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CLINICAL MICROBIOLOGY AND INFECTIOUS DISEASE Case Report Corneal Ulcer Due to Exserohilum longirostratum CHERIE L. BOUCHON, MT(ASCP)SM/ DONALD L. GREER, P H D , 2 AND CHARLES F. GENRE, MD 3 A 47-year-old man sustained a traumatic injury to the left eye, and a corneal ulcer subsequently developed. Histologic examination of the corneal tissue revealed septate hyphae with acute angle branching. The mycologic and bacterial cultures yielded a dematiaceous fungus with ellipsoidal pigmented macroconidia borne sympodially on geniculate conidiophores. The multicellular macroconidia had prominent, protruding, truncated hila. The shorter macroconidia averaged 5-7 septa, and the longer conidia 13-21 septa. Growth on V-8 agar, alternating between fluorescent light and the dark, produced macroconidia more than 200 pm in length. The isolate was identified as Exserohilum longirostratum. This is believed to be the first documented case of mycotic keratitis caused by the phaeohyphomycete E longirostratum. (Key words: Exserohilum; Mycotic keratitis; Phaeohyphomycosis) Am J Clin Pathol 1994;101:452-455. In our environment are many dematiaceous (pigmented) fungi, which can cause a variety of infections in humans. At present, there are more than 63 species of dematiaceous fungi known to cause disease in humans and animals. 1 These infections, called phaeohyphomycoses, are characterized by the presence of pigmented hyphae, pseudohyphae, and yeast-like cells in tissue. Phaeohyphomycosis occurs in both healthy and immunocompromised hosts and may present clinically as cutaneous, subcutaneous, or systemic disease. Among this group of "black" molds are the genera Bipolaris, Drechslera, Exserohilum, and Helminthosporium. In the past, several cases of human phaeohyphomycosis have been attributed to species of the genera Drechslera2'9 and Helminthosporium. 10~13 After reviewing these cases, McGinnis and colleagues' concluded that the true pathogenic species were actually of the genera Bipolaris and Exserohilum. Only species contained in these genera represented well-documented cases of phaeohyphomycosis. Many reports have documented mycotic keratitis caused by the species Drechslera3, '•' and Helminthosporium.10'1213 These isolates have since been reclassified as either Bipolaris or Exserohilum species.1,14 In a recent review of these agents in neutropenic patients, Douer and colleagues15 did not identify a single case caused by E longirostratum. We describe here the first documented case of human phaeohyphomycotic keratitis caused by E longirostratum. CASE REPORT A 47-year-old man, a native of Honduras, came to the Ochsner Clinic, New Orleans, Louisiana, on July 1 with a referral diagnosis of a perforated left corneal ulcer. The illness began in April, when the patient was on a train and was struck in the left eye by an unknown foreign object that entered through an open window. The patient was seen by physicians in Honduras and treated with antibacterial agents. Because of a slow, progressive illness, he was told that a corneal transplant was needed, but corneal tissue was unavailable. The patient was referred to the Ochsner Clinic for possible corneal transplant. When seen in the outpatient clinic in July, the patient had a normal right eye. The left eye showed a 3 x 3 mL central corneal defect. The conjunctiva was severely inflamed, and microcystic changes were noted on the corneal epithelium surrounding the ulcer. More than 75% of the anterior chamber was filled with a dense hypopyon. The remainder of the anterior chamber contained fibrin and numerous inflammatory cells. The initial diagnosis was mycotic keratitis, approximately 3 months old. In addition, severe uveitis and possible endophthalmitis were noted. At this time, corneal scrapings were sent to the microbiology laboratory for culture. The patient was treated with topical antibiotics and oral ketoconazole and placed on treatment to lower intraocular pressure. After 3 days of therapy, the anterior chamber filtrate increased, and intraocular From the Departments of 'Microbiology and ^Pathology, Alton pressure began to rise. On July 4, a corneal graft and anterior chamber Ochsner Medical Foundation, Jefferson, Louisiana; and ''•Departmentdebridement was performed. Numerous cultures of both areas were of Dermatology, Louisiana State University Medical Center, New Or- taken. Frozen section of the native cornea showed branching hyphal leans, Louisiana. elements thought to be consistent with a zygomycete. Because of nonviability of the eye and the high mortality rate of zygomycosis, the best Manuscript received October 20, 1992; revision accepted June 24, course of action was determined to be enucleation. An extensive exami1993. nation at the time of surgery revealed no sinus or extraorbital involveAddress reprint requests to Ms. Bouchon: Department of Microbiolment. As a result, systemic