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Triple Retinal Infection with Human Immunodeficiency Virus Type 1, Cytomegalovirus, and Herpes Simplex Virus Type 1
Triple Retinal Infection with Human Immunodeficiency Virus Type 1, Cytomegalovirus, and Herpes Simplex Virus Type 1 Light and Electron Microscopy, Immunohistochemistry, and In Situ Hybridization Volker Rummelt, MD,1 Carmen Rummelt,l Gerhard Jahn, MD,2 Hartmut Wenkel/ Christian Sinzger, MD,2 Ursula M. Mayer, MD,l Gottfried O. H. Naumann, MDl Purpose: This report describes the histopathologic and virologic findings of the retina from a 55-year-old bisexual patient with the acquired immune deficiency syndrome (AIDS), who had concurrent human immunodeficiency virus type 1 (HIV-1), cytomegalovirus (CMV), and herpes simplex virus type 1 (HSV-1) retinitis, and was treated with ganciclovir. Methods: The eyes were obtained at autopsy and processed for light microscopy and transmission electron microscopy. Immunohistochemical stains for HSV-1, CMV, HIV-1, varicella zoster virus, and glial fibrillary acidic protein were carried out using the peroxidase-anti peroxidase and streptavidin-biotin-alkaline phosphatase techniques. For in situ hybridization, a radiolabeled CMV DNA probe (Eco-RI-Y fragment of strain AD 169) was used. Results: Results of histopathologic examination showed a full-thickness necrotizing retinitis with cytomegalic and herpes viral intranuclear inclusions in cells of the neurosensory retina, retinal vascular endothelium, and the retinal pigment epithelium. Some areas of the retina were replaced by glial tissue. The choroid contained only a few chronic inflammatory cells. Immunoperoxidase studies disclosed CMV antigens diffusely distributed throughout all layers of the retina and the retinal pigment epithelium. Herpes simplex virus type 1 antigens were present in retinal cells and the retinal vascular endothelium. Human immunodeficiency virus type 1 antigens were found in mononuclear cells in all layers of the sensory retina. Dual infections with HIV-1 and CMV of individual multinucleated giant cells of glial origin were demonstrated immunohistochemically.
Originally received: March 17, 1993. Revision accepted: August 19, 1993. I Department of Ophthalmology, University of Erlangen-Niirnberg, Erlangen, Germany. 2 Institute of Clinical and Molecular Virology, University of ErlangenNiirnberg, Erlangen, Germany.
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Presented in part at the ARVO Annual Meeting, Sarasota, May 1991. Supported by grant Ru 464/3-1 of the Deutsche Forschungsgemeinschaft, Bonn, Germany. Reprint requests to Volker Rummeit, MD, Department of Ophthalmology, University of Erlangen-Niirnberg, Schwabachanlage 6, D-91054 Erlangen, Germany.
Rummelt et al . Triple Retinal Infection Transmission electron microscopy showed herpes viral particles in the vascular endothelium of the retinal vessels and the choriocapillaris. Human immunodeficiency virus particles were identified in the endothelium of the choriocapillaris. Conclusions: The possibility of multiple viral infections of the retina, mimicking classic CMV retinitis, should be considered in the clinical and histologic differential diagnosis of necrotizing retinitis in patie.nts with AIDS. Ophthalmology 1994;101:270-279
Cytomegalovirus (CMV) retinitis is the most common ophthalmic opportunistic infection and cause of blindness in patients with the acquired immune deficiency syndrome (AIDS). 1-4 Infections of the retina and the choroid with multiple pathogenic organisms (i.e., bacteria, fungi, viruses) have been reported recently. 5-8 Proven pathogens causing viral infections of retinal tissue in patients with AIDS have included CMV,4.5.9-20 herpes simplex virus type 1 (HSV-l), 15,21,22 varicella zoster virus (VZV),23-25 human immunodeficiency virus type 1 (HIV_l),9- 12,26,27 and human herpes virus type 6. 9.27 Co-infections of the retina with HIV-l and CMV,II .12 HSV I5 and CMV,15 HIV-l and human herpes virus type 6,9.27 and with HIV-l , CMV, and human herpes virus type 6,27 respectively, have been demonstrated. These studies have implicated that possible interactions between different viruses, especially HIV-l and herpes group viruses in dually infected cells, may influence the clinical course and severity of retinal disease in patients with AIDS. II We now report on the clinical and histopathologic findings in the retina of a 55-year-old bisexual man with AIDS. Atriple infection of the retina with HIV-I, CMV, and HSV -1 was identified using immunohistochemistry, DNA in situ hybridization, and transmission electron microscopy. Our findings provide additional information about the pathogenesis of multi viral infections of the retina in immunocompromised patients with AIDS.
Case Report A 55-year-old white bisexual man was diagnosed with AIDS in February 1986. Four months later, Kaposi sarcoma of the right foot developed. At this time, visual acuity in both eyes was 20/ 25, and there was no evidence of viral retinitis in either eye. A mild hyperpigmentation and hypopigmentation of the retinal pigment epithelium (RPE) in the macula was observed. Serologic findings showed an antibody-positive HIV -I enzyme-linked immunoassay, verified by Western blot analyses. The complement fixation titer was 1: 16 for HSV, and no antibodies against VZV and CMV were detected by complement fixation titer at this time. On his first examination at our hospital on January 4, 1990, he complained of floaters and blurred vision in the right eye. Visual acuity at this time was 20/50 in the right eye and 20/25 in the left. Results of ophthalmoscopic examination disclosed an area of necrotizing retinitis in the right eye, extending from the optic disc and between the superotemporal and superonasal retinal vessel arcades to the equator (Fig 1). This area appeared as dry, white, opacified retinal lesions with interspersed hem-
orrhages. A cotton-wool spot was identified inferotemporally. Vitreous inflammation was absent, and the anterior segment was unremarkable. There was no evidence of retinitis in the left eye. Serologic testing showed a complement fixation titer of 1: 16 for HSV, 1:32 for CMV, and 1:4 for VZV. Human immunodeficiency virus type 1 was isolated by a standard lymphocyte co-culture technique. Because there was a strong suspicion of unilateral CMV retinitis, an induction dosage of intravenous ganciclovir (5 mg/kg twice daily for 2 weeks) was started. The patient was then given a maintenance therapy of ganciclovir of 6 mg/kg daily. On January 25, 1990, complement fixation titers were 1: 16 for CMV and 1:4 for HSV. The retinal necroses were stable. Unfortunately, the ganciclovir therapy had to be stopped due to severe side effects with almost total myelosuppression. The patient refused any further treatment and died on February 12, 1990, 1month after the initial diagnosis of CMV retinitis. An autopsy was performed immediately after death. The main pathologic findings were (I) CMV pneumonia, (2) CMV encephalitis, (3) disseminated Kaposi sarcoma, and (4) cerebral malignant non-Hodgkin lymphoma.
