- Email: [email protected]
Corneal microsporidiosis
Corneal Microsporidiosis Report of Case, including Electron Microscopic Observations Ramon L. Font, MD,1 Ameed N. Samaha, MD,2 Mary J. Keener,1 Patricia Chevez-Barrios, MD,1 John D. Goosey, MD2 Objective: To report a case of corneal stromal infection caused by a protozoon of the genus Microsporidia, including clinical, histopathologic, and electron microscopic observations. Design: Case report. Methods: Light and electron microscopy studies were performed on keratectomy specimens from a 67year-old immunocompetent man who had a unilateral chronic stromal keratitis that was refractory to medical treatment. Initial corneal biopsy followed by lamellar and penetrating keratoplasty were performed on the patient. All the specimens were studied histopathologically. Results: Light microscopy of the corneal biopsy and the subsequent keratectomy specimens demonstrated myriad small, round to oval microsporidial organisms measuring 3.5 to 5.0 m in length that stained positively with the periodic acid–Schiff, Grocott-methenamine silver, and acid-fast methods and were gram positive. Electron microscopic observations demonstrated viable blastospores that had a thin osmiophilic outer cell wall and contained 11 to 13 coils of the filament. The light and electron microscopic features, the tinctorial characteristics, and the selective corneal stromal involvement are consistent with microsporidial keratitis. Conclusions: Microsporidiosis should be considered in the differential diagnosis of a culture-negative stromal keratitis refractory to medical treatment. The diagnosis can be easily established based on the morphologic features of the protozoa in the keratectomy specimens. No effective medical treatment for the stromal disease is available. Full-thickness keratoplasty is suggested because, in our patient, lamellar keratoplasty did not preclude recurrence of the disease. Ophthalmology 2000;107:1769 –1775 © 2000 by the American Academy of Ophthalmology. Microsporidia are small (3.5–5.0 m in length by 2.0 –3.0 m in width), oval obligate intracellular eukaryotic protozoan parasites that belong to the phylum Microspora. Microsporidiosis, the disease caused by these organisms, has been recognized in both vertebrates and invertebrates. More than 1000 species have been classified into approximately 100 genera, and at least 13 species have been reported to infect mammals.1– 4 Phylogenetically, the microsporidia are early eukaryotic organisms because they have a true nucleus, possess prokaryote-like ribosomes, and lack mitochondria.3 Five genera (Enterocytozoon species, Septata species, Pleistophora species, Encephalitozoon species, and
Originally received: August 5, 1999. Accepted: May 5, 2000. Manuscript no. 99539. 1 Department of Ophthalmology, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas. 2 Department of Ophthalmology and Visual Sciences, University of Texas Health Science Center, Houston, Texas. Supported in part by grants from the Retina Research Foundation, Houston, 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, Houston, TX 77030. E-mail: [email protected]. © 2000 by the American Academy of Ophthalmology Published by Elsevier Science Inc.
Nosema species) as well as unclassified microsporidia (collectively referred to as Microsporidium) have been associated with human disease involving mostly immunocompromised patients.1– 4 The affected patients may exhibit broad clinical manifestations including intestinal, pulmonary, ocular, muscular, and renal disease. Only two species, Nosema and Encephalitozoon, are known to cause ocular infections. The first report of corneal involvement in humans occurred in 1973.5 Two other cases involving the corneal stroma were reported in 1981 and 1990 by Pinnolis et al6 and Davis et al,7 respectively. Reports of superficial punctate epithelial keratopathy and conjunctivitis caused by Microsporidia have increased.8 –18 Two clinical presentations of ocular microsporidiosis are observed: a corneal stromal keratitis, which occurs in immunocompetent patients and is caused by Nosema corneum (renamed Vittaforma corneae19) and a superficial punctate keratoconjunctivitis occurring in acquired immune deficiency syndrome (AIDS) patients caused by Encephalitozoon species. We report an additional case of microsporidial stromal keratitis caused by N. corneum (V. cornea), which was studied using special stains and electron microscopy. ISSN 0161-6420/00/$–see front matter PII S0161-6420(00)00285-2
1769
Ophthalmology Volume 107, Number 9, September 2000
Case Report Clinical Summary and Histopathologic Findings A 67-year-old Hispanic man was referred from Monterey, Mexico because of an 8-month history of persistent stromal keratitis and progressively decreased visual acuity in his right eye. Initially, he was treated with various topical antibiotics, and later this was managed as a case of herpes simplex viral stromal keratitis with topical steroids (prednisolone acetate or fluorometholone) and antivirals (Viroptic and acyclovir topical and systemic) without resolution of the condition. Ophthalmologic examination in September 1997 revealed an uncorrected visual acuity of 20/80 in the right eye and 20/20 in the left eye. The right cornea showed dense grayish white stromal infiltrates involving its temporal aspect and extending across the visual axis, sparing only the nasal perilimbal area (Fig 1). The infiltrates centrally had a crystalline-like pattern. Superficial corneal vascularization was present temporally to approximately 3 mm from the limbus. The overlying epithelium was spared. The conjunctiva was not injected, and no follicular or papillary reactions were noted. The anterior chamber was quiet in the right eye. The left eye was entirely normal. After discontinuing the topical steroids (fluorometholone), the ophthalmologic examination revealed significant conjunctival injection and increased corneal vascularization peripherally (Fig 2). The appearance of the corneal infiltrates was essentially unchanged. The deep stroma up to the level of Descemet’s membrane was involved with the infiltrative process (Fig 3). Scattered infiltrates were noted on the endothelial surface. The results of conjunctival and corneal swabs and scrapings for bacterial, chlamydial, fungal, and herpes virus cultures were all negative. A corneal punch biopsy was performed on September 17, 1997. A histopathologic diagnosis of microsporidial stromal keratitis was made, and subsequently the patient began oral albendazole 400 mg three times daily and fluorometholone four times daily. Topical fumagillin bicyclohexylammonium salt (Fumadil B) 3 mg/ml, which is equivalent to 70 g/ml of fumagillin, was started by mid-October 1997 every hour while the patient was awake for 2 weeks, then six times daily for 2 weeks. After 6 and 4 weeks of treatment with albendazole and fumagillin bicyclohexylammonium salt, respectively, the right cornea showed no clinical