SECTION VII: MICROBIAL KERATITIS-VIRAL

Abstracts of Other Conference Presentations and Posters

HERPES SIMPLEX VIRUS LATENCY AND RECURRENCE

D.L. Easty, I C. Shimeld, I J. Whiteland, I S. M. Nicholls, I and T. J. Hilf

IDepartment of Ophthalmology, Bristol Eye Hospital, UK and 2Department of Pathology and Microbiology, University of Bristol, UK

Purpose: To study HSV-l reactivation and recurrent disease in mice. Methods: We have made quantitative observations in situ on the immunological events in the cornea during the development of recurrent corneal disease induced by UV irradiation of latently infected mice. Eyes with such disease were examined by immunohistochemistry using monoclonal antibodies to identity mouse immune cells and a polyclonal antibody to detect virus antigen. Results: On day 4, virus antigens were seen in the corneal epithelium of all mice, and in some animals antigens were also present in the iris and/or the conjunctival epithelium. The number of foci of infection ranged from 1-5. At this time, granulocytes were the predominant infiltrating cells; they were present throughout the corneas with large numbers associated with epithelial lesions. By day 7, in some corneas, ulcers had healed and associated stromal disease was limited to slight focal oedema and/or cellular infiltration(miid disease). In others, ulcers remained and stromal disease was severe with opacification and vessel ingress. On day 7, in corneas with mild disease there was a sig­nificant infiltrate of T cells and granulocytes were rare. In contrast, T cells were sparse in corneas with severe disease and large number of granulocytes were still present. These differences persisted until at least day 10. Conclusions: We have shown considerable variation in the amount of antigen in eyes with recurrent disease which may influence the magnitude of the immune response. The early presence of granulocytes, suggests that these cells playa role in initial clearance of virus. In this method, the severity of immu­nopathology is associated with the continued presence of large numbers of granulocytes rather than T cells. In addition we have shown that the surgical trauma of corneal grafting appears to be the most potent stimulus of recurrent disease after transplantation. Virus oc­casionally recurs in the recipient epithelium, but does not penetrate the basement mem­brane to the stroma. The graft-host junction appears to be a 'weak spot' where antigen readily reaches the stroma, perhaps, from nerve endings severed in the operation. Infiltrat-

485

486 Abstracts

ing cells then act as a conduit to the endothelium, which may become infected and preju­dice the graft.

CLINICAL PRESENTATION OF OCULAR HERPES SIMPLEX INFECTION

Andrew Tullo

The cardinal features of ocular herpes simplex were well described long before the discovery of the causative agent in the 1920's. Despite many years of shared clinical expe­rience it could be argued that we have made limited progress in managing clinical mani­festations despite a vastly improved knowledge of herpes simplex virus (HSV). We still rely heavily on clinical signs for diagnosis. We do not know if the epidemiology of herpes simplex keratitis (HSK) is changing significantly. We recognise several influential host factors including the fact that HSK is more common in men than women, but do not know why. We "understand the ability of HSV to establish latent infection in sensory neurones and possibly the cornea, but have as yet been unable to use this knowledge in prevention of disease. We acknowledge the importance of the immune system in pathogenesis but do not yet know how to best manipulate it. Topical steroids have been, and still are used in­appropriately, and antiviral agents have their limitations. We are now faced with new in­formation from molecular biological techniques which demonstrate viral DNA where we do and do not expect it. By acknowledging our limitations this may further stimulate ap­plication of laboratory knowledge in coping with HSK which continues to present a major challenge in management.

HSV: CLINICAL TRIALS

c. Dawson

The Herpetic Eye Disease Study (HEDS) addresses problems in the management of ocular herpes simplex virus infections. The HEDS trials are randomized, masked and pla­cebo controlled. They include three therapeutic and two preventive trials, and a study of factors triggering HSV ocular recurrences.