Materials and Methods Light Microscopy and Immunohistochemistry The eyes were obtained at autopsy within 8 hours after death and immediately fixed in a neutral-buffered solution of 4% paraformaldehyde and I % glutaraldehyde. They were sectioned vertically, submitting a pupil-optic nerve head section involving the area of retinal necrosis. The globes then were processed in standard histologic fashion. Hematoxylin-eosin, periodic acid-Schiff, Gomori's methenamine silver, Gram's, and Ziehl-Neelson stains of 4~m sections thick were performed. Formaldehyde-fixed and paraffin-embedded sections of the globes were deparaffinized in xylene and rehydrated in a series of graded alcohols. Endogenous peroxidase was quenched by incubation in 3% hydrogen peroxide. Immunohistochemical stains were carried out using antibodies against the following antigens: HSV-1 (polyclonal, rabbit, anti-HSV-I antibody; DAKO Diagnostika, Hamburg, Germany), VZV (5B7 monoclonal, mouse, antiVZV antibody; BioMerieux, Paris, France), CMV (E 13, a monoclonal antibody against an immediate-early CMV protein; Biosoft, Paris, France; and XP-l , a monoclonal antibody against a late CMV protein, pp I 50-courtesy of G. Jahn)28-3o, HIV-I (monoclonal, murine, anti-p24 antibody, and monoclonal, murine, anti-gp 120 antibody-courtesy of G. Jahn), and glial fibrillary acidic
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Top, Figure 1. Clinical aspect of the right fundus of a 55-year-old bisexual patient with AIDS (February 1990). Area of necrotizing retinitis with interspersed hemorrhage extending from the optic nerve, between the superotemporal and superonasal retinal vessel arcades, to the equator. Visual acuity was 20/50 in the right. Center and Bottom, Figure 2. Histopathologic findings of concurrent necrotizing human immunodeficiency virus type 1, cytomegalovirus, and herpes simplex virus type 1 retinitis. Center left (A), fuJI-thickness necrotizing retinitis (SR = sensory retina) with cytomegalic viral inclusions in retinal ceJls and the retinal pigment epithelium (arrows). Notice the paucity of chronic inflammatory ceJls in the choroid (Ch) (original magnification, Xl60). Center right (B), cytomegalic ceJls with an "owl's eye" appearance and intracytoplasmic and intranuclear viral inclusions in apparently glial ceJls of the sensory retina (original magnification, X400). Bottom (C), cytomegalic viral inclusions in retinal pigment epithelial (arrows) ceJls (original magnification, XBOO).
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Rummelt et al . Triple Retinal Infection protein (polyclonal, rabbit, anti-GFAP; DAKO Diagnostika). Commercially available versions of the peroxidaseantiperoxidase method (PAP; DAKO Diagnostika) and the streptavidin-biotin alkaline phosphatase (SBAP) technique (DAKO Diagnostika) were used. For color development, a 3,3-diaminobenzidine-hydrogen peroxide solution or aminoethylcarbazol-hydrogen peroxide solution was used. All sections were counterstained with hematoxylin. Formaldehyde-fixed and paraffin-embedded human foreskin fibroblast cultures, infected with HSV-I, HSV-2, VZV, CMV, and human brain and lung tissue, culture-positive for HSV-I, CMV, or HIV-I, provided the necessary positive and negative controls for sensitivity, specificity, and cross-reactivity of the different viral antisera. Globe sections of patients with malignant uveal melanomas and no previous history of viral infections were carried along in every series as negative controls.
DNA In Situ Hybridization For in situ hybridization, tissue sections were deparaffinized and pretreated with proteinase K and hydrochloric acid. After the pretreatment, single globe sections were digested either by DNAse or RNAse for approximately I hour to differentiate between CMV DNA and CMV RNA. The EcoRI-Y fragment ofCMV (strain AD 169) cloned into the plasmid pBR 322 was used as specific probeY The insert was isolated and radiolabeled by nick-translation using sulfur 35-labeled adenosine triphosphate. The probe showed no hybridization to the DNA ofHSV-1 or VZV genome in Southern blot hybridization. The HIND III restriction enzyme fragments A and C of VZV (strain 80-2) and the plasmid DNA ofpBR 322 served as control probes. After incubation for 17 hours at 40 0 C, the slides were washed with different concentrations of standard sodium citrate. Sections were dipped in NTB2 nuclear track emulsion (Eastman Kodak, Stuttgart, Germany), developed after 14 days of exposure at 4 0 C, stained with hematoxylin, and evaluated by light microscopy in the bright and dark field. Paraffin-embedded human foreskin fibroblast cultures infected with HSV-I, CMV, and VZV served as adequate positive and negative controls. Globe sections of patients with malignant uveal melanomas and no previous history of ocular viral infections were carried along in every series as well as one positive (CMV) and two negative (HSV-I, VZV) cell culture sections to exclude nonspecific signals.
S-7000 electron microscope (Hitachi Scientific, Tokyo, Japan).
Results Histopathologic Examination On gross examination, both eyes measured 24 X 23 X 23 mm with 5 mm of attached optic nerves. The clear cornea measured 11.5 X II mm. Both eyes showed mild hyperpigmentation and hypopigmentation of the retinal pigment epithelium (RPE) in the macula. The right eye showed a dense, white plaque replacing the retinal structures from the optic nerve and along the superotemporal and superonasal retinal vessel arcades to the equator. Scattered retinal hemorrhages were present in this area. The vitreous was clear, and the retina was flat. The retinal vessels, the choroid, and the optic nerves appeared unremarkable. On microscopic examination, the left eye was unremarkable. The right eye showed a circumscribed fullthickness necrosis of the neurosensory retina (Fig 2A). In this area, retinal tissue was replaced by lymphocytes, macrophages, occasional "multinucleated giant cells," and acute inflammatory cells. Some extravasated erythrocytes also were seen. There was a focal replacement of the sensory retina by glial tissue adjacent to the area of acute necrosis. Bruch's membrane and the inner-limiting membrane of the retina were intact. A serous retinal detachment was present in the area of retinal necrosis. Many intranuclear and intracytoplasmic cytomegalic viral inclusions were seen in cells of the necrotic retinal tissue and in glial cells of apparently normal sensory retinal structures (Fig 2B). In addition, eosinophilic intranuclear viral inclusions (Cowdry A-type) suggestive for herpesvirus were present in retinal cells near cytomegalic cells with an "owl's eye" appearance. Some retinal vessels in the necrotic area showed narrowing of their lumen as a result of mural lymphocytic infiltration and swelling of vascular endothelial cells, which occasionally contained intranuclear eosinophilic viral inclusions. The RPE underlying the necrotic retina showed the viral infection in a patchy pattern (Fig 2C), and where necrotic, the subjacent choroid contained only a few chronic inflammatory cells. There was minimal acute inflammatory reaction to the vitreous. The optic nerve and the anterior segment were unremarkable. Special stains for bacteria, fungi, and acid-fast bacilli were negative in both eyes.