improvement and the visual acuity remained unchanged. Fumagillin bicyclohexylammonium salt was well tolerated during the course of treatment with no adverse side effects. Stool, urine, and sputum specimens tested negative for Microsporidia on two occasions. The patient was seronegative for the human immunodeficiency virus. On November 5, 1997, an eccentric lamellar keratoplasty 9.5 mm in diameter was performed. The lamellar stromal dissection was carried deep just anterior to Descemet’s membrane. After surgery, albendazole and fumagillin bicyclohexylammonium salt six times daily were resumed with the addition of ofloxacin (Ocuflox) four times daily to the right eye. Complete epithelialization of the lamellar graft was achieved on the fifth day after surgery. One week after surgery, recurrence of infiltrates at different loci in the interface were noted (Figs 4 and 5). One month later, the interface infiltrates and opacities had spread anteriorly and started to involve the graft. On December 17, 1997, a penetrating keratoplasty was performed. The patient was last examined on May 22, 1998. At that time, the graft remained clear without evidence of recurrence (Fig 6). On November 5, 1997, a deep lamellar keratectomy of the right eye was performed. Microscopically, the lamellar graft showed numerous microsporidial organisms involving the deep corneal stroma (Fig 7). The possibility of recurrence in the lamellar bed
1770
was anticipated. Some of the organisms were birefringent under polarized light (Fig 8). On December 17, 1997, a penetrating graft was performed. Microscopically, the corneal epithelium showed moderate intracellular edema. Bowman’s layer appeared intact. The stroma was moderately edematous with focal areas of scarring. One of the fragments corresponded only to the deep corneal stroma, including Descemet’s membrane and the endothelium. The deep stroma contained numerous oval-shaped organisms that measured 3.5 to 5 m in length by 2.5 to 3.0 m in width. The organisms stained positively with periodic acid–Schiff and Grocott-methenamine silver methods (Figs 9 and 10) and were gram positive. Some of the organisms were also acid-fast positive (Fig 11). Descemet’s membrane was focally ruptured. Numerous epithelioid histiocytes and scattered multinucleated giant cells were clinging to Descemet’s membrane at the site of rupture. In this area, numerous pigmentladen macrophages and free melanin granules were also observed.
Electron Microscopy Findings The formalin-fixed keratectomy specimen was placed in 2% glutaraldehyde, postfixed in 1% osmium tetroxide, and processed for conventional electron microscopy. Thick section (1 m) revealed masses of round to oval sporoblasts, measuring 3.5 to 5.0 m in length by 2.5 to 3.0 m in width, dissecting amongst the corneal lamellae (Fig 12). Electron microscopy disclosed scattered viable sporoblasts, each surrounded by a thick capsule. Many degenerated organisms without internal structures were also present (Fig 13). The latter demonstrated a thin, osmiophilic outer layer and a thicker, lucent inner layer. The sporoblasts appeared as free extracellular organisms that were not surrounded by a parasitophorous vacuole. Precursor forms (meronts) were interspersed among the sporoblasts (Fig 13). Under higher magnification, the mature sporoblasts disclosed from 11 to 13 coils of the filament (Fig 14). The collagen fibrils surrounding the organisms appeared intact. Some sporoblasts disclosed diplokaryotic arrangement of the nuclei, which is believed to be the only character that conforms to the genus Nosema (Fig 15).19
Discussion Microsporidia are becoming increasingly recognized as opportunistic infectious pathogens in patients with AIDS. They are ubiquitous in nature and are widely distributed in vertebrates and invertebrates. Bilateral diffuse punctate epithelial keratopathy and conjunctivitis10 –11,13,17,18 characterize ocular microsporidiosis in AIDS patients. Some documented cases have shown only mild conjunctivitis but no keratopathy.16,17 In contrast, ocular microsporidiosis in immunocompetent patients usually shows unilateral corneal stromal involvement without epitheliopathy or iritis. Of the four reported patients with severe stromal microsporidial keratitis, corneal ulcers occurred in two,6,7 and one eye was enucleated because of a perforated corneal ulcer.6 The source of infection for humans and the routes of transmission are unknown; however, they are thought to be either orofecal, resulting from direct inoculation, or occurring after trauma.5 Direct inoculation may occur with close contacts with domestic animals such as cats and birds20,21; it may also spread from other infected persons.22,23 In our patient, such contacts were reported by the patient not to
Font et al 䡠 Corneal Microsporidiosis
Figure 1. Clinical appearance of the right cornea at time of initial examination showing the dense, whitish-gray stromal infiltrates temporally with a crystalline-like pattern as they progress centrally. Minimal conjunctival injection is present.
Figure 3. A slit-lamp view of the stromal infiltrates depicted in Figure 2 shows deep stromal involvement reaching the plane of Descemet’s membrane.
Figure 5. A cross-sectional slit-lamp view of the lamellar graft in Figure 4 demonstrating a clear graft with the recurrent infiltrates that are localized at the interface.
Figure 2. The clinical appearance of the same cornea 9 days after tapering and stopping the steroid eye drops. The bulbar conjunctiva is significantly injected. Prominent superficial peripheral corneal vascularization is observed temporally. The stromal infiltrates are essentially similar to those observed in Figure 1.
Figure 4. Clinical appearance of the right eye 1 week after the lamellar keratoplasty. Note the recurrence of the stromal infiltrates in the interface peripherally as well as the residual opacities (infiltrates) just in front of Descemet’s membrane. The lamellar graft has been fully epithelialized.
Figure 6. Clinical appearance of the right cornea 6 months after the penetrating keratoplasty shows a clear eccentric graft with no evidence of recurrence of the infection. A loose suture with a mucous plug is noted at the 5:30-o’clock position. Best-corrected visual acuity at this time was 20/40.
1771
Ophthalmology Volume 107, Number 9, September 2000
Figure 7. Deep lamellar keratectomy specimen containing masses of Nosema organisms within the deep corneal stroma. Descemet’s membrane appears intact. The endothelial cells are decreased in number. Scattered macrophages are observed along the posterior corneal surface (stain, hematoxylin– eosin; original magnification, ⫻100).
Figure 9. High-power view disclosing numerous round to oval, periodic acid–Schiff-positive sporoblasts in the deep corneal stroma. An artifactitious splitting of the anterior fetal portion from the posterior adult portion of Descemet’s membrane is observed (stain, periodic acid–Schiff; original magnification, ⫻252).