One completed HEDS therapeutic trial compared topical corticosteroid to placebo for patients with HSV stromal keratitis who also received prophylactic trifluridine drops. In this trial, the 10 week tapering course of topical corticosteroid drops was significantly better than placebo in reducing the progression or persistence of HSV stromal keratitis and shortening its duration. However, 22% of patients (11/49) on placebo also resolved during the 16 week course of the trial.

A second HEDS therapeutic trial compared 10 weeks of oral acyclovir (200 mg cap­sules) to placebo (2 capsules 5 times daily) for patients with herpetic stromal keratitis re­ceiving a 10 week tapering course of corticosteroid and trifluridine drops. In this trial there was no beneficial effect of acyclovir.

Section VII: Microbial Keratiti!l-Viral 487

Recruitment is still in progress for a trial of oral acyclovir or placebo (2 capsules twice daily) for one year to prevent HSV ocular recurrences. Patients in this trial may also enter a study on the risk factors for ocular HSV recurrences.

Other HEDS trials include a therapeutic study of oral acyclovir or placebo for steroidtrifluridine treated HSV iridocyclitis which is currently in press. Recruitment has also stopped in another HEDS trial comparing oral acyclovir to placebo capsules for triflu­ridine treated HSV epithelial keratitis to prevent subsequent stromal keratitis or iridocycli­tis. Follow-up visits are still under way in this trial.

PREVENTION OF RECURRENT HSV KERATITIS

Herbert E Kaufman, Bryan M. Gebhardt, Emily D. Varnell, and James M. Hill

Louisiana State University Eye Center, New Orleans, Louisiana

In genital and labial herpes simplex virus, multiplication of the virus at the muco-cu­taneous surface occurs and is necessary for viral pathology. Several studies have demon­strated that drugs such as acyclovir are effective in preventing recurrences of genital and labial HSV. The significant differences between infections at these sites and ocular HSV infection is that minimal to no viral replication is necessary for recrudescent disease on the ocular surface. Acyclovir is relatively ineffective in preventing ocular recurrences, probably because viral replication takes place in the distant nervous reservoir and infec­tious particles are transported to the ocular surface to set up the secondary infection. Re­cent studies in our laboratories have revealed that the beta adrenergic blocking agent, propranolol, can prevent viral reactivation in heat-stressed mice. We found that there was a statistically significant lower incidence of ocular shedding of virus in animals treated with propranolol as compared to those treated with placebo. In another series of experi­ments, we have also shown that recently identified inhibitors of viral thymidine kinase can also prevent viral reactivation in the trigeminal ganglion of heat-stressed mice. In a series of comparative experiments, we found that acyclovir is ineffective at preventing gangli­onic viral reactivation in this mouse model. We believe that these results support our hy­pothesis that ganglionic reactivation is not prevented by acyclovir because the number of viral particles reaching the ocular surface are adequate to cause recurrent disease. On the other hand, it appears that pharmacologic intervention, such as with the use of beta adren­ergic receptor blockers, may be an effective, new approach to preventing viral reactivation in the nervous system.

BRIDGING THE GAP BETWEEN THE BIOLOGY AND THE CLINICAL DISEASES OF VARICELLA ZOSTER VIRUS

Paul R. Kinchington

Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania

Herpes Zoster Ophthalmicus (HZO) and most of the clinical manifestations caused by Varicella Zoster Virus (VZV) are well-documented and for the most part are readily recognizable in the clinic and are straightforward to diagnose. The diseases caused by this

488 Abstracts

member of the herpesvirus family is largely seen in two phases of life; either as a conse­quence of a primary infection, which most often occurs during childhood, or as a result of virus which has reacted from a dormant or latent state within the host. Disease from this reactivated virus is usually seen in late adulthood and the retired population. The impor­tance of Zoster and its complications have recently been highlighted by the AIDS epi­demic and the increased use of immunosuppression, because zoster incidence increases dramatically in such individuals.