Transmission Electron Microscopy For transmission electron microscopy, parts of necrotic retina and of normal-appearing retina were fixed in a neutral-buffered 2.5% glutaraldehyde solution. Retinal tissue was postfixed in a solution of I % buffered osmium tetroxide, dehydrated, and embedded in standard fashion. Thick (l-Jlm) sections were stained with toluidine bluebasic fuchsin solution. Thin sections were stained with uranyl acetate-lead citrate and examined with the Hitachi
Immunoperoxidase Studies Immunohistochemical stains were reproducible, sensitive, and specific for the antibodies used. There was no cross-reactivity between the different viral antisera. Human immunodeficiency virus type I antigens (p24 and gp 120) were found in all layers of the neurosensory retina within mononuclear cells and "multinucleated giant cells" (Figs 3A and 3B). Many of these giant cells
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Figure 3. (continued). Top left (G), herpes simplex virus type 1 antigen (arrows) in retinal cells diffusely distributed throughout all layers of the retina (pAP; original magnification, X240). Top right (H), herpes simplex virus type 1 antigen in retinal vascular endothelium (arrows) (PAP; original magnification, X400). Bottom (I), in situ hybridization of CMV DNA (EcoRI-Y fragment). The positive reaction is confined to the nucleus and the cytoplasm of the infected retinal cells. Notice the patchy distribution of infected cells. V = vitreous; SR = sensory retina; Ch = choroid (original magnification, X240).
also stained positive for GFAP (Fig 3C), indicating their glial origin. In addition, HIV -1 antigens were found in conjunctival epithelial cells (Fig 3D). No HIV-l antigens were demonstrated in any vascular endothelium or in the RPE. Cytomegalovirus antigens (immediate-early and late antigen pp 150) were found in all layers of the retina of
the right eye within cytomegalic cells with an "owl's eye" appearance and in many nonmegalic cells without inclusions, as well as in apparently normal sensory retinal structures (Figs 3E and 3F). Retinal pigment epithelial cells stained positive only for the CMV immediate-early antigen, not for the late antigen. No CMV antigens were found in any vascular endothelium, the choroid, the optic
Figure 3. Immunohistochemistry and in situ hybridization of triple infection of the retina. Top left (A), human immunodeficiency virus type 1 antigen (p24) in multinucleated giant cell in the sensory retina (arrow). Notice cytomegalic viral inclusion (arrowhead) (peroxidase-anti-peroxidase [PAP]; original magnification, X400). Top right (B), human immunodeficiency virus type 1 antigen (p24) in mononuclear cells diffusely distributed in the sensory retina (arrows) (PAP; original magnification, X400). Center left (C), GFAP antigen in multinucleated giant cells of the sensory retina (arrows) indicating its glial origin (PAP; original magnification, X400). Second row right (D), human immunodeficiency virus type 1 antigen (p24) in conjunctival epithelial cells (arrows) (PAP; original magnification, X400). Third row right (E), immediate-early cytomegalovirus (CMV) antigen (E 13) in patchy areas of retinal cells (streptavidln-biotin alkaline phosphatase [SBAP]; original magnification, X 240). Bottom (F), immediate-early CMV antigen (E 13) in retinal pigment epithelial cells and retinal cells (SBAP; original magnification, X240). (Fig 3 continues.)
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nerve, or the anterior segment of the eyes. Dual infections of individual cells with HIV-l and CMV were demonstrable in multinucleated cells of the retina (Fig 3A), which also stained positive for GFAP, using serial sections of the same tissue. Herpes simplex virus type 1 antigens were present in all layers of the neurosensory retina (Figs 3G and 3H) but not in the RPE. Whereas CMV antigens were usually identified in focal patches of cells, HSV -1 antigens evenly were distributed throughout all layers of the sensory retina. In many instances, cytomegalic cells, which did not stain for HSV-1 antigens, were interspersed in the same microscopic field among cells bearing the HSV -1 antigen. We also detected HSV -1 antigens in endothelial cells of the retinal vessels (Fig 3H).
DNA In Situ Hybridization Corresponding to the immunohistochemical findings, CMV DNA could be detected in focal patches of cells in all layers of neurosensory retina (Fig 31). The strongly positive reaction was confined to both the nucleus and cytoplasm of the cell. No viral DNA was present in any vascular endothelium, the optic nerve, or the anterior segment.
Transmission Electron Microscopy Electron microscopic studies disclosed numerous herpes viral particles with a central nucleocapsid 100 nm in diameter in endothelial cells of retinal vessels and the choriocapillaris and in disrupted retinal cells (Fig 4A). Cytomegalovirus-like particles were identified in cells of the necrotic sensory retina (Fig 4A) and the RPE. In addition, HIV viral particles with a central tubular core nucleoid surrounded by a capsid 120 nm in diameter9,32 were present in endothelial cells of the choriocapillaris (Fig 4B). We detected a dual infection of these individual cells with herpesvirus and HIV particles, The inner-limiting membrane of the retina and Bruch's membrane were intact.