Figure 11. The vast majority of the intact sporoblasts are acid-fast positive. Scattered swollen degenerated organisms are acid-fast negative (stain, acid-fast bacilli; original magnification, ⫻252).
1772
Figure 8. Same field shown in Figure 7, depicting that some of the organisms are birefringent under examination with polarized light (stain, hematoxyilin– eosin; original magnification, ⫻100).
Figure 10. Similar field to that depicted in Figure 9 showing the round to oval sporoblasts measuring 3.0 to 5.0 m in length involving the deep corneal stroma. Few organisms did not stain with this technique (stain, Grocott-methenamine silver; original magnification, ⫻252).
Figure 12. Thick section (1 m) shows myriad round to oval sporoblasts dissecting among the corneal lamellae. Few empty carcasses of degenerated organisms are also observed (stain, Toluidine blue; original magnification, ⫻322).
Font et al 䡠 Corneal Microsporidiosis
Figure 13. This electron micrograph depicts numerous degenerated sporoblasts (black asterisk) that disclose an osmiophilic thin outer layer and a thicker, inner lucent layer. Several meronts (m) are interspersed among the degenerated sporoblasts. A viable sporoblast is identified (white asterisk). Mature collagen fibers (C) are present (original magnification, ⫻16,200).
have occurred, making the mode of transmission difficult to determine. Microsporidia are small, obligate intracellular parasites that produce infective spores. They are fastidious organisms that are difficult to culture. The organism has been isolated using special tissue culture techniques, which are available only in a few specialized laboratories, making this method impractical for routine diagnosis.12,22 Alcohol-fixed cytologic samples of scrapings from the conjunctiva, corneal epithelium, or both or biopsy specimens have proven very useful for demonstrating microsporidial blastospores.7,16 Often conjunctival scrapings alone provide a satisfactory specimen for cytologic diagnosis in microsporidial epithelial punctate keratoconjunctivitis. Cytologic findings demonstrate small, oval organisms within the epithelial cells, stromal keratocytes, and histiocytes as well as free extracellular structures.6,7,9,11,17 These spores have a uniform oval shape and are nonbudding, which help to differentiate them from bacteria and yeasts.23,24 The spores stain strongly with gram stain.9,11,13,15,17,25 Giemsa stains have also been successfully used to demonstrate microsporidial spores.5,9,11,23 Occasionally, these spores stain poorly or not at all with routine stains (hematoxylin– eosin or the Papanicolaou methods), making the organisms easily overlooked in biopsy specimens or cytologic preparations.18,24 Definitive genus identification of microsporidial ocular
Figure 14. Higher magnification of the mature sporoblasts shown in Figure 13 demonstrating 11 to 13 coils of the filament (arrowheads). Intact mature collagen fibers (C) are observed (original magnification, ⫻40,600).
infections requires examination of corneal or conjunctival biopsy specimens, or both, by electron microscopy to demonstrate the number of coils of the filament.6,9,11,13,15,17,18,25 The differentiation of Nosema species from Encephalitozoon is based on several electron microscopic features. First, Nosema are larger than Encephalitozoon (Nosema organisms measure approximately 3.5–5.0 m in length versus 2.0 –3.0 m in length for Encephalatozoon organisms). Second, the absence of a parasitophorous vacuole surrounding the organism within the host cell is more consistent with Nosema. Third, the coils of the filament range
Figure 15. Electron micrograph depicting a sporoblast displaying two abutted nuclei (N), which is highly characteristic of Nosema species (Vittaforma corneae). The arrow outlines the nuclear membranes of the abutted nuclei (original magnification, ⫻45,000).
1773
Ophthalmology Volume 107, Number 9, September 2000 from 11 to 13 in Nosema versus 4 to 7 in Encephalitozoon.6 The electron microscopy findings are consistent with Nosema species. In our patient, the presence of some sporoblasts displaying two abutted nuclei is highly characteristic of Nosema corneum.19 At present, there is no known definite medical treatment of deep microsporidial corneal stromal infections. Some previous reports have suggested that treatment with topical propamidine isethionate 0.1% (Brolene)10,18 or systemic itraconazole9 may be effective against microsporidial superficial keratoconjunctivitis. Yee et al9 reported subjective improvement with debulking and a combination of topical neomycin, bacitracin, and polymyxin B (Neosporin) antibiotics in a patient with bilateral epithelial keratopathy caused by Encephalitozoon, however, complete resolution was achieved only after the administration of oral itraconazole (an oral Triazole antifungal agent). Friedberg et al11 warned against corneal scrapings as well as the use of topical steroids or bandage contact lenses in such patients because they may result in secondary infection and penetration of the organisms into the deeper stroma. Recent reports have documented the successful treatment of microsporidial superficial keratoconjunctivitis with topical fumagillin.15,26 –30 Fumagillin is a naturally secreted, water-insoluble antibiotic of Aspergillus fumigatus and is noted to possess an inhibitory effect on some intestinal protozoa including Entamoeba histolytica.29,31 Fumagillin bicyclohexylammonium salt is the water-soluble form of fumagillin used commercially to control nosematosis of honeybees.30 The mechanisms of action of fumagillin are not clearly understood. There are preliminary data suggesting that the drug may alter DNA content or inhibit RNA synthesis in the organism.31,32 Shadduck28 observed the inhibitory effect of fumagillin on the in vitro multiplication from rabbit and canine isolates of Encephalitozoon cuniculi. Some mature spores have survived after clinically successful treatment, which suggests an inhibitory rather than a parasiticidal action.15 In one study, Gritz et al33 reported a case of superficial punctate keratoconjunctivitis and sinus microsporidial infection in an AIDS patient who was cured with systemic albendazole. In their study, ocular symptoms recurred every time topical fumagillin was discontinued until albendazole was used. In our case, both topical fumagillin bicyclohexylammonium salt and oral albendazole failed to improve or control the progression of the infection after lamellar keratoplasty. This can be explained, in part, by the presumed inadequate drug penetration into the deep corneal stroma. The deep opacities (infiltrates) first noted at the level of Descemet’s membrane and endothelial cells proved to be viable infective organisms rather than only an inflammatory reaction, as we had initially presumed clinically. This ultimately necessitated a full-thickness corneal transplant for complete control of the infection. The patient was last examined on May 22, 1998 (6 months after penetrating graft) and was found to have no signs of recurrence of the infection in the graft (Fig 6). Currently, he is maintained only on topical prednisolone
1774
acetate 1% once daily. His uncorrected visual acuity is 20/70 and the corneal graft has remained clear (Fig 6). In conclusion, microsporidial ocular infections should be considered in the differential diagnosis of culture-negative stromal keratitis or keratoconjunctivitis refractory to conventional medical treatment. Stromal disease occurs in immunocompetent patients. Because these organisms are morphologically distinctive by light and electron microscopy, we would suggest corneal biopsy for histopathologic diagnosis of stromal infections. So far, no successful medical treatment is available for microsporidial corneal stromal infections. Penetrating graft rather than lamellar keratoplasty is recommended to treat deep stromal microsporidiosis to avoid any chance of recurrence in the lamellar bed. A lamellar keratoplasty may be preferable if the infection involves the anterior or mid stroma. Topical fumagillin, which has been shown to be effective in epithelial disease, was ineffective in our case with stromal involvement. We noted fumagillin to be well tolerated by the ocular surface with no adverse side effects; in our patient, complete epithelialization of the graft occurred while he was using fumagillin drops. Further studies regarding the drug delivery and dosage of fumagillin are necessary to establish its efficacy and toxicity.
