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Capnocytophaga Keratitis
Capnocytophaga Keratitis A Clinicopathologic Study of Three Patients, Including Electron Microscopic Observations Ramon L. Font, MD,1,2 Venita Jay, MD,1 Raghunath P. Misra, MD, 1 Dan B. Jones, MD,1 Kirk R. Wilhelmus, MD1 Background: Histopathologic studies of this unusual keratitis caused by Capnocytophaga species have not been reported previously. Methods: The authors report the light microscopic and ultrastructural findings of three patients with a distinctive necrotizing keratitis caused by an anaerobic gramnegative bacillus. In three patients, ages 19, 81, and 91 years, a necrotizing stromal keratitis developed; two of these patients had a previous penetrating keratoplasty for pseudophakic bullous keratopathy. The first patient did not have ocular surgery previously and was treated initially for presumed Acanthamoeba keratitis. Results: By light microscopy, all three keratectomy specimens were strikingly similar and showed a necrotizing and/or suppurative stromal keratitis displaying myriad slender, fusiform, gram-negative bacilli located anterior to Descemet's membrane and extending into the deep corneal stroma, assuming a "picket fence" appearance. Cultures of the cornea in case 1 grew Capnocytophaga ochracea. For the remaining two patients, a diagnosis presumptively was made based on characteristic histopathologic features. Results of electron microscopic examination showed numerous bacilli that were mostly extracellular; occaSional organisms were phagocytosed by macrophages. Conclusion: The histopathologic features of Capnocytophaga keratitis are unique; therefore, a presumptive diagnosis can be made based on the morphology and location of the bacilli in the keratectomy specimens. To the authors' knowledge, this is the first study describing the typical histopathologic and electron microscopic findings of Capnocytophaga keratitis. Ophthalmology 1994; 101: 1 929-1 934
Capnocytophaga is a gram-negative bacillus that has rarely been associated with keratitis, occurring usually in preOriginally received: March 11, 1994. Revision accepted: June 14, 1994. I Department of Ophthalmology, Cullen Eye Institute, Baylor College of Medicine, Houston. 2 Department of Pathology, The Methodist Hospital, Baylor College of Medicine, Houston. Supported in part by grants from the Retina Research Foundation and the Lions Eye Bank, Houston, Texas, The Sid W. Richardson Foundation, Fort Worth, Texas, and Research to Prevent Blindness, Inc, New York, New York. Reprint requests to Ramon L. Font, MD, Ophthalmic Pathology Laboratory, Cullen Eye Institute, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
viously diseased or traumatized corneas and in immunocompromised patients. I - 9 In this report, we document the light microscopic and ultrastructural observations of three patients with Capnocytophaga keratitis and describe the unique histopathologic findings that are characteristic of this entity.
Case Reports Case 1. A 91-year-old white woman underwent a penetrating keratoplasty for pseudophakic bullous keratopathy 4 months previously. Subsequently, over a 2-week interval, necrosis and thinning of the graft developed inferiorly (Fig I). A partial conjunctival flap was performed, and topical neomycin-
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polymyxin B-dexamethasone was begun. However, progressive ulceration led to partial wound dehiscence. Reconstructive keratoplasty with removal of the anterior chamber intraocular lens was performed. The corneal button was submitted for histopathologic and microbiologic studies. Postoperative treatment included topical 0.5% erythromycin and 1% prednisolone acetate. The corneal graft remained clear for 3 years postoperatively. Vision was limited to counting fingers due to age-related macular degeneration. Cultures from the ground cornea on Schaedler's blood and chocolate agar plates grew Capnocytophaga ochracea. The isolated strain was highly sensitive to penicillin and clindamycin and moderately sensitive to erythromycin. Minimal inhibitory concentrations (JLg/mlj) of the C. ochracea were 0.12 or less of penicillin, 8.00 or less of carbenicillin, 0.50 or less of cephalothin, 8.00 or less of cefoxitin, 0.50 or less of cefotaxime, 0.50 or less of erythromycin, 0.25 or less of clindamycin, 0.50 or less of tetracycline, and more than 64.00 of metronidazole. The specimens included conjunctival debris, the intraocular lens, and a 7 X 5-mm corneal button, which showed ulceration and dense opacification. Microscopically, the conjunctival tissue showed no significant abnormalities. The corneal button showed a deep ulceration with perforation with a necrotic stroma infiltrated by cellular debris and neutrophils. The endothelium was focally absent paracentrally. Gram stains showed numerous slender fusiform gram-negative bacilli oriented perpendicular to the plane of Descemet's membrane (Fig 2). Case 2. A 19-year-old white man was admitted with a 2month history of necrotizing stromal keratitis in the right eye of undetermined etiology (Fig 3). He was treated previously with numerous topical antibiotics and antiviral medications. No history of trauma or known risk factors for human immunodeficiency virus were available. A presumptive diagnosis of Acanthamoeba keratitis was considered. Corneal scrapings, cultures, and biopsies were repeatedly negative. He was treated with oral ketaconazole (200 mg twice daily), topical 1% atropine four times daily, and topical 1% prednisolone every hour. Penetrating keratoplasty was performed for progressive ulceration. The keratectomy specimen was submitted for microbiologic, histopathologic, and ultrastructural studies. The corneal graft was clear 3 years and 10 months after keratoplasty. Microscopically, the corneal epithelium was thinned and attenuated centrally with focal disruption of the Bowman's layer by a fibrovascular pannus peripherally. There