Discussion We found evidence of a multiviral necrotizing retinitis in a patient with AIDS. The concurrent HIV -1, HSV -1, and CMV retinitis in this patient may have resulted from reactivation oflatent HIV-l, HSV-l, and/or CMV infection with exogenous strains of HSV-l or CMV, reactivation of latent HSV by superinfection with CMV, or hematogenous dissemination of the viruses during intermittent viremia in this immunocompromised patient. The detection of HSV-1 antigen in retinal vascular endothelium and of herpesvirus and HIV particles in the vascular endothelium ofthe choriocapillaris in our patient suggests that blood-borne dissemination of these viruses to the retina may have played a role in the pathogenesis of the disease. 4,15 Subsequent CMV viremia may have led to di-
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rect retinal infection via the damaged vascular endothelium. Different pathomechanisms may account for the destruction of the sensory retina in this patient, including (1) a direct viral invasion of retinal structures with ensuing cytolytic effects of the viruses themselves l ,4,15,17 (CMV, HSV-1), (2) ischemia, secondary to an occlusive vasculitis observed in patients with CMV retinitis and the acute retinal necrosis syndrome,4,31,33 (3) a virus-triggered immune-complex reaction to the retinal vessel walls potentiating the destruction of the sensory retina,33,34 and (4) interactions between HIV-l and HSV-l and CMV,35-38 respectively. In vitro experiments have raised the possibility of a bidirectional interaction between HIV-1 and herpes group viruses resulting in an increased viral replication of both types of viruses in dually infected cells. 36 Several mechanisms of interaction between the viruses were considered, including transactivation of enhancer/ promoters by cellular or viral transcription factors,36-38 and phenotypic mixing or alterations in binding affinity.35-38 These viral interactions were thought to be responsible for the rapid and devastating progression of necrotizing retinitis in some patients with AIDS. II Although we demonstrated a triple infection of the retina with HIV-l, CMV, and HSV-l and dual infections of single cells of the retina with HIV-l and CMV, there was no clinical evidence of progression of retinitis in our patient. Cytomegalovirus 1,2,4 and HSV_1 39 have been shown to spread in the retina by cell-to-cell transmission, leading to a brush-fire-like necrotizing retinitis. Although HIV1 infection of the retina has been reported previously, it may not, by itself, represent a cause of necrotizing retinitis. 9- 13 ,26,27 However, HIV-l may play an important role in the development of vascular changes, leading to occlusion of retinal vessels and/or the choriocapillaris with ensuing ischemia of the blood-supported area. 11,40 Herpesvirus and HIV particles and HSV-l antigen were seen in retinal and choroidal vascular endothelium of our patient's retina and choroid (Figs 3H and 4B). The lumen of some of these vessels was obstructed as a result of mural lymphocytic infiltration and swelling of endothelial cells. We did not document any evidence of immune-complex response in the retinal vascular walls of this patient. Because we have observed similar histopathologic and immunohistochemical findings in a patient with a VZV-induced bilateral acute retinal necrosis syndrome,31 one may assume that similar pathomechanisms may lead to retinal necrosis in herpesvirus-induced retinitis. In addition to direct viral cytolytic effects to the cells of the sensory retina, viral invasion of a vessel wall might lead, via activation of the immune-complex system,33,34 to a granulomatous or nongranulomatous occlusive vasculitis with ensuing ischemia and/or necrosis of the blood-supported area (i.e, the sensory retina). Matsuo and co-workers33 have shown that the visual outcome in patients with acute retinal necrosis and occlusive vasculitis was worse than in patients with herpesvirus-induced acute retinal necrosis without occlusive vasculopathy. They assumed that circulating
Rummelt et al . Triple Retinal Infection
Figure 4. Transmission electron microscopy of the retina. A , viral particle with nucleocapsid characteristic for herpesvirus (arrow) in disrupted retina cell (bar = 0.3 /Lm). B, viral particle with central tubular nucleoid surrounded by capsid (arrow) suggestive for human immunodeficiency virus in vascular endothelial cell (bar = 0.5 /Lm) . Ch = choriocapillaris.
immune complexes are either the cause or the result of extensive inflammation in retinal vessel walls. Circulating immune complexes are common in patients with AIDS, and arteriolar deposition of immunoglobulins has been described in the retina of such patients. 34 However, Forster and co-workers25 recently demonstrated that retinal arteritis or periphlebitis in progressive outer retinal necrosis
were minimal or absent in immunocompromised patients with AIDS, when the cellular and the humoral immune response was markedly decreased. Thus, retinal and choroidal microvasculopathy may have played an important role in the development of retinal necrosis in our patient, because he showed a nongranulomatous vasculitis of the retinal vessels in the area of necrotizing retinitis.
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Of interest in this case was the study of the retinal cell types infected with the different viruses. Immunoperoxidase studies showed that many "multinucleated giant cells" which stained positive for HIV-1 antigen, were also positive for GFAP antigen, indicating their glial origin (Muller cells; Fig 3C). However, some of the multinucleated cells appeared to be of monocytic origin, because HIV-l had infected mononuclear cells in the sensory retina, morphologically consistent with macrophages. Cells infected with HIV-1 are known to form syncytia, thereby resembling multinucleated giant cells. In our patient's retina, some of these cells also contained cytomegalic viral inclusions, which stained positive for CMV antigen. These results support previous studies that demonstrated that HIV-1 and CMV have an affinity to glial tissue,!2 also in the brain of patients with AIDS.12,4!·42 We identified CMV DNA and viral antigen in all layers of the neurosensory retina and in Muller cells and the RPE, but not in any vascular endothelium. Cells susceptible to HSV-1 were diffusely distributed throughout the retina and retinal vascular endothelium. However, we were not able to demonstrate the HSV -1 antigen in the RPE, the choriocapillaris, or the optic nerve. These findings confirm previous studies that showed affinity of HSV-1 to vascular endothelium in the retina and the brain of patients with AIDS. 4!,42 Our results support the assumption that viral spread to the retina occurs via the hematogenous route rather than by the neural pathway (i.e., the optic nerve and/or posterior ciliary nerves). Skolnik and co-workers!! reported an acute inflammatory cell response in the vitreous, retina, and choroid in half of their autopsy cases with CMV retinitis in AIDS. They suggested that granulocyte function in patients with AIDS was less compromised than in patients without AIDS with a secondary granulocyte defect such as transplant patients and patients with malignancies. We did not find a marked acute inflammatory cell response in the vitreous of our patient. In addition, only a few chronic inflammatory cells (lymphocytes and macrophages) were present in the choroid. This discrepancy in our patient with AIDS can be explained by a secondary lymphopenia and granulocytopenia due to the bone marrow toxicity to ganciclovir,43 which led to the cessation of therapy. Therefore, the therapeutic regimen has an influence on the histopathologic outcome of the disease. Human immunodeficiency virus has been cultured from the cornea, the conjunctiva, and the tear film of patients with AIDS.44 We demonstrated the detection of HIV-1 antigen (p24) in conjunctival epithelial cells (Fig 3D). This observation is interesting, because HIV may be transmitted from one patient to another from the cornea and/or the conjunctiva during applanation tonometry if no disinfection or sterilization occurs. However, no proven transmission of the virus via this pathway has been reported so far. In addition, the possible role of HIV-l in the development of conjunctival Kaposi sarcoma45 ,46 needs further study. Although our patient showed the typical clinical signs ofa CMV-induced necrotizing retinitis, a triple infection
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of the retina with HIV-l, CMV, and HSV-l was detected histopathologically and immunohistochemically. The ophthalmologist should be aware of concurrent viral infections of the retina that may occur in patients with AIDS, mimicking classic CMV retinitis or typical and/or atypical acute retinal necrosis syndrome. This fact is of practical interest, because the possibility of retinal infections with more than one virus should be considered in the clinical differential diagnosis of an apparently therapyresistant necrotizing retinitis in immunocompromised patients.