References 1. Shadduck JA, Greely E. Microsporodia and human infections. Clin Microbiol Rev 1989;2:158 – 65. 2. Shadduck JA. Human microsporidiosis and AIDS. Rev Infect Dis 1989;11:203–7. 3. Didier ES, Snowden KF, Shadduck JA. Biology of microsporidian species infecting mammals [review]. Adv Parasitol 1998;40:283–320. 4. Weber R, Bryan RT, Schwartz DA, Owen RL. Human microsporidian infections. Clin Microbiol Rev 1994;7:426 – 61. 5. Ashton N, Wirasinha PA. Encephalitozoonosis (nosematosis) of the cornea. Br J Ophthalmol 1973;57:669 –74. 6. Pinnolis M, Egbert PR, Font RL, Winter FC. Nosematosis of the cornea. Case report, including electron microscopic studies. Arch Ophthalmol 1981;99:1044 –7. 7. Davis RM, Font RL, Keisler MS, Shadduck JA. Corneal microsporidiosis. A case report including ultrastructural observations. Ophthalmology 1990;97:953–7. 8. Bryan RT, Cali A, Owen RL, Spencer HC. Microsporidia: opportunistic pathogens in patients with AIDS. In: Sun T, ed. Progress in Clinical Parasitology, 2nd ed. Philadelphia: Field and Wood, 1991;1–26. 9. Yee RW, Tio FO, Martinez JA, et al. Resolution of microsporidial epithelial keratopathy in a patient with AIDS. Ophthalmology 1991;98:196 –201. 10. Metcalfe TW, Doran RM, Rowlands PL, et al. Microsporidial keratoconjunctivitis in a patient with AIDS. Br J Ophthalmol 1992;76:177– 8. 11. Friedberg DN, Stenson SM, Orenstein JM, et al. Microsporidial keratoconjunctivitis in acquired immunodeficiency syndrome. Arch Ophthalmol 1990;108:504 – 8. 12. Didier ES, Didier PJ, Friedberg DN, et al. Isolation and characterization of a new human microsporidian, Encephalitozoon hellum (n.sp.) from three AIDS patients with keratoconjunctivitis. J Infect Dis 1991;163:617–21. 13. Lowder CY, Meisler M, McMahon JT, et al. Microsporidia
Font et al 䡠 Corneal Microsporidiosis
14. 15. 16.
17.
18. 19.
20. 21. 22. 23.
infection of the cornea in a man seropositive for human immunodeficiency virus. Am J Ophthalmol 1990;109:242– 4. Cali A. General microsporidian features and recent findings on AIDS isolates. J Protozool 1991;38:625–30. Diesenhouse MC, Wilson LA, Corrent GF, et al. Treatment of microsporidial keratoconjunctivitis with topical fumagillin. Am J Ophthalmol 1993;115:293– 8. Schwartz DA, Visvesvara GS, Diesenhouse MC, et al. Pathologic features and immuno-fluorescent antibody demonstration of ocular microsporidiosis (Encephalitozoon hellum) in seven patients with acquired immunodeficiency syndrome. Am J Ophthalmol 1993;115:285–92. Weber R, Kuster H, Visvesvara GS, et al. Disseminated microsporidiosis due to Encephalitozoon hellum: pulmonary colonization, microhematuria, and mild conjunctivitis in a patient with AIDS. Clin Infect Dis 1993;17:415–9. McCluskey PJ, Goonan PV, Marriott DJ, Fields AS. Microsporidial keratoconjunctivitis in AIDS. Eye 1993;7:80 –3. Silveria H, Canning EU. Vittaforma corneae n. comb. for the human microsporidium Nosema corneum based on its ultrastructure in the liver of experimentally infected athymic mice. J Eukaryot Microbiol 1995;42:158 – 65. Botha WS, van Dellen AF, Stewart CG. Canine Encephalitozoonosis in South Africa. J S Afr Vet Assoc 1979;50:135– 44. Gannon J. A survey of Encephalitozoon cuniculi in laboratory animal colonies in the United Kingdom. Lab Anim 1980;14: 91– 4. Shadduck JA, Meccoli RA, Davis R, Font RL. Isolation of a microsporidian from a human patient. J Infect Dis 1990;162: 773– 6. Schwartz DA, Bryan RT, Visvesvara GS. Diagnostic ap-
24.
25.
26.
27.
28. 29. 30. 31.