was a large area of ulceration centrally in which the epithelium and Bowman's layer were absent. The stromal lamellae were focally necrotic and showed areas of scarring with fibroblastic proliferation. The deep corneal stroma was markedly necrotic with swollen necrotic keratocytes intermixed with macrophages and acute and chronic inflammatory cells with scattered nuclear debris (Fig 4). Descemet's membrane was slightly wrinkled, but thickness was normal and the membrane was intact throughout. The endothelial cells showed marked degeneration. No cysts or trophozoites of Acanthamoeba were identified in the sections examined. Gram stains demonstrated the presence of gram-negative filamentous organisms located just anterior to Descemet's membrane (Fig 5). These organisms were stained with the Gomori methenamine silver stain but were acid-fast-negative. Scattered macrophages containing intracellular gram-negative organisms with similar morphologic features were present in the posterior layers of the stroma. Cultures of a portion of the keratectomy specimen (ground-up technique) were negative. Case 3. An 81-year-old white man underwent a penetrating keratoplasty and secondary intraocular lens implantation for
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necrotizing stromal keratitis and aphakia in the right eye. Four years previously, he had a penetrating keratoplasty and removal of an intraocular lens for pseudophakic bullous keratopathy in the same eye. Preoperative cultures of corneal scrapings for bacteria and fungi from the right eye were negative. The right corneal button was submitted for histopathologic examination. The corneal graft remained clear 8 months postoperatively. The patient was lost to follow-up. The specimen consisted of a cloudy corneal button which was submitted for histopathologic and ultrastructural studies. Microscopically, the corneal epithelium, basement membrane, and Bowman's layer were focally absent. The preserved epithelium was thinned and atrophic. The stromal lamellae were edematous and necrotic. Stromal scarring was present peripherally at the host-donor interface. Numerous gram-negative, acid-fast-negative, filamentous organisms were observed primarily in the deep stroma, just anterior to a slightly thickened Descemet's membrane (Fig 6). A thin collagenous membrane was present posterior to the Descemet's membrane. The endothelium was markedly attenuated.
Electron Microscopic Studies Formalin-fixed tissue from each keratectomy specimen was submitted for conventional transmission electron microscopy. Thick sections (±1 JLm) were stained with toluidine blue or paragon (Figs 6 and 7 [inset]). Thin sections were stained with uranyl acetate and lead citrate and mounted on copper grids for examination. Ultrastructural studies showed bacilli lying mostly extracellularly and located anterior to Descemet's membrane with scattered intracellular bacilli which had been phagocytosed mostly by macrophages and occasional neutrophils (Figs 7 and 8). The bacilli had a trilaminar membrane with an irregular, slightly fuzzy coat attached to their outer surfaces. Depending on the plane of sectioning, some bacilli were cut on cross-section or obliquely, whereas others were cut longitudinally. The longitudinally cut bacilli had a characteristic fusiform appearance with tapering ends (Fig 9). Some bacilli had binary fission (Fig 10).
Discussion Capnocytophaga (formerly Bacteroides ochracea and Centers for Disease Control and Prevention group DF-l) is a gram-negative fusiform bacillus known for its gliding motility and capnophilic (C0 2-loving) metabolism. IO- 14 Capnocytophaga is part of the normal human oral flora and is sometimes found in sputum and throat specimens. Three species have been found in human infections, namely C. ochracea, Capnocytophaga sputigena, and Capnocytophaga gingivalis. 10 It is a frequent cause of periodontitis and has been implicated in a variety of infections in immunocompromised and neutropenic patients. 15- 19 In this setting, it is presumed that the breakdown of the normal mucosal barrier with entry of the organism into the bloodstream may lead to bacteremia. Less commonly, Capnocytophaga infection has been described in the nonimmunocompromised host.2o
Font et al . Capnocytophaga Keratitis
Top left, Figure 1. Case 1. Clinical appearance of the left eye shows diffuse haziness and thinning of the graft inferiorly. The patient underwent a penetrating keratoplasty for pseudophakic bullous keratopathy 4 months previously. Top right, Figure 2. Case 1. Depicts numerous slender filamentous gram-negative bacilli, most of which are oriented perpendicular to the plane of Descemet's membrane (Brown and Hopps modified gram stain; original magnification, X433). Center left, Figure 3. Case 2. Clinical appearance of the right eye 1 week after onset of topical steroids. Notice the central stromal opacity with diffuse haze. Three 2-mm trephine biopsies of the peripheral cornea were performed to rule out Acanthamoeba keratitis. Center right, Figure 4. Case 2. Posterior half of keratectomy specimen depicts marked necrosis of the stromal lamellae with prominent neutrophilic infiltrate. Scattered activated fibroblasts and macrophages are observed mostly in front of Descemet's membrane which is thin and intact (hematoxylineosin; original magnification, X40). Bottom left, Figure 5. Case 2. Numerous gram-negative filamentous bacilli are shown with a "picket fence" appearance extending perpendicularly from the anterior portion of Descemet's membrane into the deep corneal stroma (Brown and Hopps modified gram stain; original magnification, X252). Bottom right, Figure 6. Case 3. View depicts deep corneal stroma containing numerous bacilli cut in different planes of section intermixed with macrophages and scattered neutrophils. Descemet's membrane is slightly thickened. A collagenous layer extends behind Descemet's membrane (Paragon stain; original magnification, X252).