antifungal chemotherapy was not initiated. ogy, Alton Ochsner Medical Foundation, 1516 Jefferson Highway, Jefferson, LA 70121. The patient's postoperative course was uneventful and he recovered 452 BOUCHON, GREER, AND GENRE Corneal Ulcer Due 453 E longirostratum acid-SchifF and Gomori-Grocott methenamine silver. All tissue slides were read and interpreted by the anatomic pathologist. RESULTS Direct Examination The gram stain of the corneal scrapings, the corneal tissue obtained during surgery, and the exudate from the anterior chamber showed only rare polymorphonuclear leukocytes. No microorganisms were observed. Histologic Findings Tissue sections of the cornea stained with hematoxylin and eosin revealed focally compressed and necrotic stroma. The epithelium was effaced, and Bowman's membrane could not be identified. The periodic acid-Schiff stain revealed fungal hyphae in the necrotic zones. These elements appeared broad, septate, and pleomorphic with acute angle branching, but pigment was not visible in their walls (Fig. 1). FIG. 1. Tissue section of eye showing period acid-Schiff positive, pleomorphic hyphal elements compatible with phaeohyphomycosis (X400). well. The patient was placed on topical therapy and returned to Honduras to be followed by ophthalmology services there. Microbiologic Findings Two colonies of a dematiaceous fungus grew on the blood agar from the corneal scrapings taken in the ophthalmology clinic. No growth occurred on the chocolate agar. All cultures of the anterior chamber exudate and the corneal tissue obtained from surgery were negative for all organisms. The plates were held for 7 days. MATERIALS A N D METHODS Materials On admission from the outpatient clinic, the infected cornea was scraped, and fragments were inoculated onto trypticase soy agar plates containing 5% rabbit blood and chocolate agar (Becton-Dickinson Microbiology System, Cockeysville, MD). These routine cultures were processed in the general bacteriology laboratory. In addition, a Sabouraud's dextrose agar plate containing only .5 mg/mL of chloramphenicol (Becton-Dickinson Microbiology System, Cockeysville, MD) was inoculated and incubated in the mycology laboratory. At the time of surgery, corneal tissue debris and anterior chamber exudate were plated on agar plates as above and cultured for bacteria and fungi. Fragments of corneal tissue were also placed in formalin and submitted for histologic examinations. Methods All routine cultures for bacteria were incubated at 37 °C in 5% to 10% carbon dioxide for 7 days. The Sabouraud's dextrose media for fungi was incubated at 25 °C in the dark and held for 30 days. Fungal growth from both bacterial and fungal media was identified by their morphologic features using standard laboratory procedures. Speciation of the dematiacious mold isolated was according to McGinnis and colleagues.1 Surgical specimens were routinely stained with hematoxylin and eosin. Special stains for fungal structures included periodic Vol. 101 FIG. 2. Cylindro-ellipsoidal macroconidia with 5-8 septa produced on V-8 Agar. Note protruding hila and darkly pigmented basal and distal septa (XI60). No. 4 454 CLINICAL MICROBIOLOGY AND INFECTIOUS DISEASE Case Report TABLE 1. CHARACTERISTICS OF PRESENT ISOLATE COMPARED WITH THOSE OF BIPOLARIS, DRECHSLERA, AND EXSEROHILUM Conidia Reported as Pathogen Cortidiation Average Size (lim) + Profuse 8X26 Poor Bipolaris Drechslera Germ lubes No. of Septa Hilum Origin 3-5 Protrudes slightly 16X65 3-5 Does not protrude One or both end cells, adjacent to hilum Intermediate and end cells, not adjacent to hilum One or both end cells, adjacent to hilum; often other cells One or both end cells, adjacent to hilum, often other cells Exserohilum + Profuse 14 X90 5-12 Protrudes strongly Present + Profuse 14X90 5-12 Protrudes strongly Orientation from Basal Cell Along axis of conidium Perpendicular to conidial axis Along axis of conidium Along axis of conidium Modified from Larone.16 On Sabouraud's dextrose agar, colonies of a dematiaceous, woolly mold grew in 5 days from all fragments of the corneal scrapings received from the outpatient clinic. No growth occurred on either Sabouraud's dextrose agar or Mycosel from either surgical specimen (corneal tissue or anterior chamber exudate). These cultures were held for 30 days. A cellophane tape preparation mounted in lactophenol cotton blue was made of the fungus from the original corneal scrapings. The preparation revealed a pigmented, filamentous mold with cylindrical macroconidia compatible with a species of Drechslera. For further identification, slide cultures were prepared on potato dextrose and 15% V-8 juice agar.1 After 1 FIG. 3. Elongated macroconidia seen on V-8 Agar cultures exposed to intermittent fluorescent light. Length varied from 196-260 iim (X64). week incubation at room temperature (25-28 °C), both cultures produced abundant macroconidia. On V-8 juice agar, brown