued after surgery, but the intravenous amphotericin B subsequently had to be discontinued after an intermittent 9-day course (240 mg total) because of rising blood creatinine levels. Because of a moderate anterior chamber reaction, topical prednisolone acetate (l %, four times daily) was added to the drug regimen. After 19 days in the hospital, the patient was discharged home with a clear graft and a visual acuity of 20/300. The topical natamycin and prednisolone acetate were continued.

Light microscopic examination results of the keratoplasty specimen obtained 8 days after the injury showed a central zone of inflammation extending to the midstroma and an adjacent shallow ring-like paracentral anterior stromal ulceration (Fig 2). Within and especially just beneath Bowman layer and in the deeper areas of inflammation, numerous irregular tubulocystic nonseptate hyphae measuring 3 to 20 j.tm in width were identified (Fig 3). The fungi were most readily seen with the Grocott stain (Fig 4) and with fluorescence with calcofluor white (Figs 5, 6), but also were identified with hematoxylin and eosin (Fig 3), periodic acid-Schiff, the gram stain, toluidine blue, and stains for acid fast bacilli. The inflamed stroma contained acute and chronic inflammatory cells and necrotic debris. Descemet membrane was separated focally from the endothelium by a nidus of acute inflammatory cells, but otherwise appeared uninvolved by the infectious

Figure 8. Higher magnification of a hypha that is almost completely engulfed by a macrophage with a persistent cleft (arrow) leading to the organism. The organism has a multilaminar cell wall and degenerated intracellular organelles. Variably sized homogeneous electron-dense structures are evident in close apposition to the cell wall. The cell membrane covering these structures belongs to an engulfing macrophage (original magnification, x36,OOO).

1109

Ophthalmology

Volume 104, Number 7, July 1997 The corneal button obtained 1 year after presentation showed moderate epithelial edema, severe stromal edema, absence of the endothelium, and no evidence of microorganisms. These findings are consistent with the clinical diagnosis of graft rejection.

Discussion

Figure 9. Longitudinal section of a hypha that i sextens ively engulfed by macrophages (original magnification, x9000).

process. The adjacent lamellar and deep scleral biopsy specimens displayed acute and chronic inflammation but no organisms. The Prussian blue stain for iron was negative. Transmission electron microscopy of the inflamed cornea showed variably shaped organisms in the corneal stroma (Figs 7 -9). A rare organism was seen cut lengthwise and measured approximately 28 J1m in length (Fig 9). The majority of the organisms were cut in cross or oblique sections and measured between 1 to 7 J1m in width. The organisms had a multilaminar cell wall that measured approximately 0.25 J1m in thickness. In many areas, electron-dense round-to-oval-shaped, fairly homogeneous structures ranging in size from 0.1 to 0.25 J1m in diameter were seen in close apposition to the exterior of portions of the cell wall of most of the organisms (Figs 8, 9). The organisms appeared quite degenerated occasionally with only remnants of the cytoplasm and of intracellular organelles including mitochondria. They showed no septa. Rare organisms were found among corneal stromal lamellae surrounded only by granular debris. Most of the hyphae appeared almost completely or partially engulfed by macrophages with the cell membrane of the macrophage in close apposition to the cell wall of the organisms, usually with a persistent cleft formed in the external cell wall of the macrophage leading to the organism (Figs 7-9). Occasionally among the collagen lamellae, distinct from the organisms and inflammatory cells, elongated areas of granular electron-dense material were seen possibly representing degenerated nuclear and other cellular debris (Fig 7). Immunoelectron microscopic examination results showed kappa light chains concentrated in the cell wall of the fungi and, to a much lesser extent, internal to the plasmalemma. There was only minimal irregular reactivity within the electron-dense structures on the exterior surface of the cell wall of the organisms.

