Pseudodendritic fungal epithelial keratitis in an extended wear contact lens user.

PURPOSE: Pseudodendritic keratitis in a contact lens wearer is generally associated with acanthamoeba keratitis. We report a case of isolated pseudodendritic fungal epithelial keratitis that occurred in an extended wear contact lens user. METHODS: A 48-year-old woman was evaluated in our clinic for a 36-hour history of left eye pain. She wore extended wear soft contact lenses and frequently rinsed her eyes with tap water. Her left cornea had a paracentral 3-mm area of epithelium with raised ridges in a pseudodendritic pattern. The underlying corneal stroma was normal. A therapeutic and diagnostic corneal scraping of the lesion was performed and sent for Gomori methenamine silver (GMS) staining. The clinical concern was for epithelial acanthamoeba keratitis. RESULTS: The GMS staining revealed septate fungal hyphae within sheets of corneal epithelium. The patient was started on frequent alternating natamycin (5%) and amphotericin B (0.15%) antifungal eyedrops and exhibited a rapid clinical response. Her keratitis completely resolved, and her vision returned to her baseline of 20/25. Corneal fungal cultures showed no growth. CONCLUSIONS: Our case is an extremely unusual presentation of fungal keratitis, which rarely presents as a pseudodendritic epithelial keratitis. There are two previous similar case reports initially misdiagnosed as acanthamoeba keratitis. Clinicians should be aware that isolated fungal epithelial keratitis can present as a distinct entity and should be considered in the differential diagnosis of pseudodendritic keratitis. The GMS staining is an excellent diagnostic test in a patient presenting with pseudodendritic keratitis because it allows rapid diagnosis of acanthamoeba and fungal infections.

Ocular disease pattern induced by herpes simplex virus is genetically determined by a specific region of viral DNA.

The pattern of ocular disease produced in the rabbit eye by HSV-1 (F) and HSV-1(MP) strains and recombinants F(MP)A, F(MP)B, F(MP)C, F(MP)D, F(MP)E, and F(MP)F was studied. The characteristics of ocular herpetic disease such as morphology of dendritic ulcers, severity of epithelial disease and incidence and duration of stromal disease produced in the rabbit eye are genetically determined by the virus strain. Our studies show that transfer of a defined part of the genome of the stromal disease-producing virus, HSV-1(MP), to the genome of an epithelial disease-producing virus, HSV-1(F), yielded recombinants with one or more of the disease characteristics of the donor strain. Specifically, recombinant F(MP)D produced lesions characteristic of the donor HSV-1(MP) strain; recombinants F(MP)C and F(MP)E produced stromal disease approaching the severity of the disease produced by the donor HSV-1(MP) strain, and only recombinants F(MP)A and F(MP)B retained the typically elongate lesions of the recipient HSV-1(F), whereas the recombinant strain F(MP)F produced no disease. The viral functions pertaining to the ocular disease pattern map between 0.70 and 0.83 map units in HSV-1 DNA within the BglII F DNA fragment. The pattern of stromal disease is independent of the production of glycoprotein C and fusion of HEp-2-infected cells. The functions relating to these aspects of ocular disease segregate but are closely linked.

Unusual dendritic keratitis.

Bilateral pseudo-dendritic keratitis in infancy can be due to tyrosinemia, a rare metabolic disorder. Ocular involvement may be the earliest presenting manifestation of this disease. Early diagnosis is essential because dietary modifications can result in complete reversal of the manifestations of this disorder. This disease must be suspected in all cases of non-responsive dendritic keratitis in the pediatric age group, especially if it is associated with cutaneous lesions such as patmoplantar keratosis. Serum tyrosine levels must be done in these cases.

Patterns of herpes simplex keratitis in inbred mice.