pigmented, septate hyphae were produced on geniculate conidiophores with cylindro-ellipsoidal macroconidia. The conidia had strongly protruding truncate hila. The shorter conidia averaged 5-9 septa. The basal and distal septa were darkly pigmented, and occasional warty projections were observed (Fig. 2). These findings identified the isolate as a species of Exserohilum (Table 1). Additional duplicate cultures on V-8 agar were incubated at 25 °C totally in the dark and at 25 °C with periodic exposure to fluorescent light.1 Cultures that had been exposed to intermittent fluorescent light produced unusually long, thin, rostrate macroconidia, 196-260 nm in length, with 13-21 septations (Fig. 3). These studies suggested that the isolate was E longirostratum (Table 2). Germination was studied by emulsifying a teased portion of the fungal growth in sterile water and allowing the suspension to incubate at 25 °C for 24 hours.' After incubation, germ tube formation was observed in direct wet preparations. Macroconidia produced germ tubes from the ends of the conidia along the conidial axis. The germ tube arose adjacent to the hilum of the basal cells and displaced it to one side. Based on our results and the reports by McGinnis and colleagues,1 the isolate was identified as E longiroslratum. DISCUSSION Mycotic keratitis is an opportunistic infection that usually follows trauma to the cornea. The present case illustrates that a previously unreported, usually saprophytic dematiaceous fungal species could cause a serious infection in an otherwise healthy host after trauma to the cornea. The only microorganism isolated from the specimens was the dematiaceous fungus E longiroslratum. Tissue sections stained with hematoxylin and eosin showed an acute infectious process. The mycotic infection was confirmed further by the observation of septate, branching hyphae in the native cornea. The failure to recover the fungal agent from the surgical specimens is unexplained. The small amount of specimen received may not have contained any viable fungal elements, or perhaps the topical antibiotics given before surgery inhibited the growth of the fungus in culture. AJ.C.P. • April 1994 455 BOUCHON, GREER, AND GENRE Corneal Ulcer Due to E longirostratum TABLE 2. CHARACTERISTICS OF THE MATURE CONIDIA OF PRESENT ISOLATE COMPARED WITH THOSE OF OTHER SPECIES OF EXSEROHILUM Anamorph Shape Septation Size (urn) 9-11 13X83 7-9 15X73 E longirostratum Straight, curved, or slightly bent; ellipsoidal; strongly protruding, truncate hilum; walls with irregular warty projections Same as E mcginnisii except rostrate, septum above hilum thickened and dark, walls often finely roughened Same as E rosiratum Present Same as E longirostratum E mcginnisii E rosiratum Long conidia, 13-21; short conidia, 5-9 Long conidia, 12-19; short conidia, 5-9 Long conidia, 196-260; short conidia, 16x61 Long conidia, 192-260; short conidia, 16X61 Modified from McGinnis1. An increase in phaeohyphomycotic infections has been observed in recent years. This increase is due partly to the heightened awareness of the dematiaceous fungi as agents of infection, and partly to an increase in the population of immunocompromised patients. At the Alton Ochsner Medical Foundation, we have observed that solid organ transplant patients are particularly vulnerable to infections caused by this group of fungi. Recently, the taxonomy and nomenclature of this large group of fungi have been clarified.1,17"'9 Specific identification of these fungi becomes vital, because they play a large role as infectious agents in both hospitalized patients and healthy persons. The clinician must be aware of the invasive capabilities of these fungi. This growing knowledge will aid in a more rapid diagnosis and initiation of appropriate therapy. 6. McAleer R, Kroenert DB, Elder J L, Froudist JH. Allergic bronchopulmonary disease caused by Curvularia lunata and Drechslera hawaiiensis. Thorax 1981;36:338-344. 7. Rolston K.VI, Hopfer RL, Larson DL. Infections caused by Drechslera species: Case report and review of the literature. Rev Infect Dis 1985;7:525-529. 8. Yoshimori RN, Moore RA, Itabashi HH, Fijikawa DG. Phaeohyphomycosis of brain: Granulomatous encephalitis caused by Drechslera spicifera. Am J Clin Pathol 1982;77:363-370. 9. Zapater RC, Albesi EJ, Garcia GH. Mycotic keratitis by Drechslera spicifera. Sabouraudia 1975;13:295-298. 10. Chin GN. Corneal perforation due to Helminthosporium and Mima polymorpha. Ann Opthalmol 1978; 10:607-609. 11. Dolan CT, Weed LA, Dines DE. Bronchopulmonary Helminthosporiosis. Am J Clin Pathol 1970;53:235-242. 12. Harris R, Smith RE, Wood TR, Biddle M. Helminthosporium corneal ulcers. Ann Opthalmol 1978; 10:729-733. 13. Krachmer JH, Anderson RL, Binder PS, Waring GO, Rousey JJ, Acknowledgment. The authors thank Dr. G. W. Willis, Department Meeks ES. Helminthosporium corneal ulcers. 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