32. 33.
proaches for encephalitozoon infections in patients with AIDS [review]. J Eukaryot Microbiol 1994;41:59S– 60S. Schwartz DA, Sobottka I, Leitch GJ, et al. Pathology of microsporidiosis. Emerging parasitic infections in patients with the acquired immunodeficiency syndrome [review]. Arch Pathol Lab Med 1996;120:173– 88. Cali A, Meisler DM, Rutherford I, et al. Corneal microsporidiosis in a patient with AIDS. Am J Trop Med Hyg 1991; 44:463– 8. Rosberger DF, Serdarevic ON, Erlandson RA, et al. Successful treatment of microsporidial keratoconjunctivitis with topical fumagillin in a patient with AIDS. Cornea 1993;12:261–5. Mccowen MC, Callender ME, Lawlis JF Jr. Fumagillin (H-3), a new antibiotic with amebicidal properties. Science 1951; 113:202–3. Shadduck JA. Effect of fumagillin on in vitro multiplication of Encephalitozoon cuniculi. J Protozool 1980;27:202– 8. Killough JH, Magill GB, Smith RC. The treatment of amebiasis with fumagillin. Science 1952;115:70. Katznelson H, Jamieson CA. Control of Nosema disease of honey bees with fumagillin. Science 1952;115:70 –1. Hartwig A, Przelecka A. Nucleic acids in intestine of Apis mellifica infected with Nosema apis and treated with fumagillin DCH: cytochemical and autoradiographic studies. J Invertebr Pathol 1971;18:331– 6. Jaronski ST. Cytochemical evidence for RNA synthesis inhibition by fumagillin. J Antibiot (Tokyo) 1972;25:327–31. Gritz DC, Holsclaw DS, Neger RE, et al. Ocular and sinus microsporidial infection cured with systemic albendazole. Am J Ophthalmol 1997;124:241–3.
1775
Originally received: August 5, 1999. Accepted: May 5, 2000. Manuscript no. 99539. 1 Department of Ophthalmology, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas. 2 Department of Ophthalmology and Visual Sciences, University of Texas Health Science Center, Houston, Texas. Supported in part by grants from the Retina Research Foundation, Houston, 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, Houston, TX 77030. E-mail: [email protected]. © 2000 by the American Academy of Ophthalmology Published by Elsevier Science Inc.
Nosema species) as well as unclassified microsporidia (collectively referred to as Microsporidium) have been associated with human disease involving mostly immunocompromised patients.1– 4 The affected patients may exhibit broad clinical manifestations including intestinal, pulmonary, ocular, muscular, and renal disease. Only two species, Nosema and Encephalitozoon, are known to cause ocular infections. The first report of corneal involvement in humans occurred in 1973.5 Two other cases involving the corneal stroma were reported in 1981 and 1990 by Pinnolis et al6 and Davis et al,7 respectively. Reports of superficial punctate epithelial keratopathy and conjunctivitis caused by Microsporidia have increased.8 –18 Two clinical presentations of ocular microsporidiosis are observed: a corneal stromal keratitis, which occurs in immunocompetent patients and is caused by Nosema corneum (renamed Vittaforma corneae19) and a superficial punctate keratoconjunctivitis occurring in acquired immune deficiency syndrome (AIDS) patients caused by Encephalitozoon species. We report an additional case of microsporidial stromal keratitis caused by N. corneum (V. cornea), which was studied using special stains and electron microscopy. ISSN 0161-6420/00/$–see front matter PII S0161-6420(00)00285-2
1769
Ophthalmology Volume 107, Number 9, September 2000
Case Report Clinical Summary and Histopathologic Findings A 67-year-old Hispanic man was referred from Monterey, Mexico because of an 8-month history of persistent stromal keratitis and progressively decreased visual acuity in his right eye. Initially, he was treated with various topical antibiotics, and later this was managed as a case of herpes simplex viral stromal keratitis with topical steroids (prednisolone acetate or fluorometholone) and antivirals (Viroptic and acyclovir topical and systemic) without resolution of the condition. Ophthalmologic examination in September 1997 revealed an uncorrected visual acuity of 20/80 in the right eye and 20/20 in the left eye. The right cornea showed dense grayish white stromal infiltrates involving its temporal aspect and extending across the visual axis, sparing only the nasal perilimbal area (Fig 1). The infiltrates centrally had a crystalline-like pattern. Superficial corneal vascularization was present temporally to approximately 3 mm from the limbus. The overlying epithelium was spared. The conjunctiva was not injected, and no follicular or papillary reactions were noted. The anterior chamber was quiet in the right eye. The left eye was entirely normal. After discontinuing the topical steroids (fluorometholone), the ophthalmologic examination revealed significant conjunctival injection and increased corneal vascularization peripherally (Fig 2). The appearance of the corneal infiltrates was essentially unchanged. The deep stroma up to the level of Descemet’s membrane was involved with the infiltrative process (Fig 3). Scattered infiltrates were noted on the endothelial surface. The results of conjunctival and corneal swabs and scrapings for bacterial, chlamydial, fungal, and herpes virus cultures were all negative. A corneal punch biopsy was performed on September 17, 1997. A histopathologic diagnosis of microsporidial stromal keratitis was made, and subsequently the patient began oral albendazole 400 mg three times daily and fluorometholone four times daily. Topical fumagillin bicyclohexylammonium salt (Fumadil B) 3 mg/ml, which is equivalent to 70 g/ml of fumagillin, was started by mid-October 1997 every hour while the patient was awake for 2 weeks, then six times daily for 2 weeks. After 6 and 4 weeks of treatment with albendazole and fumagillin bicyclohexylammonium salt, respectively, the right cornea showed no clinical improvement and the visual acuity remained unchanged. Fumagillin bicyclohexylammonium salt was well tolerated during the course of treatment with no adverse side effects. Stool, urine, and sputum specimens tested negative for Microsporidia on two occasions. The patient was seronegative for the human immunodeficiency virus. On November 5, 1997, an eccentric lamellar keratoplasty 9.5 mm in diameter was performed. The lamellar stromal dissection was carried deep just anterior to Descemet’s membrane. After surgery, albendazole and fumagillin bicyclohexylammonium salt six times daily were resumed with the addition of ofloxacin (Ocuflox) four times daily to the right eye. Complete epithelialization of the lamellar graft was achieved on the fifth day after surgery. One week after surgery, recurrence of infiltrates at different loci in the interface were noted (Figs 4 and 5). One month later, the interface infiltrates and opacities had spread anteriorly and started to involve the graft. On December 17, 1997, a penetrating keratoplasty was performed. The patient was last examined on May 22, 1998. At that time, the graft remained clear without evidence of recurrence (Fig 6). On November 5, 1997, a deep lamellar keratectomy of the right eye was performed. Microscopically, the lamellar graft showed numerous microsporidial organisms involving the deep corneal stroma (Fig 7). The possibility of recurrence in the lamellar bed
1770
was anticipated. Some of the organisms were birefringent under polarized light (Fig 8). On December 17, 1997, a penetrating graft was performed. Microscopically, the corneal epithelium showed moderate intracellular edema. Bowman’s layer appeared intact. The stroma was moderately edematous with focal areas of scarring. One of the fragments corresponded only to the deep corneal stroma, including Descemet’s membrane and the endothelium. The deep stroma contained numerous oval-shaped organisms that measured 3.5 to 5 m in length by 2.5 to 3.0 m in width. The organisms stained positively with periodic acid–Schiff and Grocott-methenamine silver methods (Figs 9 and 10) and were gram positive. Some of the organisms were also acid-fast positive (Fig 11). Descemet’s membrane was focally ruptured. Numerous epithelioid histiocytes and scattered multinucleated giant cells were clinging to Descemet’s membrane at the site of rupture. In this area, numerous pigmentladen macrophages and free melanin granules were also observed.