The authors have investigated the course of herpes simplex type 1 (HSV) keratitis in three different inbred strains of mice infected with four different HSV isolates. Severity of ocular disease and mortality is dependent upon both the virus isolate and the host strain. In particular, the likelihood of progression from self-limited dendritic keratitis to severe necrotizing stromal keratitis varies markedly among the virus-host strain combinations tested. When mice from strains resistant to stromal disease are crossed with mice from strains susceptible to stromal disease, the F1 offspring are resistant, suggesting that the gene(s) controlling resistance is dominant. Corneal stromal keratocytes and embryo fibroblasts from inbred mice differ significantly in their ability to support the replication of HSV in vitro. HSV replicates more efficiently in vitro in keratocytes from mice susceptible to stromal keratitis than it does in keratocytes from mice resistant to stromal keratitis. These findings provide evidence in an animal model for both virus- and host-related mechanisms that determine susceptibility to stromal keratitis.

Role of T-lymphocytes in the pathogenesis of herpetic stromal keratitis.

Our study was designed to investigate the mechanism of the stromal reaction in experimental ocular infection of murine eyes with herpes simplex virus (HSV). Severe stromal keratitis with scarring occurred in BALB/c mice after infection of the scarified cornea but similar reactions did not occur in athymic mice. However, if athymic mice were given adoptive transfers of lymphoid cells, a severe necrotizing and ulcerative keratitis accompanied by scarring resulted. The lesion progressed more quickly in recipients of lymphoid cells specifically immune to HSV and containing cytotoxic T-lymphocyte activity. In such mice, necrosis and ulceration were marked on the sixth day after transfer compared with 9-12 days for those given nonimmune cells. Removal of T-lymphocytes from the immune lymphoid population by treatment with specific antiserum and complement abrogated the adoptive transfer of the stromal reaction. Our results further demonstrate that stromal keratitis represents a host immunopathologic response to HSV infection in which T-lymphocytes are essential participants. Multiple mechanisms of T-cell immunopathology appear to be operating, including a reaction mediated by cytotoxic T-lymphocytes.

Histological and electron microscopic studies of experimental herpetic keratitis in the rabbit.

Histological and electron microscopic observations, together with virus cultures, were made in the eyes of 50 New Zealand white rabbits which received bilateral intrastromal inoculation with the RE strain of herpes simplex virus. Virus cultures of whole corneas were positive for the first 2 weeks following inoculation, but were consisently negative thereafter. An inflammatory response to HSV infection, consisting of polymorphonuclear leukocytes and lymphocytes, was seen in the limbus within 7 hours after inoculation of the cornea. A massive accumulation of lymphocytes and plasma cells appeared in the limbus, suggesting that the limbus may behave as a lymphoid tissue, where differentiation and maturation of lymphoid cells occur as they acquire immunocompetence. Neovascularization of the cornea was associated with a heavy infiltration of the surrounding stroma with polymorphonuclear leukocytes, as well as numerous plasma cells and a few lymphocytes and macrophages. Numerous abnormal, pleomorphic keratocytes were found in the stroma. Lymphocytes were frequently found closely adhering to these abnormal keratocytes, suggesting a T-cell attack on a target cell. A model which describes the mechanism by which herpes virus infection leads to corneal scarring is suggested.

Resolution of HSV corneal infection in the absence of delayed-type hypersensitivity.

The role of delayed-type hypersensitivity (DTH) in the resolution of herpes simplex virus type 1 (HSV-1) ocular infection was examined. Infection of Balb/c mice on the sacrificed cornea with HSV-1 resulted in sensitization for DTH. This response, demonstrable by swelling of the ear following inoculation with ultraviolet-irradiated virus, was optimal 7 days postinfection. The reaction was immunologically specific and characterized histologically by a predominately mononuclear cell infiltrate. DTH responsiveness could be completely abrogated if the mice were inoculated intravenously with an attenuated strain of HSV-17 days before corneal infection. DTH-unresponsive mice were, nevertheless, resistant to corneal challenge with sublethal or lethal doses of HSV-1. Resistance was accompanied by a greater than 30-fold reduction in infectious virus in the eye 24 hr post challenge. A cellular infiltrate characteristic of a DTH response was not observed within the cornea during virus clearance. Tolerance was restricted to DTH, as antibodies to HSV antigens could be readily demonstrated 6-7 days after intravenous virus immunization. These antibodies may have contributed to the resistance observed. The results establish that neither a systemic nor local DTH response is required by the host to resist HSV-1 ocular infection.