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Figure 7. Case 2. Electron micrograph displays numerous bacilli located in front of Descemet's membrane (DM). Most bacilli are cut on crosssection (circle), whereas others are cut longitudinally (small arrowheads). Occasional macrophages (large arrowhead) have phagocytosed bacilli within cytoplasmic membrane-bound vacuoles (uranyl acetate and lead citrate; original magnification, X4590). Inset, thick section (±1 /Lm) demonstrates adjacent field depicting the bacilli cut on cross-section (circle) as well as longitudinally (arrowhead) within the deep corneal stroma (toluidine blue, original magnification, X 1280).
Ocular manifestations of Capnocytophaga infections reported to date are keratitis, canaliculitis, conjunctivitis, endophthalmitis, and dacryocystitis. 1-9.20,21 The predisposing factors for keratitis have included corneal edema, ocular trauma, and long-term use of topical steroids, How the organisms gain access to the eye is not definitively known, but the association with periodontitis suggests a mouth-to-hand-to-eye transmission, The infection usually involves a partially devitalized cornea either from trauma, chronic edema, or multidrug therapy, Corneal infection by Capnocytophaga was first reported by Roussel et al, 6 with the predisposing factors in their patients being corneal edema and trauma. Heidemann et al 3 reported three cases of necrotizing keratitis with stromal ring infiltrate, all occurring in a previously diseased or traumatized cornea in patients who received topical steroids. In two of these patients, the infection led to corneal perforation. One patient (case 1) reported by Heidemann et al 3 (similar to our case 2) was treated for presumed Acanthamoeba keratitis. In two of their three patients, no organisms were identified by cultures or histopathologic studies, whereas in a third patient, cultures of both cornea and contact lens yielded C. ochracea. Ormerod et al4 described a patient with severe keratitis treated with oral and topical steroid therapy that was due to C. sputigena. Pamel et al 5 reported positive cultures for C. ochracea from corneal scrapings in a 35year-old man with ocular trauma and presumed fungal keratitis. Corneal infection by Capnocytophaga canimorsus, a normal inhabitant in a dog's mouth, closely resembling the Capnocytophaga sp in humans, has been reported in a veterinarian who was struck in the eye during a tooth extraction of a poodle with severe gingivitis. 22 Rarely, Capnocytophaga infection has been reported in patients with no obvious systemic or local predisposing 1932
Figure 8. Case 3. Electron micrograph discloses numerous bacilli, some of which have been phagocytosed within macrophages. Some bacilli have been incorporated in membrane-bound lysosomes (arrowheads). Asterisk = Descemet's membrane (uranyl acetate and lead citrate; original magnification, X9000).
Font et al . Capnocytophaga Keratitis
Figure 9. Case 2. Highpower view of bacilli cut longitudinally and oriented along the anterior banded portion of Descemet's membrane (asterisk). The bacilli show a striking fusiform appearance with tapering ends (uranyl acetate and lead citrate; original magnification, X22,OOO).
factors. 2 In case 1 of our series, there was no previous surgery and no known history of trauma. Capnocytophaga keratitis has been reported in patients infected with the human immunodeficiency virus (HIV).1,7,BTicho et af reported a corneal ulcer secondary to Capnocytophaga in an intravenous drug abuser with positive HIV serology and poor dentition. Aristimuno et
Figure 10. Case 1. Deep corneal stroma shows a bacillus cut longitudinally and displays binary fission, a trilaminar membrane, and numerous particles of ribonucleic acid. Several bacilli cut on cross-section also are observed (uranyl acetate and lead citrate; original magnification, X28,900).