Electron Microscopy Findings The formalin-fixed keratectomy specimen was placed in 2% glutaraldehyde, postfixed in 1% osmium tetroxide, and processed for conventional electron microscopy. Thick section (1 m) revealed masses of round to oval sporoblasts, measuring 3.5 to 5.0 m in length by 2.5 to 3.0 m in width, dissecting amongst the corneal lamellae (Fig 12). Electron microscopy disclosed scattered viable sporoblasts, each surrounded by a thick capsule. Many degenerated organisms without internal structures were also present (Fig 13). The latter demonstrated a thin, osmiophilic outer layer and a thicker, lucent inner layer. The sporoblasts appeared as free extracellular organisms that were not surrounded by a parasitophorous vacuole. Precursor forms (meronts) were interspersed among the sporoblasts (Fig 13). Under higher magnification, the mature sporoblasts disclosed from 11 to 13 coils of the filament (Fig 14). The collagen fibrils surrounding the organisms appeared intact. Some sporoblasts disclosed diplokaryotic arrangement of the nuclei, which is believed to be the only character that conforms to the genus Nosema (Fig 15).19
Discussion Microsporidia are becoming increasingly recognized as opportunistic infectious pathogens in patients with AIDS. They are ubiquitous in nature and are widely distributed in vertebrates and invertebrates. Bilateral diffuse punctate epithelial keratopathy and conjunctivitis10 –11,13,17,18 characterize ocular microsporidiosis in AIDS patients. Some documented cases have shown only mild conjunctivitis but no keratopathy.16,17 In contrast, ocular microsporidiosis in immunocompetent patients usually shows unilateral corneal stromal involvement without epitheliopathy or iritis. Of the four reported patients with severe stromal microsporidial keratitis, corneal ulcers occurred in two,6,7 and one eye was enucleated because of a perforated corneal ulcer.6 The source of infection for humans and the routes of transmission are unknown; however, they are thought to be either orofecal, resulting from direct inoculation, or occurring after trauma.5 Direct inoculation may occur with close contacts with domestic animals such as cats and birds20,21; it may also spread from other infected persons.22,23 In our patient, such contacts were reported by the patient not to
Font et al 䡠 Corneal Microsporidiosis
Figure 1. Clinical appearance of the right cornea at time of initial examination showing the dense, whitish-gray stromal infiltrates temporally with a crystalline-like pattern as they progress centrally. Minimal conjunctival injection is present.
Figure 3. A slit-lamp view of the stromal infiltrates depicted in Figure 2 shows deep stromal involvement reaching the plane of Descemet’s membrane.
Figure 5. A cross-sectional slit-lamp view of the lamellar graft in Figure 4 demonstrating a clear graft with the recurrent infiltrates that are localized at the interface.
Figure 2. The clinical appearance of the same cornea 9 days after tapering and stopping the steroid eye drops. The bulbar conjunctiva is significantly injected. Prominent superficial peripheral corneal vascularization is observed temporally. The stromal infiltrates are essentially similar to those observed in Figure 1.
Figure 4. Clinical appearance of the right eye 1 week after the lamellar keratoplasty. Note the recurrence of the stromal infiltrates in the interface peripherally as well as the residual opacities (infiltrates) just in front of Descemet’s membrane. The lamellar graft has been fully epithelialized.
Figure 6. Clinical appearance of the right cornea 6 months after the penetrating keratoplasty shows a clear eccentric graft with no evidence of recurrence of the infection. A loose suture with a mucous plug is noted at the 5:30-o’clock position. Best-corrected visual acuity at this time was 20/40.
1771
Ophthalmology Volume 107, Number 9, September 2000
Figure 7. Deep lamellar keratectomy specimen containing masses of Nosema organisms within the deep corneal stroma. Descemet’s membrane appears intact. The endothelial cells are decreased in number. Scattered macrophages are observed along the posterior corneal surface (stain, hematoxylin– eosin; original magnification, ⫻100).
Figure 9. High-power view disclosing numerous round to oval, periodic acid–Schiff-positive sporoblasts in the deep corneal stroma. An artifactitious splitting of the anterior fetal portion from the posterior adult portion of Descemet’s membrane is observed (stain, periodic acid–Schiff; original magnification, ⫻252).
Figure 11. The vast majority of the intact sporoblasts are acid-fast positive. Scattered swollen degenerated organisms are acid-fast negative (stain, acid-fast bacilli; original magnification, ⫻252).
1772
Figure 8. Same field shown in Figure 7, depicting that some of the organisms are birefringent under examination with polarized light (stain, hematoxyilin– eosin; original magnification, ⫻100).
Figure 10. Similar field to that depicted in Figure 9 showing the round to oval sporoblasts measuring 3.0 to 5.0 m in length involving the deep corneal stroma. Few organisms did not stain with this technique (stain, Grocott-methenamine silver; original magnification, ⫻252).
Figure 12. Thick section (1 m) shows myriad round to oval sporoblasts dissecting among the corneal lamellae. Few empty carcasses of degenerated organisms are also observed (stain, Toluidine blue; original magnification, ⫻322).