A role for T lymphocytes in preventing experimental herpes simplex virus type 1-induced retinitis.

We have previously shown that herpes simplex virus Type 1 (HSV-1) inoculated into the anterior chamber (AC) of one eye of BALB/c mice results in retinal destruction in the opposite eye while retinas in virus-injected eyes are preserved. In the present study, immunodeficient mice (athymic BALB/c or normal BALB/c which had received either gamma-irradiation [450 R] or cyclophosphamide [150 mg/kg treatment]), demonstrated bilateral retinal destruction upon unilateral AC inoculation of HSV-1. Reconstitution of these immunodeficient mice with spleen cells obtained from days 10-21 AC-inoculated donor mice, prior to AC inoculation of HSV-1, prevented retinal necrosis in more than 80% of both eyes. In contrast, donor cells from mice inoculated subcutaneously (SC) with HSV-1 preserved only about 30% of recipient retinas, regardless of the cell transfer time after donors received HSV-1. Normal unsensitized syngeneic donor spleen cells failed to prevent bilateral retinal necrosis in either athymic or irradiated BALB/c mice, although they eliminated recipient mortality. T cell depletion of AC- or SC-inoculated donor cells removed their retinal protective effects completely. These studies demonstrate an active role for T lymphocytes in controlling the extent of disease in a murine model of HSV-induced retinitis.

The normal and abnormal human corneal epithelial surface: a scanning electron microscope study.

The surface morphology of 108 corneal buttons obtained at keratoplasty showed specific patterns for each disease process. The surface over a traumatically scarred cornea was identical to that of undamaged sites, showing microvilli and microplicae in various numbers and combinations. Keratoconus specimens showed many dark cells, frequently noted to have surface blebs 0.25 to 3 micrometer in size over the entire cone in the nipple type, and in a broad basal band inside the cone or over the entire button in the sagging-cone type. Some blebs contained cytoplasm and 250 A glycogen-like granules. In larger, dark cells, holes were found in the blebs and the plasma membrane was degenerated. Corneal epithelial edema was manifested by a large irregular surface caused by the anterior bulge of edematous cells, many attached by only a small area, and variable-sized depressions, often the size of epithelial cells. More than a year after stromal scarring from herpetic keratitis, many epithelial cells lay loosely on the surface whereas other epithelial cells were edematous and partially detached from the surface cell sheet. Localized heaping of rounded epithelial and inflammatory cells persisted in some areas.

The dichotomy between herpes simplex virus type 1-induced ocular pathology and systemic immunity.

Herpes simplex virus Type 1 (HSV-1) was injected into the mouse eye by the intravitreal (into the vitreous chamber; VC) or translimbal (across the limbus into the anterior chamber; TxL) route. These routes were compared for ocular pathology and systemic immunity. After VC inoculation, the virus-injected eye developed a viral infection, with the majority of opposite, uninjected eyes remaining intact and free of virus over the 4 week period of observation. In contrast, following translimbal inoculation, the entire virus-injected eye developed infection and inflammation together with subsequent chorioretinitis in the opposite uninjected eye. Systemic immunity induced by VC or TxL virus inoculation was similar to the effects of anterior chamber (AC) inoculation of the same dose of HSV-1: T cell-mediated DTH responses were suppressed while levels of anti-HSV neutralizing antibody were enhanced, compared to subcutaneously primed positive control mice. These findings demonstrate that HSV-1-induced ocular pathology does not necessarily correlate directly with systemic immunity.

Superiority of antibody versus delayed hypersensitivity in clearance of HSV-1 from eye.