all described a perforating corneal ulcer with a severe corneosclerallimbal keratitis which was unresponsive to topical antibiotics in a 29-year-old woman with a history of intravenous drug abuse and positive HIV serology. Thus, Capnocytophaga keratitis adds to the spectrum of opportunistic ocular manifestations of patients with acquired immune deficiency syndrome. In the case reported by Hemady et al 8 of bilateral corneal infections in a patient infected with HIV, multiple infectious organisms were implicated, including Capnocytophaga, Candida, and other bacteria. Capnocytophaga are known to release proteolytic enzymes capable of degrading IgA and IgG and may release factors altering local neutrophil migration. These virulence factors decrease local defenses and contribute to tissue damage. 23,24 With periodontal disease, the organism is thought to act synergistically with other gram-negative anaerobic bacteria to cause destruction of alveolar bone. 16 The differential diagnosis of necrotizing keratitis caused by Capnocytophaga includes Acanthamoeba. herpes simplex, and endotoxin-producing gram-negative bacteria and fungi. Capnocytophaga may not be isolated frequently from clinical specimens by standard laboratory techniques. 17, 18 Capnocytophaga is a facultative anaerobe and requires a COr enriched atmosphere over a prolonged period for growth. Failure to grow the organism also may be due to the fact that it resides deeply within the corneal stroma and cannot be retrieved by superficial scrapings. In case 1, cultural identification of the organism in appropriate anaerobic media showed C. ochracea. While no organisms were cultured in cases 2 and 3, we retrospectively diag-
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nosed presumed CapnocylOphaga keratitis in the latter two patients. This assumption was based on the morphologic features, staining qualities, pre-Descemetic location, and ultrastructural findings of the organisms. In addition, we are not aware of any other gram-negative rod orienting itself just anterior to Descemet's membrane, giving a "picket fence" appearance. The capnophilic properties of this organism may provide the basis of its peculiar affinity for the deepest layers of the corneal stroma just anterior to Descemet's membrane. Studies25 ,26 on oxygen tension distribution and oxygen consumption in the cornea suggest that the predescemetic region contains lower oxygen tension and presumably the highest CO2 tension optimal for rapid growth of this anaerobic organism, Capnocytophaga is usually resistant to conventional antibiotic therapy and responds to antimicrobial agents that have the greatest activity against anaerobes. In vitro sensitivity testing under anaerobic conditions has demonstrated sensitivity to clindamycin, penicillin, chloramphenicol, tetracycline, and carbenicillin, a variable sensitivity to cephalosporins, and a relative resistance to aminoglycosides, polymyxins, and vancomycin. 2o,27,28 In summary, a progressive suppurative keratitis may be caused by Capnocytophaga infection. This may occur after surgery, trauma, or in immunocompromised states. The histopathologic hallmark of this infection is a suppurative and/or necrotizing keratitis containing myriad slender, fusiform, gram-negative bacilli typically extending from the anterior portion of Descemet's membrane into the deep stroma and assuming a characteristic "picket fence" appearance. Acknowledgments The authors thank John W. Reed,
MD, for contributing case 2 and Warren Cross, MD, for contributing case 3.
References 1. Aristimufio B, Nirankari VS, Hemady RK, Rodrigues MM. Spontaneous ulcerative keratitis in immunocompromised patients. Am J Ophthalmol 1993; 115:202-8. 2. Eiferman RA, Levartovsky S, Box JD. Anaerobic Capnocytophaga corneal ulcer [letter]. Am J OphthalmoI1988;105: 427. 3. Heidemann DG, Pflugfelder SC, Kronish J, et ai, Necrotizing keratitis caused by Capnocytophaga ochracea. Am J Ophthalmol 1988; 105:655-60, 4, Ormerod LD, Foster CS, Paton BG, et aL Ocular Capnocytophaga infection in an edentulous, immunocompetent host. Cornea 1988;7:218-22, 5, Pamel GJ, Buckley DJ, Frucht J, et al. Capnocytophaga keratitis [letter], Am J Ophthalmol 1989; 107: 193-4. 6, Roussel TJ, Osato MS, Wilhelm us KR. Capnocytophaga keratitis. Br J Ophthalmol 1985;69: 187-8. 7, Ticho BH, Urban RC Jr, Safran MJ, Saggau DD, Capnocytophaga keratitis associated with poor dentition and human immunodeficiency virus infection [letter]. Am J Ophthalmol 1990; 109:352-3.