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tope encoded by exon 2 of the major immediate early gene. J Gen virol 1992;73:2699-703. Rummelt V, Wenkel H, Rummelt C, et al. Detection of varicella zoster virus DNA and viral antigen in the late stage of bilateral acute retinal necrosis syndrome. Arch Ophthalmol 1992; 11 0: 1132-6. Kostianovsky M, Grimley PM. Ultrastructural findings in the acquired immunodeficiency syndrome (AIDS). Ultrastruct Pathol 1985;8: 123-30. Matsuo T, Morimoto K, Matsuo N. Factors associated with poor visual outcome in acute retinal necrosis. Br J Ophthalmol 1991;75:450-4. Gupta S, Licorish K. Circulating immune complexes in AIDS [letter]. N Eng! J Med 1984;310:1530-1. Hirsch MS, Schooley RT, HO DD, Kaplan Je. Possible viral interactions in the acquired immunodeficiency syndrome (AIDS). Rev Infect Dis 1984;6:726-31. Skolnik PR, Kosloff BR, Hirsch MS. Bidirectional interactions between human immunodeficiency virus type 1 and cytomegalovirus. J Infect Dis 1988;157:508-14. Mosca JD, Bednarik DP, Raj NBK, et al. Herpes simplex virus type-1 can reactivate transcription of latent human immunodeficiency virus. Nature 1987;325:67-70. Davis MG, Kenney SC, Kamine J, et al. Immediate-early gene region of human cytomegalovirus transactivates the promoter of human immunodeficiency virus. Proc Natl Acad Sci USA 1987;84:8642-6. Pepose JS, Kreiger AE, Tomiyasu U, et al. Immunocytologic localization of herpes simplex type 1 viral antigens in herpetic retinitis and encephalitis in an adult. Ophthalmology 1985;92: 160-6. O'Donnell 11, Jacobson MA. Cotton-wool spots and cytomegalovirus retinitis in AIDS. Int Ophthalmol Clin 1989;29: 105-7. Nelson JA, Reynolds-Kohler C, Oldstone MBA, Wiley CA. HIV and HCMV coinfect brain cells in patients with AIDS. Virology 1989; 165:286-90. Wiley CA, Schrier RD, Nelson JA, et al. Cellular localization of human immunodeficiency virus infection within the brains of acquired immune deficiency syndrome patients. Proc Nat! Acad Sci USA 1986;83:7089-93. Millar AB, Miller RF, Patou G, et al. Treatment of cytomegalovirus retinitis with zidovudine and ganciclovir in patients with AIDS: outcome and toxicity. Genitourin Med 1990;66: 156-8. Fujikawa LS, Salahuddin SZ, Ablashi D, et al. HTLV-III in the tears of AIDS patients. Ophthalmology 1986;93: 1479-81. Visser OH, Bos PJ. Kaposi's sarcoma of the conjunctiva and CMV-retinitis in AIDS. Doc Ophthalmol 1986;64:77-85. Reichert CM, O'Leary TL, Levens DL, et al. Autopsy pathology in the acquired immune deficiency syndrome. Am J PathoI1983;112:357-82.
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Originally received: March 17, 1993. Revision accepted: August 19, 1993. I Department of Ophthalmology, University of Erlangen-Niirnberg, Erlangen, Germany. 2 Institute of Clinical and Molecular Virology, University of ErlangenNiirnberg, Erlangen, Germany.
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Presented in part at the ARVO Annual Meeting, Sarasota, May 1991. Supported by grant Ru 464/3-1 of the Deutsche Forschungsgemeinschaft, Bonn, Germany. Reprint requests to Volker Rummeit, MD, Department of Ophthalmology, University of Erlangen-Niirnberg, Schwabachanlage 6, D-91054 Erlangen, Germany.
Rummelt et al . Triple Retinal Infection Transmission electron microscopy showed herpes viral particles in the vascular endothelium of the retinal vessels and the choriocapillaris. Human immunodeficiency virus particles were identified in the endothelium of the choriocapillaris. Conclusions: The possibility of multiple viral infections of the retina, mimicking classic CMV retinitis, should be considered in the clinical and histologic differential diagnosis of necrotizing retinitis in patie.nts with AIDS. Ophthalmology 1994;101:270-279
Cytomegalovirus (CMV) retinitis is the most common ophthalmic opportunistic infection and cause of blindness in patients with the acquired immune deficiency syndrome (AIDS). 1-4 Infections of the retina and the choroid with multiple pathogenic organisms (i.e., bacteria, fungi, viruses) have been reported recently. 5-8 Proven pathogens causing viral infections of retinal tissue in patients with AIDS have included CMV,4.5.9-20 herpes simplex virus type 1 (HSV-l), 15,21,22 varicella zoster virus (VZV),23-25 human immunodeficiency virus type 1 (HIV_l),9- 12,26,27 and human herpes virus type 6. 9.27 Co-infections of the retina with HIV-l and CMV,II .12 HSV I5 and CMV,15 HIV-l and human herpes virus type 6,9.27 and with HIV-l , CMV, and human herpes virus type 6,27 respectively, have been demonstrated. These studies have implicated that possible interactions between different viruses, especially HIV-l and herpes group viruses in dually infected cells, may influence the clinical course and severity of retinal disease in patients with AIDS. II We now report on the clinical and histopathologic findings in the retina of a 55-year-old bisexual man with AIDS. Atriple infection of the retina with HIV-I, CMV, and HSV -1 was identified using immunohistochemistry, DNA in situ hybridization, and transmission electron microscopy. Our findings provide additional information about the pathogenesis of multi viral infections of the retina in immunocompromised patients with AIDS.
Case Report A 55-year-old white bisexual man was diagnosed with AIDS in February 1986. Four months later, Kaposi sarcoma of the right foot developed. At this time, visual acuity in both eyes was 20/ 25, and there was no evidence of viral retinitis in either eye. A mild hyperpigmentation and hypopigmentation of the retinal pigment epithelium (RPE) in the macula was observed. Serologic findings showed an antibody-positive HIV -I enzyme-linked immunoassay, verified by Western blot analyses. The complement fixation titer was 1: 16 for HSV, and no antibodies against VZV and CMV were detected by complement fixation titer at this time. On his first examination at our hospital on January 4, 1990, he complained of floaters and blurred vision in the right eye. Visual acuity at this time was 20/50 in the right eye and 20/25 in the left. Results of ophthalmoscopic examination disclosed an area of necrotizing retinitis in the right eye, extending from the optic disc and between the superotemporal and superonasal retinal vessel arcades to the equator (Fig 1). This area appeared as dry, white, opacified retinal lesions with interspersed hem-
orrhages. A cotton-wool spot was identified inferotemporally. Vitreous inflammation was absent, and the anterior segment was unremarkable. There was no evidence of retinitis in the left eye. Serologic testing showed a complement fixation titer of 1: 16 for HSV, 1:32 for CMV, and 1:4 for VZV. Human immunodeficiency virus type 1 was isolated by a standard lymphocyte co-culture technique. Because there was a strong suspicion of unilateral CMV retinitis, an induction dosage of intravenous ganciclovir (5 mg/kg twice daily for 2 weeks) was started. The patient was then given a maintenance therapy of ganciclovir of 6 mg/kg daily. On January 25, 1990, complement fixation titers were 1: 16 for CMV and 1:4 for HSV. The retinal necroses were stable. Unfortunately, the ganciclovir therapy had to be stopped due to severe side effects with almost total myelosuppression. The patient refused any further treatment and died on February 12, 1990, 1month after the initial diagnosis of CMV retinitis. An autopsy was performed immediately after death. The main pathologic findings were (I) CMV pneumonia, (2) CMV encephalitis, (3) disseminated Kaposi sarcoma, and (4) cerebral malignant non-Hodgkin lymphoma.