The contribution that antibody and delayed type hypersensitivity (DTH) make in promoting HSV-1 clearance from the infected cornea was investigated. Balb/c mice were immunized intravenously or subcutaneously with an attenuated strain of HSV-1 to generate hosts which were antibody-producing DTH-tolerant or antibody-producing DTH-responsive. Anti-mu serum treated mice were likewise sensitized intravenously or subcutaneously to obtain hosts which were antibody depressed-DTH tolerant or antibody-depressed DTH-responsive. Eight days after sensitization, these four sensitized groups and unsensitized controls were infected on scarified corneas with a stromal keratitis inducing strain of HSV-1, and the extent of virus replication was determined 1, 3, and 7 days later. Very different results were obtained depending upon the host's immune status. Virus proliferated extensively (greater than 3-4 logs) in the eyes of nonimmune mice and antibody-depressed DTH-tolerant hosts during the first 3 days after infection. In striking contrast, HSV-1 could not be detected even 24 hr post challenge in antibody-producing DTH-tolerant mice. In fact, such mice cleared virus from the eye as efficiently as immunologically intact hosts. However, in mice with the reverse immune status, ie antibody-depressed DTH-responsive, virus growth was clearly evident (greater than 2-3 logs) during days 1-3, and only thereafter did complete clearance occur. These results indicate that in the sensitized host antibody is both independent of and significantly more effective than DTH in promoting HSV-1 eradication from the infected eye.

Bilateral alterations of the ERG and retinal histology following unilateral HSV-1 inoculation.

The physiological condition of the retinas of BALB/c mice inoculated unilaterally in the anterior chamber with the KOS strain of herpes simplex virus type 1 (HSV-1) was monitored by ERG recordings. After the ERG recordings, the retinas were examined for histopathological changes. In the inoculated eye, depressed ERGs were recorded on day 2 PI and abolished ERGs on day 4 PI. The changes in the ERGs were complete by day 5-6 PI. Of the 53 inoculated eyes followed for longer than day 6 PI, four (7.5%) remained normal, 30 (56.6%) had reduced ERGs and 19 (35.8%) had abolished ERGs. In the contralateral eyes, the first changes were noted on day 8 PI, and abolished ERGs were recorded on day 9 PI. Of the 55 contralateral eyes followed for longer than 10 days, 15 (27.3%) remained normal, four (7.2%) had reduced ERGs and 36 (65.4%) had abolished ERGs. The percentage of eyes with depressed ERGs was significantly higher in the inoculated than in the uninoculated eyes, and the percentage of eyes with abolished ERGs was significantly higher in the uninoculated eyes than in the inoculated eyes. The histopathological alterations were different for the two eyes. In the inoculated eyes, the changes were mainly in the outer retina, with characteristic folds in the photoreceptor and outer nuclear layer interspersed with normal appearing retina. The pigment epithelium was also abnormal. In the uninoculated eyes, the changes began in the inner retina but rapidly spread to all layers of the retina. This panretinal necrosis accounted for the higher percentage of abolished ERGs in the uninoculated eyes. The differences in the alterations of the ERG and the histopathological changes may be related to the underlying mechanism of action of the HSV-1 during the evolution of the experimental retinopathy.

Immunologic modulation of virus-induced pathology in a murine model of acute herpetic retinal necrosis.

Unilateral inoculation of herpes simplex virus Type 1 (KOS strain) into the anterior chamber of BALB/c eyes produces an ocular disease with a distinctive differential pattern of retinal pathology. Specifically, the retina of the inoculated eye remains histologically intact, whereas the contralateral retina becomes necrotic. We demonstrate that retinal necrosis in opposite uninjected eyes directly correlates with the presence of herpes simplex viral antigens, whereas the intact retinas of virus-injected eyes are devoid of immunocytochemically detectable viral antigens. Immunosuppression or lack of a thymus results in bilateral retinal necrosis, with positive immunoperoxidase staining for viral antigens in both eyes. We have shown previously that retinal protection in both eyes can be restored to irradiated recipients by adoptive transfer of spleen cells from mice primed by AC injection of HSV. Our results with reconstituted and normal mice suggest that virus-mediated cytopathic effects underlie contralateral retinal necrosis since HSV antigens are localized to areas of retinal necrosis and their presence precedes the local inflammatory response; immunosuppression does not alter the development of contralateral retinal necrosis. They also indicate that ipsilateral retinal preservation reflects T cell-mediated inhibition of viral spread to retinas of injected eyes. Reconstitution of irradiated recipients with AC primed donor cells prevents immunohistochemically detectable virus and retinal necrosis in both eyes. In all experimental groups we failed to detect viral antigens in the absence of retinal pathology.