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8. Hemady RK, Griffin N, Aristimuno B. Recurrent corneal infections in a patient with the acquired immunodeficiency syndrome. Cornea 1993;12:266-9. 9. Rubsamen PE, McLeish WM, Pflugfelder S, Miller D. Capnocytophaga endophthalmitis, Ophthalmology 1993;100: 456-9, 10, Leadbetter ER, Holt SC, Socransky SS. Capnocytophaga: new genus of gram-negative gliding bacteria, L General characteristics, taxonomic considerations and significance, Arch Microbiol 1979;122:9-16. 11. Newman MG, Sutter VL, Pickett MJ, et aL Detection, identification, and comparisons of Capnocytophaga, Bacteroides ochraceous, and DF-1. J Clin Microbiol 1979;10:557-62, 12, Williams BL, Hollis D, Holdeman LV, Synonymy of strains of Center for Disease Control group DF-I with species of Capnocytophaga. J Clin Microbiol 1979; 10:550-6, 13, Socransky SS, Holt SC, Leadbetter ER, et al, Capnocytophaga: a new genus of gram-negative gliding bacteria, III, Physiological characterization, Arch Microbiol 1979; 122: 29-33, 14, Bernard K, Cooper C, Tessier S, Ewan EP. Use of chemotaxonomy as an aid to differentiate among Capnocytophaga species, CDC group DF-3, and aerotolerant stains of Leptotrichia buccalis. J Clin Microbiol 1991 ;29:2263-5, 15, Warren JS, Allen SD, Clinical, pathogenetic, and laboratory features of Capnocytophaga infections, Am J Clin Pathol 1986;86:513-18, 16, Newman MG. The role of Bacteroides melaninogenicus and other anaerobes in peridontal infections. Rev Infect Dis 1979; 1:313-24. 17, Forlenza SW, Newman MG, Lipsey AI, et al. Capnocytophaga sepsis: a newly recognised clinical entity in granulocytopenic patients. Lancet 1980;1:567-8, 18, Gilligan PH, McCarthy LR, Bissett BK. Capnocytophaga ochracea septicemia, J Clin Microbiol 1981;13:643-5, 19, Hawkey PM, Malnick H, Glover SA, et aL Capnocytophaga ochracea infection: two cases and a review of the published work. J Clin Pathol 1984;37:1066-70, 20, Parenti DM, Snydman DR, Capnocytophaga species: infections in nonimmunocompromised and immunocompromised hosts, J Infect Dis 1985;151:140-7, 21. Jones DB, Robinson NM. Anaerobic ocular infections, Trans Am Acad Ophthalmol Otolaryngol 1977;83:0P30931. 22, de Smet MD, Chan CC, Nussenblatt RB, Palestine AG, Capnocytophaga canimorsus as the cause of a chronic corneal infection [letter]. Am J Ophthalmol 1990;109:240-2, 23. Fumarola D, Miragliotta G. Virulence factors of Capnocytophaga: newer contributions [letter]. Am J Clin Pathol 1982;73:425-6, 24, Shurin SB, Socransky SS, Sweeney E, Stossel TP, A neutrophil disorder induced by Capnocytophaga, a dental microorganism. N Engl J Med 1979;301:849-54. 25, Freeman RD, Oxygen consumption by the component layers of the cornea. J Physiol (Lond) 1972;225: 15-32. 26, Fatt I, Freeman RD, Lin D. Oxygen tension distributions in the cornea: a re-examination, Exp Eye Res 1974;18:35765, 27, Forlenza SW, Newman MG, Horikoshi AL, Blachman U. Antimicrobial susceptibility of Capnocytophaga. Antimicrob Agents Chemother 1981; 19: 144-6, 28, Sutter VL, Pyeatt D, Kwok yy, In vitro susceptibility of Capnocytophaga strains to 18 antimicrobial agents, Antimicrob Agents Chemother 1981 ;20:270-1,
Capnocytophaga is a gram-negative bacillus that has rarely been associated with keratitis, occurring usually in preOriginally received: March 11, 1994. Revision accepted: June 14, 1994. I Department of Ophthalmology, Cullen Eye Institute, Baylor College of Medicine, Houston. 2 Department of Pathology, The Methodist Hospital, Baylor College of Medicine, Houston. Supported in part by grants from the Retina Research Foundation and the Lions Eye Bank, Houston, Texas, The Sid W. Richardson Foundation, Fort Worth, Texas, and Research to Prevent Blindness, Inc, New York, New York. Reprint requests to Ramon L. Font, MD, Ophthalmic Pathology Laboratory, Cullen Eye Institute, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
viously diseased or traumatized corneas and in immunocompromised patients. I - 9 In this report, we document the light microscopic and ultrastructural observations of three patients with Capnocytophaga keratitis and describe the unique histopathologic findings that are characteristic of this entity.