Materials and Methods Light Microscopy and Immunohistochemistry The eyes were obtained at autopsy within 8 hours after death and immediately fixed in a neutral-buffered solution of 4% paraformaldehyde and I % glutaraldehyde. They were sectioned vertically, submitting a pupil-optic nerve head section involving the area of retinal necrosis. The globes then were processed in standard histologic fashion. Hematoxylin-eosin, periodic acid-Schiff, Gomori's methenamine silver, Gram's, and Ziehl-Neelson stains of 4~m sections thick were performed. Formaldehyde-fixed and paraffin-embedded sections of the globes were deparaffinized in xylene and rehydrated in a series of graded alcohols. Endogenous peroxidase was quenched by incubation in 3% hydrogen peroxide. Immunohistochemical stains were carried out using antibodies against the following antigens: HSV-1 (polyclonal, rabbit, anti-HSV-I antibody; DAKO Diagnostika, Hamburg, Germany), VZV (5B7 monoclonal, mouse, antiVZV antibody; BioMerieux, Paris, France), CMV (E 13, a monoclonal antibody against an immediate-early CMV protein; Biosoft, Paris, France; and XP-l , a monoclonal antibody against a late CMV protein, pp I 50-courtesy of G. Jahn)28-3o, HIV-I (monoclonal, murine, anti-p24 antibody, and monoclonal, murine, anti-gp 120 antibody-courtesy of G. Jahn), and glial fibrillary acidic
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Top, Figure 1. Clinical aspect of the right fundus of a 55-year-old bisexual patient with AIDS (February 1990). Area of necrotizing retinitis with interspersed hemorrhage extending from the optic nerve, between the superotemporal and superonasal retinal vessel arcades, to the equator. Visual acuity was 20/50 in the right. Center and Bottom, Figure 2. Histopathologic findings of concurrent necrotizing human immunodeficiency virus type 1, cytomegalovirus, and herpes simplex virus type 1 retinitis. Center left (A), fuJI-thickness necrotizing retinitis (SR = sensory retina) with cytomegalic viral inclusions in retinal ceJls and the retinal pigment epithelium (arrows). Notice the paucity of chronic inflammatory ceJls in the choroid (Ch) (original magnification, Xl60). Center right (B), cytomegalic ceJls with an "owl's eye" appearance and intracytoplasmic and intranuclear viral inclusions in apparently glial ceJls of the sensory retina (original magnification, X400). Bottom (C), cytomegalic viral inclusions in retinal pigment epithelial (arrows) ceJls (original magnification, XBOO).
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Rummelt et al . Triple Retinal Infection protein (polyclonal, rabbit, anti-GFAP; DAKO Diagnostika). Commercially available versions of the peroxidaseantiperoxidase method (PAP; DAKO Diagnostika) and the streptavidin-biotin alkaline phosphatase (SBAP) technique (DAKO Diagnostika) were used. For color development, a 3,3-diaminobenzidine-hydrogen peroxide solution or aminoethylcarbazol-hydrogen peroxide solution was used. All sections were counterstained with hematoxylin. Formaldehyde-fixed and paraffin-embedded human foreskin fibroblast cultures, infected with HSV-I, HSV-2, VZV, CMV, and human brain and lung tissue, culture-positive for HSV-I, CMV, or HIV-I, provided the necessary positive and negative controls for sensitivity, specificity, and cross-reactivity of the different viral antisera. Globe sections of patients with malignant uveal melanomas and no previous history of viral infections were carried along in every series as negative controls.
DNA In Situ Hybridization For in situ hybridization, tissue sections were deparaffinized and pretreated with proteinase K and hydrochloric acid. After the pretreatment, single globe sections were digested either by DNAse or RNAse for approximately I hour to differentiate between CMV DNA and CMV RNA. The EcoRI-Y fragment ofCMV (strain AD 169) cloned into the plasmid pBR 322 was used as specific probeY The insert was isolated and radiolabeled by nick-translation using sulfur 35-labeled adenosine triphosphate. The probe showed no hybridization to the DNA ofHSV-1 or VZV genome in Southern blot hybridization. The HIND III restriction enzyme fragments A and C of VZV (strain 80-2) and the plasmid DNA ofpBR 322 served as control probes. After incubation for 17 hours at 40 0 C, the slides were washed with different concentrations of standard sodium citrate. Sections were dipped in NTB2 nuclear track emulsion (Eastman Kodak, Stuttgart, Germany), developed after 14 days of exposure at 4 0 C, stained with hematoxylin, and evaluated by light microscopy in the bright and dark field. Paraffin-embedded human foreskin fibroblast cultures infected with HSV-I, CMV, and VZV served as adequate positive and negative controls. Globe sections of patients with malignant uveal melanomas and no previous history of ocular viral infections were carried along in every series as well as one positive (CMV) and two negative (HSV-I, VZV) cell culture sections to exclude nonspecific signals.
S-7000 electron microscope (Hitachi Scientific, Tokyo, Japan).
Results Histopathologic Examination On gross examination, both eyes measured 24 X 23 X 23 mm with 5 mm of attached optic nerves. The clear cornea measured 11.5 X II mm. Both eyes showed mild hyperpigmentation and hypopigmentation of the retinal pigment epithelium (RPE) in the macula. The right eye showed a dense, white plaque replacing the retinal structures from the optic nerve and along the superotemporal and superonasal retinal vessel arcades to the equator. Scattered retinal hemorrhages were present in this area. The vitreous was clear, and the retina was flat. The retinal vessels, the choroid, and the optic nerves appeared unremarkable. On microscopic examination, the left eye was unremarkable. The right eye showed a circumscribed fullthickness necrosis of the neurosensory retina (Fig 2A). In this area, retinal tissue was replaced by lymphocytes, macrophages, occasional "multinucleated giant cells," and acute inflammatory cells. Some extravasated erythrocytes also were seen. There was a focal replacement of the sensory retina by glial tissue adjacent to the area of acute necrosis. Bruch's membrane and the inner-limiting membrane of the retina were intact. A serous retinal detachment was present in the area of retinal necrosis. Many intranuclear and intracytoplasmic cytomegalic viral inclusions were seen in cells of the necrotic retinal tissue and in glial cells of apparently normal sensory retinal structures (Fig 2B). In addition, eosinophilic intranuclear viral inclusions (Cowdry A-type) suggestive for herpesvirus were present in retinal cells near cytomegalic cells with an "owl's eye" appearance. Some retinal vessels in the necrotic area showed narrowing of their lumen as a result of mural lymphocytic infiltration and swelling of vascular endothelial cells, which occasionally contained intranuclear eosinophilic viral inclusions. The RPE underlying the necrotic retina showed the viral infection in a patchy pattern (Fig 2C), and where necrotic, the subjacent choroid contained only a few chronic inflammatory cells. There was minimal acute inflammatory reaction to the vitreous. The optic nerve and the anterior segment were unremarkable. Special stains for bacteria, fungi, and acid-fast bacilli were negative in both eyes.