Font et al 䡠 Corneal Microsporidiosis
Figure 13. This electron micrograph depicts numerous degenerated sporoblasts (black asterisk) that disclose an osmiophilic thin outer layer and a thicker, inner lucent layer. Several meronts (m) are interspersed among the degenerated sporoblasts. A viable sporoblast is identified (white asterisk). Mature collagen fibers (C) are present (original magnification, ⫻16,200).
have occurred, making the mode of transmission difficult to determine. Microsporidia are small, obligate intracellular parasites that produce infective spores. They are fastidious organisms that are difficult to culture. The organism has been isolated using special tissue culture techniques, which are available only in a few specialized laboratories, making this method impractical for routine diagnosis.12,22 Alcohol-fixed cytologic samples of scrapings from the conjunctiva, corneal epithelium, or both or biopsy specimens have proven very useful for demonstrating microsporidial blastospores.7,16 Often conjunctival scrapings alone provide a satisfactory specimen for cytologic diagnosis in microsporidial epithelial punctate keratoconjunctivitis. Cytologic findings demonstrate small, oval organisms within the epithelial cells, stromal keratocytes, and histiocytes as well as free extracellular structures.6,7,9,11,17 These spores have a uniform oval shape and are nonbudding, which help to differentiate them from bacteria and yeasts.23,24 The spores stain strongly with gram stain.9,11,13,15,17,25 Giemsa stains have also been successfully used to demonstrate microsporidial spores.5,9,11,23 Occasionally, these spores stain poorly or not at all with routine stains (hematoxylin– eosin or the Papanicolaou methods), making the organisms easily overlooked in biopsy specimens or cytologic preparations.18,24 Definitive genus identification of microsporidial ocular
Figure 14. Higher magnification of the mature sporoblasts shown in Figure 13 demonstrating 11 to 13 coils of the filament (arrowheads). Intact mature collagen fibers (C) are observed (original magnification, ⫻40,600).
infections requires examination of corneal or conjunctival biopsy specimens, or both, by electron microscopy to demonstrate the number of coils of the filament.6,9,11,13,15,17,18,25 The differentiation of Nosema species from Encephalitozoon is based on several electron microscopic features. First, Nosema are larger than Encephalitozoon (Nosema organisms measure approximately 3.5–5.0 m in length versus 2.0 –3.0 m in length for Encephalatozoon organisms). Second, the absence of a parasitophorous vacuole surrounding the organism within the host cell is more consistent with Nosema. Third, the coils of the filament range
Figure 15. Electron micrograph depicting a sporoblast displaying two abutted nuclei (N), which is highly characteristic of Nosema species (Vittaforma corneae). The arrow outlines the nuclear membranes of the abutted nuclei (original magnification, ⫻45,000).
1773
Ophthalmology Volume 107, Number 9, September 2000 from 11 to 13 in Nosema versus 4 to 7 in Encephalitozoon.6 The electron microscopy findings are consistent with Nosema species. In our patient, the presence of some sporoblasts displaying two abutted nuclei is highly characteristic of Nosema corneum.19 At present, there is no known definite medical treatment of deep microsporidial corneal stromal infections. Some previous reports have suggested that treatment with topical propamidine isethionate 0.1% (Brolene)10,18 or systemic itraconazole9 may be effective against microsporidial superficial keratoconjunctivitis. Yee et al9 reported subjective improvement with debulking and a combination of topical neomycin, bacitracin, and polymyxin B (Neosporin) antibiotics in a patient with bilateral epithelial keratopathy caused by Encephalitozoon, however, complete resolution was achieved only after the administration of oral itraconazole (an oral Triazole antifungal agent). Friedberg et al11 warned against corneal scrapings as well as the use of topical steroids or bandage contact lenses in such patients because they may result in secondary infection and penetration of the organisms into the deeper stroma. Recent reports have documented the successful treatment of microsporidial superficial keratoconjunctivitis with topical fumagillin.15,26 –30 Fumagillin is a naturally secreted, water-insoluble antibiotic of Aspergillus fumigatus and is noted to possess an inhibitory effect on some intestinal protozoa including Entamoeba histolytica.29,31 Fumagillin bicyclohexylammonium salt is the water-soluble form of fumagillin used commercially to control nosematosis of honeybees.30 The mechanisms of action of fumagillin are not clearly understood. There are preliminary data suggesting that the drug may alter DNA content or inhibit RNA synthesis in the organism.31,32 Shadduck28 observed the inhibitory effect of fumagillin on the in vitro multiplication from rabbit and canine isolates of Encephalitozoon cuniculi. Some mature spores have survived after clinically successful treatment, which suggests an inhibitory rather than a parasiticidal action.15 In one study, Gritz et al33 reported a case of superficial punctate keratoconjunctivitis and sinus microsporidial infection in an AIDS patient who was cured with systemic albendazole. In their study, ocular symptoms recurred every time topical fumagillin was discontinued until albendazole was used. In our case, both topical fumagillin bicyclohexylammonium salt and oral albendazole failed to improve or control the progression of the infection after lamellar keratoplasty. This can be explained, in part, by the presumed inadequate drug penetration into the deep corneal stroma. The deep opacities (infiltrates) first noted at the level of Descemet’s membrane and endothelial cells proved to be viable infective organisms rather than only an inflammatory reaction, as we had initially presumed clinically. This ultimately necessitated a full-thickness corneal transplant for complete control of the infection. The patient was last examined on May 22, 1998 (6 months after penetrating graft) and was found to have no signs of recurrence of the infection in the graft (Fig 6). Currently, he is maintained only on topical prednisolone
1774
acetate 1% once daily. His uncorrected visual acuity is 20/70 and the corneal graft has remained clear (Fig 6). In conclusion, microsporidial ocular infections should be considered in the differential diagnosis of culture-negative stromal keratitis or keratoconjunctivitis refractory to conventional medical treatment. Stromal disease occurs in immunocompetent patients. Because these organisms are morphologically distinctive by light and electron microscopy, we would suggest corneal biopsy for histopathologic diagnosis of stromal infections. So far, no successful medical treatment is available for microsporidial corneal stromal infections. Penetrating graft rather than lamellar keratoplasty is recommended to treat deep stromal microsporidiosis to avoid any chance of recurrence in the lamellar bed. A lamellar keratoplasty may be preferable if the infection involves the anterior or mid stroma. Topical fumagillin, which has been shown to be effective in epithelial disease, was ineffective in our case with stromal involvement. We noted fumagillin to be well tolerated by the ocular surface with no adverse side effects; in our patient, complete epithelialization of the graft occurred while he was using fumagillin drops. Further studies regarding the drug delivery and dosage of fumagillin are necessary to establish its efficacy and toxicity.