Case Reports Case 1. A 91-year-old white woman underwent a penetrating keratoplasty for pseudophakic bullous keratopathy 4 months previously. Subsequently, over a 2-week interval, necrosis and thinning of the graft developed inferiorly (Fig I). A partial conjunctival flap was performed, and topical neomycin-
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polymyxin B-dexamethasone was begun. However, progressive ulceration led to partial wound dehiscence. Reconstructive keratoplasty with removal of the anterior chamber intraocular lens was performed. The corneal button was submitted for histopathologic and microbiologic studies. Postoperative treatment included topical 0.5% erythromycin and 1% prednisolone acetate. The corneal graft remained clear for 3 years postoperatively. Vision was limited to counting fingers due to age-related macular degeneration. Cultures from the ground cornea on Schaedler's blood and chocolate agar plates grew Capnocytophaga ochracea. The isolated strain was highly sensitive to penicillin and clindamycin and moderately sensitive to erythromycin. Minimal inhibitory concentrations (JLg/mlj) of the C. ochracea were 0.12 or less of penicillin, 8.00 or less of carbenicillin, 0.50 or less of cephalothin, 8.00 or less of cefoxitin, 0.50 or less of cefotaxime, 0.50 or less of erythromycin, 0.25 or less of clindamycin, 0.50 or less of tetracycline, and more than 64.00 of metronidazole. The specimens included conjunctival debris, the intraocular lens, and a 7 X 5-mm corneal button, which showed ulceration and dense opacification. Microscopically, the conjunctival tissue showed no significant abnormalities. The corneal button showed a deep ulceration with perforation with a necrotic stroma infiltrated by cellular debris and neutrophils. The endothelium was focally absent paracentrally. Gram stains showed numerous slender fusiform gram-negative bacilli oriented perpendicular to the plane of Descemet's membrane (Fig 2). Case 2. A 19-year-old white man was admitted with a 2month history of necrotizing stromal keratitis in the right eye of undetermined etiology (Fig 3). He was treated previously with numerous topical antibiotics and antiviral medications. No history of trauma or known risk factors for human immunodeficiency virus were available. A presumptive diagnosis of Acanthamoeba keratitis was considered. Corneal scrapings, cultures, and biopsies were repeatedly negative. He was treated with oral ketaconazole (200 mg twice daily), topical 1% atropine four times daily, and topical 1% prednisolone every hour. Penetrating keratoplasty was performed for progressive ulceration. The keratectomy specimen was submitted for microbiologic, histopathologic, and ultrastructural studies. The corneal graft was clear 3 years and 10 months after keratoplasty. Microscopically, the corneal epithelium was thinned and attenuated centrally with focal disruption of the Bowman's layer by a fibrovascular pannus peripherally. There was a large area of ulceration centrally in which the epithelium and Bowman's layer were absent. The stromal lamellae were focally necrotic and showed areas of scarring with fibroblastic proliferation. The deep corneal stroma was markedly necrotic with swollen necrotic keratocytes intermixed with macrophages and acute and chronic inflammatory cells with scattered nuclear debris (Fig 4). Descemet's membrane was slightly wrinkled, but thickness was normal and the membrane was intact throughout. The endothelial cells showed marked degeneration. No cysts or trophozoites of Acanthamoeba were identified in the sections examined. Gram stains demonstrated the presence of gram-negative filamentous organisms located just anterior to Descemet's membrane (Fig 5). These organisms were stained with the Gomori methenamine silver stain but were acid-fast-negative. Scattered macrophages containing intracellular gram-negative organisms with similar morphologic features were present in the posterior layers of the stroma. Cultures of a portion of the keratectomy specimen (ground-up technique) were negative. Case 3. An 81-year-old white man underwent a penetrating keratoplasty and secondary intraocular lens implantation for
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necrotizing stromal keratitis and aphakia in the right eye. Four years previously, he had a penetrating keratoplasty and removal of an intraocular lens for pseudophakic bullous keratopathy in the same eye. Preoperative cultures of corneal scrapings for bacteria and fungi from the right eye were negative. The right corneal button was submitted for histopathologic examination. The corneal graft remained clear 8 months postoperatively. The patient was lost to follow-up. The specimen consisted of a cloudy corneal button which was submitted for histopathologic and ultrastructural studies. Microscopically, the corneal epithelium, basement membrane, and Bowman's layer were focally absent. The preserved epithelium was thinned and atrophic. The stromal lamellae were edematous and necrotic. Stromal scarring was present peripherally at the host-donor interface. Numerous gram-negative, acid-fast-negative, filamentous organisms were observed primarily in the deep stroma, just anterior to a slightly thickened Descemet's membrane (Fig 6). A thin collagenous membrane was present posterior to the Descemet's membrane. The endothelium was markedly attenuated.
Electron Microscopic Studies Formalin-fixed tissue from each keratectomy specimen was submitted for conventional transmission electron microscopy. Thick sections (±1 JLm) were stained with toluidine blue or paragon (Figs 6 and 7 [inset]). Thin sections were stained with uranyl acetate and lead citrate and mounted on copper grids for examination. Ultrastructural studies showed bacilli lying mostly extracellularly and located anterior to Descemet's membrane with scattered intracellular bacilli which had been phagocytosed mostly by macrophages and occasional neutrophils (Figs 7 and 8). The bacilli had a trilaminar membrane with an irregular, slightly fuzzy coat attached to their outer surfaces. Depending on the plane of sectioning, some bacilli were cut on cross-section or obliquely, whereas others were cut longitudinally. The longitudinally cut bacilli had a characteristic fusiform appearance with tapering ends (Fig 9). Some bacilli had binary fission (Fig 10).