Transmission Electron Microscopy For transmission electron microscopy, parts of necrotic retina and of normal-appearing retina were fixed in a neutral-buffered 2.5% glutaraldehyde solution. Retinal tissue was postfixed in a solution of I % buffered osmium tetroxide, dehydrated, and embedded in standard fashion. Thick (l-Jlm) sections were stained with toluidine bluebasic fuchsin solution. Thin sections were stained with uranyl acetate-lead citrate and examined with the Hitachi
Immunoperoxidase Studies Immunohistochemical stains were reproducible, sensitive, and specific for the antibodies used. There was no cross-reactivity between the different viral antisera. Human immunodeficiency virus type I antigens (p24 and gp 120) were found in all layers of the neurosensory retina within mononuclear cells and "multinucleated giant cells" (Figs 3A and 3B). Many of these giant cells
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Figure 3. (continued). Top left (G), herpes simplex virus type 1 antigen (arrows) in retinal cells diffusely distributed throughout all layers of the retina (pAP; original magnification, X240). Top right (H), herpes simplex virus type 1 antigen in retinal vascular endothelium (arrows) (PAP; original magnification, X400). Bottom (I), in situ hybridization of CMV DNA (EcoRI-Y fragment). The positive reaction is confined to the nucleus and the cytoplasm of the infected retinal cells. Notice the patchy distribution of infected cells. V = vitreous; SR = sensory retina; Ch = choroid (original magnification, X240).
also stained positive for GFAP (Fig 3C), indicating their glial origin. In addition, HIV -1 antigens were found in conjunctival epithelial cells (Fig 3D). No HIV-l antigens were demonstrated in any vascular endothelium or in the RPE. Cytomegalovirus antigens (immediate-early and late antigen pp 150) were found in all layers of the retina of
the right eye within cytomegalic cells with an "owl's eye" appearance and in many nonmegalic cells without inclusions, as well as in apparently normal sensory retinal structures (Figs 3E and 3F). Retinal pigment epithelial cells stained positive only for the CMV immediate-early antigen, not for the late antigen. No CMV antigens were found in any vascular endothelium, the choroid, the optic
Figure 3. Immunohistochemistry and in situ hybridization of triple infection of the retina. Top left (A), human immunodeficiency virus type 1 antigen (p24) in multinucleated giant cell in the sensory retina (arrow). Notice cytomegalic viral inclusion (arrowhead) (peroxidase-anti-peroxidase [PAP]; original magnification, X400). Top right (B), human immunodeficiency virus type 1 antigen (p24) in mononuclear cells diffusely distributed in the sensory retina (arrows) (PAP; original magnification, X400). Center left (C), GFAP antigen in multinucleated giant cells of the sensory retina (arrows) indicating its glial origin (PAP; original magnification, X400). Second row right (D), human immunodeficiency virus type 1 antigen (p24) in conjunctival epithelial cells (arrows) (PAP; original magnification, X400). Third row right (E), immediate-early cytomegalovirus (CMV) antigen (E 13) in patchy areas of retinal cells (streptavidln-biotin alkaline phosphatase [SBAP]; original magnification, X 240). Bottom (F), immediate-early CMV antigen (E 13) in retinal pigment epithelial cells and retinal cells (SBAP; original magnification, X240). (Fig 3 continues.)
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nerve, or the anterior segment of the eyes. Dual infections of individual cells with HIV-l and CMV were demonstrable in multinucleated cells of the retina (Fig 3A), which also stained positive for GFAP, using serial sections of the same tissue. Herpes simplex virus type 1 antigens were present in all layers of the neurosensory retina (Figs 3G and 3H) but not in the RPE. Whereas CMV antigens were usually identified in focal patches of cells, HSV -1 antigens evenly were distributed throughout all layers of the sensory retina. In many instances, cytomegalic cells, which did not stain for HSV-1 antigens, were interspersed in the same microscopic field among cells bearing the HSV -1 antigen. We also detected HSV -1 antigens in endothelial cells of the retinal vessels (Fig 3H).
DNA In Situ Hybridization Corresponding to the immunohistochemical findings, CMV DNA could be detected in focal patches of cells in all layers of neurosensory retina (Fig 31). The strongly positive reaction was confined to both the nucleus and cytoplasm of the cell. No viral DNA was present in any vascular endothelium, the optic nerve, or the anterior segment.
Transmission Electron Microscopy Electron microscopic studies disclosed numerous herpes viral particles with a central nucleocapsid 100 nm in diameter in endothelial cells of retinal vessels and the choriocapillaris and in disrupted retinal cells (Fig 4A). Cytomegalovirus-like particles were identified in cells of the necrotic sensory retina (Fig 4A) and the RPE. In addition, HIV viral particles with a central tubular core nucleoid surrounded by a capsid 120 nm in diameter9,32 were present in endothelial cells of the choriocapillaris (Fig 4B). We detected a dual infection of these individual cells with herpesvirus and HIV particles, The inner-limiting membrane of the retina and Bruch's membrane were intact.