The isolation of herpes simplex virus from rabbit corneas during latency.

Herpes simplex virus type 1 (HSV-1) latency, as operationally defined, is a state in which cell-free infectious virus cannot be demonstrated in tissue at the time of sacrifice, but infectious virus can be isolated from the same tissue after prolonged cultivation. Latent HSV has been routinely detected in sensory ganglia of the infected dermatome. We have isolated HSV-1 (RE) from the corneas of 11% of infected rabbits which harbored virus in a latent state in trigeminal ganglia. HSV-1 (RE) was isolated from 10 of 88 cultures of corneal cells established following collagenase digestion of individual corneas taken from asymptomatic animals 118 days after infection. Virus was recovered only after prolonged primary culture and in some cases serial passage of corneal cells (range 5 to 26 days to initial cytopathic effect, n = 10). Virus was isolated from 68 of 68 trigeminal ganglia from the same rabbits by cocultivation of ganglion pieces with Vero cells (range 9 to 20 days to initial cytopathic effect, n = 68) while no cell-free virus was isolated from ganglia at the time of sacrifice. Virus isolation from corneas during the latent period occurred in a manner independent of prior antiviral or antiviral plus immunosuppressive therapy. Clinical evaluation of the corneas throughout the course of acute disease, stromal disease, and at the time of sacrifice provided no evidence that could be used to predict which corneas would yield virus. These data suggest that HSV-1 can remain in a nonreplicative state characteristic of latency in cells of rabbit corneas for long periods after infection and therapy of herpetic eye disease.

Histology and immunohistology of Igh-1-restricted herpes simplex keratitis in BALB/c congenic mice.

In order to characterize the local ocular immunologic milieu of Igh-1-restricted herpes simplex keratitis (HSK), we investigated histologic and immunohistologic correlates of disease over a 21-day time course. Clinically observable keratitis began 10 days postinoculation in susceptible C.AL-20 (Igh-1d) and moderately susceptible BALB/c (Igh-1a) mice, whereas HSV-1-resistant C.B-17 (Igh-1b) mice rarely developed disease. Igh-1-restricted histologic differences were observed by day 11 postinoculation; C.AL-20 and BALB/c mice showed augmented recruitment of neutrophils and mononuclear cells in conjunctival, limbal, and corneal tissues compared to C.B-17 mice. On immunohistologic study, Lyt-1 to Lyt-2 cell ratios by day 11 postinoculation were 7:1, 2:1, and 1:8 in corneas from C.AL-20, BALB/c, and C.B-17 mice, respectively. Macrophages and neutrophils were absent in corneas from C.B-17 mice at this time, but could be found in large numbers in the corneas of susceptible mouse strains through day 21. These data demonstrate a strong relationship between Igh-1 phenotype and inflammatory cell recruitment in response to corneal infection with HSV-1, and support a role for T cell subpopulations in mediating Igh-1-restricted HSK.

HSV-2 alters retinal physiology and morphology bilaterally in mice.