Discussion Capnocytophaga (formerly Bacteroides ochracea and Centers for Disease Control and Prevention group DF-l) is a gram-negative fusiform bacillus known for its gliding motility and capnophilic (C0 2-loving) metabolism. IO- 14 Capnocytophaga is part of the normal human oral flora and is sometimes found in sputum and throat specimens. Three species have been found in human infections, namely C. ochracea, Capnocytophaga sputigena, and Capnocytophaga gingivalis. 10 It is a frequent cause of periodontitis and has been implicated in a variety of infections in immunocompromised and neutropenic patients. 15- 19 In this setting, it is presumed that the breakdown of the normal mucosal barrier with entry of the organism into the bloodstream may lead to bacteremia. Less commonly, Capnocytophaga infection has been described in the nonimmunocompromised host.2o
Font et al . Capnocytophaga Keratitis
Top left, Figure 1. Case 1. Clinical appearance of the left eye shows diffuse haziness and thinning of the graft inferiorly. The patient underwent a penetrating keratoplasty for pseudophakic bullous keratopathy 4 months previously. Top right, Figure 2. Case 1. Depicts numerous slender filamentous gram-negative bacilli, most of which are oriented perpendicular to the plane of Descemet's membrane (Brown and Hopps modified gram stain; original magnification, X433). Center left, Figure 3. Case 2. Clinical appearance of the right eye 1 week after onset of topical steroids. Notice the central stromal opacity with diffuse haze. Three 2-mm trephine biopsies of the peripheral cornea were performed to rule out Acanthamoeba keratitis. Center right, Figure 4. Case 2. Posterior half of keratectomy specimen depicts marked necrosis of the stromal lamellae with prominent neutrophilic infiltrate. Scattered activated fibroblasts and macrophages are observed mostly in front of Descemet's membrane which is thin and intact (hematoxylineosin; original magnification, X40). Bottom left, Figure 5. Case 2. Numerous gram-negative filamentous bacilli are shown with a "picket fence" appearance extending perpendicularly from the anterior portion of Descemet's membrane into the deep corneal stroma (Brown and Hopps modified gram stain; original magnification, X252). Bottom right, Figure 6. Case 3. View depicts deep corneal stroma containing numerous bacilli cut in different planes of section intermixed with macrophages and scattered neutrophils. Descemet's membrane is slightly thickened. A collagenous layer extends behind Descemet's membrane (Paragon stain; original magnification, X252).
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Figure 7. Case 2. Electron micrograph displays numerous bacilli located in front of Descemet's membrane (DM). Most bacilli are cut on crosssection (circle), whereas others are cut longitudinally (small arrowheads). Occasional macrophages (large arrowhead) have phagocytosed bacilli within cytoplasmic membrane-bound vacuoles (uranyl acetate and lead citrate; original magnification, X4590). Inset, thick section (±1 /Lm) demonstrates adjacent field depicting the bacilli cut on cross-section (circle) as well as longitudinally (arrowhead) within the deep corneal stroma (toluidine blue, original magnification, X 1280).
Ocular manifestations of Capnocytophaga infections reported to date are keratitis, canaliculitis, conjunctivitis, endophthalmitis, and dacryocystitis. 1-9.20,21 The predisposing factors for keratitis have included corneal edema, ocular trauma, and long-term use of topical steroids, How the organisms gain access to the eye is not definitively known, but the association with periodontitis suggests a mouth-to-hand-to-eye transmission, The infection usually involves a partially devitalized cornea either from trauma, chronic edema, or multidrug therapy, Corneal infection by Capnocytophaga was first reported by Roussel et al, 6 with the predisposing factors in their patients being corneal edema and trauma. Heidemann et al 3 reported three cases of necrotizing keratitis with stromal ring infiltrate, all occurring in a previously diseased or traumatized cornea in patients who received topical steroids. In two of these patients, the infection led to corneal perforation. One patient (case 1) reported by Heidemann et al 3 (similar to our case 2) was treated for presumed Acanthamoeba keratitis. In two of their three patients, no organisms were identified by cultures or histopathologic studies, whereas in a third patient, cultures of both cornea and contact lens yielded C. ochracea. Ormerod et al4 described a patient with severe keratitis treated with oral and topical steroid therapy that was due to C. sputigena. Pamel et al 5 reported positive cultures for C. ochracea from corneal scrapings in a 35year-old man with ocular trauma and presumed fungal keratitis. Corneal infection by Capnocytophaga canimorsus, a normal inhabitant in a dog's mouth, closely resembling the Capnocytophaga sp in humans, has been reported in a veterinarian who was struck in the eye during a tooth extraction of a poodle with severe gingivitis. 22 Rarely, Capnocytophaga infection has been reported in patients with no obvious systemic or local predisposing 1932
Figure 8. Case 3. Electron micrograph discloses numerous bacilli, some of which have been phagocytosed within macrophages. Some bacilli have been incorporated in membrane-bound lysosomes (arrowheads). Asterisk = Descemet's membrane (uranyl acetate and lead citrate; original magnification, X9000).