In contrast to a well characterized and comprehensive clinical picture of VZV dis­ease, considerably less is understood about the biology of the virus itself. VZV has re­mained quite refractory to growth outside the human host, despite the effort of many researchers and virologists. In particular, very little is known of the virus in its dormant state, and current knowledge is conflicting and controversial. Only recently, largely through the application of the techniques of molecular biology, have we begun to under­stand in depth the fundamental events of the viral growth cycle during the acute, or active state of infection. This knowledge is a vital prerequisite for the design of new drugs to combat the diseases caused by this virus; it is also essential to understand how the virus can be combated in both the quiescent and active states of growth in the human.

In this short talk, I will highlight recent advances in specific areas of VZV biology which may impact upon the physician and the treatment and management ofVZV disease.

EXTERNAL OCULAR EPSTEIN-BARR VIRUS (EBV) INFECTION

Stephen C. Pflugfelder

Bascom Palmer Eye Institute, University of Miami School of Medicine

Epstein-Ban virus (EBV) is a ubiquitous virus that infects the majority of humans by early adulthood. Similar to other herpesviruses, it has a lifecycle consisting of latent and replicative (lytic) phases. Two types of latent EBV infections have been identified: I) ac­tive latent infection with transcription of EBV oncoproteins that transform (or immortal­ize) B cells into lymphoblasts, 2) passive latent infection that is typically utilized by the virus after infecting small resting B cells during which transcription of only one gene (EDNA-I) has been identified. Lytic infection occurs most commonly in mucosal epi­thelial cells that shed infectious virus. EBV was initially observed in biopsies of Burkitt's lymphoma, a neoplasia that occurs predominantly in children in certain tropical regions of Africa. EBV was subsequently found to be the agent responsible for infectious mononu­cleosis (1M), a common febrile illness. EBV has also been detected in other human neo­plasias including nasopharyngeal carcinoma, thymic carcinoma, Hodgkin's disease, oral hairy leukoplakia, and B-cell lymphoproliferation in allograft recipients receiving immu­nosuppressive therapy.

There is increasing clinical and laboratory evidence suggesting that EBV is capable of infecting ocular surface epithelial cells. Expression of CD21, the putative EBV recep­tor, has been detected in conjunctival and corneal epithelial cells. EBV genomic se­quences have been detected in normal human conjunctiva and corneal epithelium. Numerous cases of conjunctivitis and keratitis have been reported in patients with 1M syn­drome. EBV can cause dendritic epithelial keratitis, although stromal keratitis is the more common form observed clinically. Among the twelve reported cases ofEBV stromal kera­titis, there appears to be thee distinct morphologic patterns of the corneal lesions. Subepi-

Section VII: Microbial Keratiti!t-Viral 489

thelial infiltrates (type I) most closely resemble those of adenovirus epidemic keratocon­junctivitis. Anterior-to-mid stromal opacities (type II) occur in two forms: small, granular, circular or ring-shaped opacities with minimal associated inflammation, or larger, blotchy, pleomorphic infiltrates with active inflammation. Full-thickness or deep stromal keratitis (type III) Is pleomorphic and blotchy, predominantly it involves the deep peripheral cor­nea, and may mimic luetic interstitial keratitis, or when unilateral, HSV stromal keratitis, direct and indirect evidence has been reported that EBV plays a pathogenic role in the lac­rimal gland pathology of Sjogrens Syndrome. EBV may persist in a latent non-pathogenic form in a small percentage of normal lacrimal glands. EBV genomes, and antigens (both active latent and lytic) have been detected in residual glandular epithelial cells and the mononuclear cells infiltrating Sjogrens Syndrome lacrimal glands. EBV genomes have been detected in 80% of tear samples obtained from Sjogrens Syndrome patients suggest­ing that this virus may be shed from the diseased lacrimal glands onto the ocular surface. The efficacy of anti-viral therapy for ocular EBV infection has not been established. It is likely that some of the ocular inflammatory conditions observed in patients with acute EBV infection, such as stromal keratitis and uveitis, are due to inflammation insighted by the virus rather than active viral replication. Corticosteroid therapy either alone, or in combination with acyclovir. has been reported to be effective in these conditions, and may be considered for severe ocular inflammation.