References 1. Shadduck JA, Greely E. Microsporodia and human infections. Clin Microbiol Rev 1989;2:158 – 65. 2. Shadduck JA. Human microsporidiosis and AIDS. Rev Infect Dis 1989;11:203–7. 3. Didier ES, Snowden KF, Shadduck JA. Biology of microsporidian species infecting mammals [review]. Adv Parasitol 1998;40:283–320. 4. Weber R, Bryan RT, Schwartz DA, Owen RL. Human microsporidian infections. Clin Microbiol Rev 1994;7:426 – 61. 5. Ashton N, Wirasinha PA. Encephalitozoonosis (nosematosis) of the cornea. Br J Ophthalmol 1973;57:669 –74. 6. Pinnolis M, Egbert PR, Font RL, Winter FC. Nosematosis of the cornea. Case report, including electron microscopic studies. Arch Ophthalmol 1981;99:1044 –7. 7. Davis RM, Font RL, Keisler MS, Shadduck JA. Corneal microsporidiosis. A case report including ultrastructural observations. Ophthalmology 1990;97:953–7. 8. Bryan RT, Cali A, Owen RL, Spencer HC. Microsporidia: opportunistic pathogens in patients with AIDS. In: Sun T, ed. Progress in Clinical Parasitology, 2nd ed. Philadelphia: Field and Wood, 1991;1–26. 9. Yee RW, Tio FO, Martinez JA, et al. Resolution of microsporidial epithelial keratopathy in a patient with AIDS. Ophthalmology 1991;98:196 –201. 10. Metcalfe TW, Doran RM, Rowlands PL, et al. Microsporidial keratoconjunctivitis in a patient with AIDS. Br J Ophthalmol 1992;76:177– 8. 11. Friedberg DN, Stenson SM, Orenstein JM, et al. Microsporidial keratoconjunctivitis in acquired immunodeficiency syndrome. Arch Ophthalmol 1990;108:504 – 8. 12. Didier ES, Didier PJ, Friedberg DN, et al. Isolation and characterization of a new human microsporidian, Encephalitozoon hellum (n.sp.) from three AIDS patients with keratoconjunctivitis. J Infect Dis 1991;163:617–21. 13. Lowder CY, Meisler M, McMahon JT, et al. Microsporidia
Font et al 䡠 Corneal Microsporidiosis
14. 15. 16.
17.
18. 19.
20. 21. 22. 23.
infection of the cornea in a man seropositive for human immunodeficiency virus. Am J Ophthalmol 1990;109:242– 4. Cali A. General microsporidian features and recent findings on AIDS isolates. J Protozool 1991;38:625–30. Diesenhouse MC, Wilson LA, Corrent GF, et al. Treatment of microsporidial keratoconjunctivitis with topical fumagillin. Am J Ophthalmol 1993;115:293– 8. Schwartz DA, Visvesvara GS, Diesenhouse MC, et al. Pathologic features and immuno-fluorescent antibody demonstration of ocular microsporidiosis (Encephalitozoon hellum) in seven patients with acquired immunodeficiency syndrome. Am J Ophthalmol 1993;115:285–92. Weber R, Kuster H, Visvesvara GS, et al. Disseminated microsporidiosis due to Encephalitozoon hellum: pulmonary colonization, microhematuria, and mild conjunctivitis in a patient with AIDS. Clin Infect Dis 1993;17:415–9. McCluskey PJ, Goonan PV, Marriott DJ, Fields AS. Microsporidial keratoconjunctivitis in AIDS. Eye 1993;7:80 –3. Silveria H, Canning EU. Vittaforma corneae n. comb. for the human microsporidium Nosema corneum based on its ultrastructure in the liver of experimentally infected athymic mice. J Eukaryot Microbiol 1995;42:158 – 65. Botha WS, van Dellen AF, Stewart CG. Canine Encephalitozoonosis in South Africa. J S Afr Vet Assoc 1979;50:135– 44. Gannon J. A survey of Encephalitozoon cuniculi in laboratory animal colonies in the United Kingdom. Lab Anim 1980;14: 91– 4. Shadduck JA, Meccoli RA, Davis R, Font RL. Isolation of a microsporidian from a human patient. J Infect Dis 1990;162: 773– 6. Schwartz DA, Bryan RT, Visvesvara GS. Diagnostic ap-
24.
25.
26.
27.
28. 29. 30. 31.
32. 33.
proaches for encephalitozoon infections in patients with AIDS [review]. J Eukaryot Microbiol 1994;41:59S– 60S. Schwartz DA, Sobottka I, Leitch GJ, et al. Pathology of microsporidiosis. Emerging parasitic infections in patients with the acquired immunodeficiency syndrome [review]. Arch Pathol Lab Med 1996;120:173– 88. Cali A, Meisler DM, Rutherford I, et al. Corneal microsporidiosis in a patient with AIDS. Am J Trop Med Hyg 1991; 44:463– 8. Rosberger DF, Serdarevic ON, Erlandson RA, et al. Successful treatment of microsporidial keratoconjunctivitis with topical fumagillin in a patient with AIDS. Cornea 1993;12:261–5. Mccowen MC, Callender ME, Lawlis JF Jr. Fumagillin (H-3), a new antibiotic with amebicidal properties. Science 1951; 113:202–3. Shadduck JA. Effect of fumagillin on in vitro multiplication of Encephalitozoon cuniculi. J Protozool 1980;27:202– 8. Killough JH, Magill GB, Smith RC. The treatment of amebiasis with fumagillin. Science 1952;115:70. Katznelson H, Jamieson CA. Control of Nosema disease of honey bees with fumagillin. Science 1952;115:70 –1. Hartwig A, Przelecka A. Nucleic acids in intestine of Apis mellifica infected with Nosema apis and treated with fumagillin DCH: cytochemical and autoradiographic studies. J Invertebr Pathol 1971;18:331– 6. Jaronski ST. Cytochemical evidence for RNA synthesis inhibition by fumagillin. J Antibiot (Tokyo) 1972;25:327–31. Gritz DC, Holsclaw DS, Neger RE, et al. Ocular and sinus microsporidial infection cured with systemic albendazole. Am J Ophthalmol 1997;124:241–3.
1775