Anterior chamber inoculation of 10(4) PFU of the MS strain of HSV-2 resulted in physiologic and morphologic changes in the retina of the inoculated and the uninoculated eyes. In the inoculated eyes, electroretinogram (ERG) depression was first detected on day 3 and abolished ERGs on day 8 postinoculation (PI). The decrease in the ERGs was rapid and the time course was similar for all of the eyes. In spite of a 90% decrease in the amplitude of the b-wave, the retinal sensitivity did not change. Of 23 eyes tested on or after day 10 PI, none had normal, 4.3% had reduced, and 95.6% had abolished ERGs. In the uninoculated eyes, ERG depression was first detected on day 8 and abolished ERGs on day 12 PI. The course of the ERG depression was more variable, and some of the eyes showed a decrease in retinal sensitivity. Of the 22 eyes tested on or after day 17 PI, 18% had normal, 32% had reduced, and 50% had abolished ERGs. The majority (17/33) of the retinas of the inoculated eyes showed panretinal necrosis, although 7 of 33 retinas had pathology confined to the outer layers of the retina. In the uninoculated eyes, only 5 of 30 retinas were necrotic and 14 of 30 retinas had pathology limited to the outer layers of the retina. These observations suggested that the physiologic and morphologic changes progress through two stages: an early stage with reduced ERGs and pathology limited to the outer retinal layers, and a second stage in which the ERG is abolished and the pathologic changes extend into the inner retina. Not all retinas progress to the second stage.

Recurrent HSV-1 corneal lesions in rabbits induced by cyclophosphamide and dexamethasone.

Herpes simplex virus type 1 (HSV-1) ocular shedding and recurrent corneal epithelial lesions were assessed following an intravenous injection of cyclophosphamide (75 mg/kg) and 24 hr later an intravenous injection of dexamethasone (4 mg/kg) in 24 eyes of 15 rabbits latently infected with HSV-1 strain McKrae. Sampling for HSV-1 ocular shedding and epithelial lesion began on the day after cyclophosphamide injection and continued for 8 consecutive days. Ocular tear film was collected on a Dacron swab with care taken to avoid swabbing the corneal epithelium. Slit-lamp biomicroscopic examination was used to observe and characterize induced HSV-1 corneal epithelial lesions as deep punctate keratitis, dendritic keratitis or geographic epithelial defects. The ratio of positive days of epithelial lesions per total days was 82/187 (44%). There were 32 deep punctate lesions, 17 dendritic lesions, and 33 geographic epithelial defects. The ratio of positive swabs per total swabs was 78/187 (42%). Of the 82 positive lesion days, 54 (66%) were associated with a positive swab. Of the 78 positive swabs, 54 (69%) were associated with an epithelial lesion. Of the 54 days of both positive lesion and swab, 16 (30%) were associated with a dendritic lesion. By chi-square analysis, there was a significant association between HSV-1 swabs and HSV-1 lesions (P less than 0.001). These results confirm that intravenous injections of cyclophosphamide and dexamethasone induce both HSV-1 ocular shedding and recurrent herpes simplex corneal lesions in rabbits latently infected with HSV-1 strain McKrae.

Induction of Fc and C3b receptors on rabbit corneal cells by herpes simplex virus.

The expression of receptors for Fc portion (FcR) of immunoglobulin G (IgG) and for a C3b component of complement (C3bR) by herpes simplex virus (HSV) was studied in primary cultures of rabbit corneal cells. Monolayer cultures of epithelial, stromal and endothelial cells of the rabbit cornea were infected with three strains each of type 1 (HSV-1) and type 2 HSV (HSV-2). Rosette methods were used to detect receptors by means of sheep erythrocytes sensitized with rabbit IgG for FcR and C3b-coated sheep erythrocytes for C3bR. The FcR were expressed regularly on epithelial, stromal and endothelial cells by all three strains of both HSV-1 and HSV-2. The C3bR, however, were expressed only by HSV-1 on epithelial and stromal cells. Little or no C3bR activities could be detected on endothelial cells infected with any strain of HSV-1 or HSV-2. The FcR and C3bR expressed on corneal cells were induced by HSV and were blocked by monoclonal antibody to HSV-1 glycoprotein E(gE) or glycoprotein C(gC) respectively, confirming findings of other investigators that gE acts as FcR and gC as C3bR.