1110

Fungi can be divided broadly into those that are predominantly filamentous, dimorphic, or yeasts.5 Yeasts such as Candida tend to cause disease, including corneal ulcers, in eyes in which there is some predisposing factor. Examples of the pathogenic dimorphic fungi, which form both yeasts and mycelia and which rarely cause keratitis, are Blastomyces, Coccidiodes, Histoplasma, and Sporothrix. The filamentous fungi are further classified as septate or nonseptate, with septated organisms such as Aspergillus, Fusarium, Acremonium (formerly called Cephalosporium), and the dematiaceous fungi as a cause of posttraumatic keratitis in otherwise healthy individuals. 5 The zygomycetes (formerly referred to as phycomycetes) are nonseptate filamentous saprophytic organisms that are divided into the orders Mucorales and Entomophthorales, which cause the diseases mucormycosis and entomophthoromycosis, respectively. 6.7 Entomophthoromycosis is a tropical disease of the subcutaneous and submucosal tissues in immunocompetent individuals and is rarely seen in North America. The order Mucorales is further divided into several families, which include the Mucoraceae, Cunninghamellae, Mortierellaceae, and Saksenaceae. The genera of the Mucoraceae family are the most frequently isolated agents of mucormycosis, with Rhizopus, Rhizomucor, Mucor, and Absidia being the most common. I 5, -7 The genera are further speciated in the laboratory, which resulted in the identification of Absidia (genus) corymbifera (species) in our patient. Keratitis as the primary manifestation of mucormycosis is quite unusual. In a review of ocular and orbital phycomycosis in 1977, Schwartz et ae disclosed that there never had been a case of a corneal ulcer caused by one of these organisms in which the fungus was cultured from the cornea and also showed in tissue sections. Since then, a case of post-traumatic keratitis caused by the genus Rhizopus (family Mucoraceae) has been reported. 4 This case involved a patient who was treated with topical corticosteroids after the corneal injury, with the organism being isolated from the cornea 2.5 months after the initial insult. Our case is unique because the patient had a fulminant course and required corneal transplantation within 9 days of his injury in conjunction with antifungal medications to eradicate the infection. Additionally, our patient had not received any preoperative corticosteroid medications, and the cultures that showed the organism were taken shortly after the injury. The light microscopic features observed in the cornea of our patient were characteristic of a zygomycotic infection. The filaments appeared nonseptate but actually are pauciseptate as occasional irregular septations may occur. 7 The overall lack of structural support of the hyphae explains their irregular twisted or folded shape and their

Marshall et al . Post-traumatic Corneal Mucormycosis variable contour when cut in cross sections. The fact that the organisms were well seen with hematoxylin and eosin also is characteristic of this type of fungus as was their large size. The Prussian blue stain for iron, which was negative, was performed because of the history of metallic trauma and because the organisms that cause mucormycosis have been shown to be more aggressive when there is an overload of iron.l,s To our knowledge, we are the first to document the ultrastructural characteristics of Absidia corymbifera. The organisms appeared quite degenerated, consistent with the 9 days of antifungal treatment resulting in difficulty in the identification of cytoplasmic organelles with only occasional mitochondria being evident. The hyphae showed characteristics of the zygomycetes including multilamellar cell walls, a lack of septations, and a large diameter. 9 Macrophages were noted to almost completely surround most of the organisms, which showed kappa light chains in their wall, showing the host's response to this infection. 6 The elongated areas of granular electron-dense material most likely represent degenerated nuclear and other cellular components resulting from this infection. The electron-dense structures on the fungal exterior similarly may represent compacted degenerated material. Mucormycosis has a tendency to be an aggressive infection, and early surgical debridement of the affected area is considered to be important.l,s Although our patient was healthy, surgical intervention at a relatively early stage clinically appeared indicated to help to eradicate the infection in conjunction with the antifungal medications. The combination of early surgical debridement and systemic amphotericin B currently is the accepted treatment for mucormycosis.I,6,s In conclusion, the agents of mucormycosis are ubiquitous organisms and could be interpreted as contaminants

in culture. They should, however, be considered potential pathogens if isolated from a case of fulminant keratitis, even in a patient who is immunocompetent. We believe that this is the first reported case of keratitis caused by Absidia corymbifera and, as such, represents an uncommon form of mucormycosis in an otherwise healthy individual. Acknowledgment. The authors thank Mr. Peter Ripstein, ART, for performing the immunoelectron microscopic studies.

References 1. Sugar AM. Mucormycosis. Clin Infect Dis 1992; 14(Suppl 1):S126-9. 2. Ferry AP, Abedi S. Diagnosis and management of rhinoorbitocerebral mucormycosis (phycomycosis). A report of 16 personally observed cases. Ophthalmology 1983; 90: 1096-104. 3. Schwartz IN, Donnelly EH, Klintworth GK. Ocular and orbital phycomycosis. Surv Ophthalmol 1977;22:3-28. 4. Schwartz LK, Loignon LM, Webster Jr RG. Posttraumatic phycomycosis of the anterior segment. Arch Ophthalmol 1978; 96:860-3. 5. Arffa RC. Grayson's diseases of the cornea, 3rd ed. St. Louis: Mosby Year Book, 1991; chap. 10. 6. Sugar AM. Agents of mucormycosis and related species. In: Mandell GL, Douglas Jr RG, Bennett JE, eds. Principles and Practice of Infectious Diseases, 3rd ed. New York: Churchill Livingstone, 1990; 1962-72. 7. Chandler FW, Watts JC. Pathologic diagnosis of fungal infections. Chicago, IL: ASCP Press, 1987;85-95. 8. Yohai RA, Bullock JD, Aziz AA, Markert RJ. Survival factors in rhino-orbital-cerebral mucormycosis. Surv Ophthalmol 1994;39:3-22. 9. Margo C, Rabinowicz 1M, Kwon-Chung KJ, Zimmerman LE. Subacute zygomycosis of the orbit. Arch Ophthalmol 1983; 101:1580-5.

1111