Font et al . Capnocytophaga Keratitis
Figure 9. Case 2. Highpower view of bacilli cut longitudinally and oriented along the anterior banded portion of Descemet's membrane (asterisk). The bacilli show a striking fusiform appearance with tapering ends (uranyl acetate and lead citrate; original magnification, X22,OOO).
factors. 2 In case 1 of our series, there was no previous surgery and no known history of trauma. Capnocytophaga keratitis has been reported in patients infected with the human immunodeficiency virus (HIV).1,7,BTicho et af reported a corneal ulcer secondary to Capnocytophaga in an intravenous drug abuser with positive HIV serology and poor dentition. Aristimuno et
Figure 10. Case 1. Deep corneal stroma shows a bacillus cut longitudinally and displays binary fission, a trilaminar membrane, and numerous particles of ribonucleic acid. Several bacilli cut on cross-section also are observed (uranyl acetate and lead citrate; original magnification, X28,900).
all described a perforating corneal ulcer with a severe corneosclerallimbal keratitis which was unresponsive to topical antibiotics in a 29-year-old woman with a history of intravenous drug abuse and positive HIV serology. Thus, Capnocytophaga keratitis adds to the spectrum of opportunistic ocular manifestations of patients with acquired immune deficiency syndrome. In the case reported by Hemady et al 8 of bilateral corneal infections in a patient infected with HIV, multiple infectious organisms were implicated, including Capnocytophaga, Candida, and other bacteria. Capnocytophaga are known to release proteolytic enzymes capable of degrading IgA and IgG and may release factors altering local neutrophil migration. These virulence factors decrease local defenses and contribute to tissue damage. 23,24 With periodontal disease, the organism is thought to act synergistically with other gram-negative anaerobic bacteria to cause destruction of alveolar bone. 16 The differential diagnosis of necrotizing keratitis caused by Capnocytophaga includes Acanthamoeba. herpes simplex, and endotoxin-producing gram-negative bacteria and fungi. Capnocytophaga may not be isolated frequently from clinical specimens by standard laboratory techniques. 17, 18 Capnocytophaga is a facultative anaerobe and requires a COr enriched atmosphere over a prolonged period for growth. Failure to grow the organism also may be due to the fact that it resides deeply within the corneal stroma and cannot be retrieved by superficial scrapings. In case 1, cultural identification of the organism in appropriate anaerobic media showed C. ochracea. While no organisms were cultured in cases 2 and 3, we retrospectively diag-
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nosed presumed CapnocylOphaga keratitis in the latter two patients. This assumption was based on the morphologic features, staining qualities, pre-Descemetic location, and ultrastructural findings of the organisms. In addition, we are not aware of any other gram-negative rod orienting itself just anterior to Descemet's membrane, giving a "picket fence" appearance. The capnophilic properties of this organism may provide the basis of its peculiar affinity for the deepest layers of the corneal stroma just anterior to Descemet's membrane. Studies25 ,26 on oxygen tension distribution and oxygen consumption in the cornea suggest that the predescemetic region contains lower oxygen tension and presumably the highest CO2 tension optimal for rapid growth of this anaerobic organism, Capnocytophaga is usually resistant to conventional antibiotic therapy and responds to antimicrobial agents that have the greatest activity against anaerobes. In vitro sensitivity testing under anaerobic conditions has demonstrated sensitivity to clindamycin, penicillin, chloramphenicol, tetracycline, and carbenicillin, a variable sensitivity to cephalosporins, and a relative resistance to aminoglycosides, polymyxins, and vancomycin. 2o,27,28 In summary, a progressive suppurative keratitis may be caused by Capnocytophaga infection. This may occur after surgery, trauma, or in immunocompromised states. The histopathologic hallmark of this infection is a suppurative and/or necrotizing keratitis containing myriad slender, fusiform, gram-negative bacilli typically extending from the anterior portion of Descemet's membrane into the deep stroma and assuming a characteristic "picket fence" appearance. Acknowledgments The authors thank John W. Reed,
MD, for contributing case 2 and Warren Cross, MD, for contributing case 3.
References 1. Aristimufio B, Nirankari VS, Hemady RK, Rodrigues MM. Spontaneous ulcerative keratitis in immunocompromised patients. Am J Ophthalmol 1993; 115:202-8. 2. Eiferman RA, Levartovsky S, Box JD. Anaerobic Capnocytophaga corneal ulcer [letter]. Am J OphthalmoI1988;105: 427. 3. Heidemann DG, Pflugfelder SC, Kronish J, et ai, Necrotizing keratitis caused by Capnocytophaga ochracea. Am J Ophthalmol 1988; 105:655-60, 4, Ormerod LD, Foster CS, Paton BG, et aL Ocular Capnocytophaga infection in an edentulous, immunocompetent host. Cornea 1988;7:218-22, 5, Pamel GJ, Buckley DJ, Frucht J, et al. Capnocytophaga keratitis [letter], Am J Ophthalmol 1989; 107: 193-4. 6, Roussel TJ, Osato MS, Wilhelm us KR. Capnocytophaga keratitis. Br J Ophthalmol 1985;69: 187-8. 7, Ticho BH, Urban RC Jr, Safran MJ, Saggau DD, Capnocytophaga keratitis associated with poor dentition and human immunodeficiency virus infection [letter]. Am J Ophthalmol 1990; 109:352-3.
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