Discussion We found evidence of a multiviral necrotizing retinitis in a patient with AIDS. The concurrent HIV -1, HSV -1, and CMV retinitis in this patient may have resulted from reactivation oflatent HIV-l, HSV-l, and/or CMV infection with exogenous strains of HSV-l or CMV, reactivation of latent HSV by superinfection with CMV, or hematogenous dissemination of the viruses during intermittent viremia in this immunocompromised patient. The detection of HSV-1 antigen in retinal vascular endothelium and of herpesvirus and HIV particles in the vascular endothelium ofthe choriocapillaris in our patient suggests that blood-borne dissemination of these viruses to the retina may have played a role in the pathogenesis of the disease. 4,15 Subsequent CMV viremia may have led to di-
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rect retinal infection via the damaged vascular endothelium. Different pathomechanisms may account for the destruction of the sensory retina in this patient, including (1) a direct viral invasion of retinal structures with ensuing cytolytic effects of the viruses themselves l ,4,15,17 (CMV, HSV-1), (2) ischemia, secondary to an occlusive vasculitis observed in patients with CMV retinitis and the acute retinal necrosis syndrome,4,31,33 (3) a virus-triggered immune-complex reaction to the retinal vessel walls potentiating the destruction of the sensory retina,33,34 and (4) interactions between HIV-l and HSV-l and CMV,35-38 respectively. In vitro experiments have raised the possibility of a bidirectional interaction between HIV-1 and herpes group viruses resulting in an increased viral replication of both types of viruses in dually infected cells. 36 Several mechanisms of interaction between the viruses were considered, including transactivation of enhancer/ promoters by cellular or viral transcription factors,36-38 and phenotypic mixing or alterations in binding affinity.35-38 These viral interactions were thought to be responsible for the rapid and devastating progression of necrotizing retinitis in some patients with AIDS. II Although we demonstrated a triple infection of the retina with HIV-l, CMV, and HSV-l and dual infections of single cells of the retina with HIV-l and CMV, there was no clinical evidence of progression of retinitis in our patient. Cytomegalovirus 1,2,4 and HSV_1 39 have been shown to spread in the retina by cell-to-cell transmission, leading to a brush-fire-like necrotizing retinitis. Although HIV1 infection of the retina has been reported previously, it may not, by itself, represent a cause of necrotizing retinitis. 9- 13 ,26,27 However, HIV-l may play an important role in the development of vascular changes, leading to occlusion of retinal vessels and/or the choriocapillaris with ensuing ischemia of the blood-supported area. 11,40 Herpesvirus and HIV particles and HSV-l antigen were seen in retinal and choroidal vascular endothelium of our patient's retina and choroid (Figs 3H and 4B). The lumen of some of these vessels was obstructed as a result of mural lymphocytic infiltration and swelling of endothelial cells. We did not document any evidence of immune-complex response in the retinal vascular walls of this patient. Because we have observed similar histopathologic and immunohistochemical findings in a patient with a VZV-induced bilateral acute retinal necrosis syndrome,31 one may assume that similar pathomechanisms may lead to retinal necrosis in herpesvirus-induced retinitis. In addition to direct viral cytolytic effects to the cells of the sensory retina, viral invasion of a vessel wall might lead, via activation of the immune-complex system,33,34 to a granulomatous or nongranulomatous occlusive vasculitis with ensuing ischemia and/or necrosis of the blood-supported area (i.e, the sensory retina). Matsuo and co-workers33 have shown that the visual outcome in patients with acute retinal necrosis and occlusive vasculitis was worse than in patients with herpesvirus-induced acute retinal necrosis without occlusive vasculopathy. They assumed that circulating
Rummelt et al . Triple Retinal Infection
Figure 4. Transmission electron microscopy of the retina. A , viral particle with nucleocapsid characteristic for herpesvirus (arrow) in disrupted retina cell (bar = 0.3 /Lm). B, viral particle with central tubular nucleoid surrounded by capsid (arrow) suggestive for human immunodeficiency virus in vascular endothelial cell (bar = 0.5 /Lm) . Ch = choriocapillaris.
immune complexes are either the cause or the result of extensive inflammation in retinal vessel walls. Circulating immune complexes are common in patients with AIDS, and arteriolar deposition of immunoglobulins has been described in the retina of such patients. 34 However, Forster and co-workers25 recently demonstrated that retinal arteritis or periphlebitis in progressive outer retinal necrosis
were minimal or absent in immunocompromised patients with AIDS, when the cellular and the humoral immune response was markedly decreased. Thus, retinal and choroidal microvasculopathy may have played an important role in the development of retinal necrosis in our patient, because he showed a nongranulomatous vasculitis of the retinal vessels in the area of necrotizing retinitis.
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Of interest in this case was the study of the retinal cell types infected with the different viruses. Immunoperoxidase studies showed that many "multinucleated giant cells" which stained positive for HIV-1 antigen, were also positive for GFAP antigen, indicating their glial origin (Muller cells; Fig 3C). However, some of the multinucleated cells appeared to be of monocytic origin, because HIV-l had infected mononuclear cells in the sensory retina, morphologically consistent with macrophages. Cells infected with HIV-1 are known to form syncytia, thereby resembling multinucleated giant cells. In our patient's retina, some of these cells also contained cytomegalic viral inclusions, which stained positive for CMV antigen. These results support previous studies that demonstrated that HIV-1 and CMV have an affinity to glial tissue,!2 also in the brain of patients with AIDS.12,4!·42 We identified CMV DNA and viral antigen in all layers of the neurosensory retina and in Muller cells and the RPE, but not in any vascular endothelium. Cells susceptible to HSV-1 were diffusely distributed throughout the retina and retinal vascular endothelium. However, we were not able to demonstrate the HSV -1 antigen in the RPE, the choriocapillaris, or the optic nerve. These findings confirm previous studies that showed affinity of HSV-1 to vascular endothelium in the retina and the brain of patients with AIDS. 4!,42 Our results support the assumption that viral spread to the retina occurs via the hematogenous route rather than by the neural pathway (i.e., the optic nerve and/or posterior ciliary nerves). Skolnik and co-workers!! reported an acute inflammatory cell response in the vitreous, retina, and choroid in half of their autopsy cases with CMV retinitis in AIDS. They suggested that granulocyte function in patients with AIDS was less compromised than in patients without AIDS with a secondary granulocyte defect such as transplant patients and patients with malignancies. We did not find a marked acute inflammatory cell response in the vitreous of our patient. In addition, only a few chronic inflammatory cells (lymphocytes and macrophages) were present in the choroid. This discrepancy in our patient with AIDS can be explained by a secondary lymphopenia and granulocytopenia due to the bone marrow toxicity to ganciclovir,43 which led to the cessation of therapy. Therefore, the therapeutic regimen has an influence on the histopathologic outcome of the disease. Human immunodeficiency virus has been cultured from the cornea, the conjunctiva, and the tear film of patients with AIDS.44 We demonstrated the detection of HIV-1 antigen (p24) in conjunctival epithelial cells (Fig 3D). This observation is interesting, because HIV may be transmitted from one patient to another from the cornea and/or the conjunctiva during applanation tonometry if no disinfection or sterilization occurs. However, no proven transmission of the virus via this pathway has been reported so far. In addition, the possible role of HIV-l in the development of conjunctival Kaposi sarcoma45 ,46 needs further study. Although our patient showed the typical clinical signs ofa CMV-induced necrotizing retinitis, a triple infection
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of the retina with HIV-l, CMV, and HSV-l was detected histopathologically and immunohistochemically. The ophthalmologist should be aware of concurrent viral infections of the retina that may occur in patients with AIDS, mimicking classic CMV retinitis or typical and/or atypical acute retinal necrosis syndrome. This fact is of practical interest, because the possibility of retinal infections with more than one virus should be considered in the clinical differential diagnosis of an apparently therapyresistant necrotizing retinitis in immunocompromised patients.
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