RECENT ADVANCES IN THE PATHOGENESIS AND TREATMENT OF ADENOVIRUS OCULAR INFECTIONS

Jerold Gorden

University of Pittsburgh, Pittsburgh, Pennsylvania

Adenovirus ocular infections are the most common external ocular infections that occur worldwide. They are usually characterized by epidemics in the community and medical facilities. Although not blinding, ocular morbidity is significant for the affected individual and the broader economic ramifications in ophthalmology, business, education, and military preparedness are considerable. Recent advances in the molecular virology of adenoviridae will be presented. An understanding of the pathogenesis of adenovirus ocu­lar infection has been advanced through the successful development on animal models that mimic human infection. The role of differences in adenovirus serotype and isolate viru­lence will be considered in terms of host specificity and clinical infections. Risk factors for the transmission of adenovirus within the ophthalmologist's office will be reviewed and appropriate recommendations made. Evaluation of current diagnostic tests including culture, enzyme immune assay, and PCR will be presented. Important advances in the de­velopment of specific antiviral agents for the treatment of adenovirus ocular infections (e.g., cidofovir) will be reviewed including the results of recent animal studies and the current status of clinical trials.

490 Abstracts

CORNEAL IMPRESSION TESTING FOR THE DIAGNOSIS OF RABIES

Andrew Billingsley and Gerald W. Zaidman

Department of Ophthalmology; Westchester County Medical Center New York Medical, College, Valhalla, New York

The antemortem diagnosis of rabies is very difficult. A confirmed diagnosis of ra­bies is usually obtained late in the disease or by post mortem brain biopsy. Once symp­toms appear the disease is usually fatal. Therefore early diagnosis is important. Early diagnosis allows isolation of infected patients. Also post exposure treatment (with human rabies immune globulin and human diploid cell vaccine) has been effective in preventing the disease if applied very soon after exposure.

A 13 year old girl was admitted with signs of acute encephalitis of unknown etiol­ogy. Viral and bacterial cultures and fluorescent antibody testing of the blood, spinal fluid and saliva as well as testing for Lyme's disease and toxoplasmosis were negative. A skin biopsy from the nape of the neck was negative. Corneal impressions were taken four days after admission. Standard glass microscope slides were gently applied to the cornea until an impression was obtained. The slides were air dried and fixed with acetone. Fluorescent antibody testing of the smear (and two additional smears taken 2 days later) were positive for rabies. Subsequently the diagnosis was confirmed by serum serology and skin biopsy.

This case demonstrates that corneal smears can help in the diagnosis of acute rabies en­cephalitis. They should be part of the routine antemortem workup for presumptive rabies.

MAINTENANCE OF DNA FROM A REPLICATION-DEFICIENT HERPES SIMPLEX VIRUS IN CORNEAL KERATOCYTES

w.J. O'Brien and J.L. Taylor

Departments of Ophthalmology and Microbiology, Medical College of Wisconsin, Milwaukee, Wisconsin

The discovery of herpes simplex virus (HSV) DNA in corneal cells during asympto­matic periods, long after acute disease has resolved, suggests that replication deficient virus may be suitable as a vector for gene delivery to corneal cells and other cells of the eye. The aim of these studies was to determine the stability and configuration of viral DNA delivered to cells by the replication-deficient mutant ofHSV-l KOS strain, 5d11.2. This virus possesses a deletion in the gene encoding infected cell protein 27 (ICP27), a gene of the immediate early class reported to be required for virus replication (McCarthy et aI, J. Virol. 63: 18, 1989). In order to evaluate the stability and structure of the viral DNA, cultures ofVero cells, cornea stromal cells, and retinal pigment epithelia cells were infected at various multiplicities with HSV 5d11.2. At various times after infection, cultures were observed and cells were har­vested. Viral DNA was analyzed by embedding infected cells in agarose rods which were di­gested with proteinase K and detergents. Slices of the agarose containing embedded DNA were subjected to pulsed-field electrophoresis to separate linear DNA from circular and other replicative intermediate DNA forms. DNA structure was analyzed by the use of rare-cutting

Section VII: Microbial Keratitir-Viral 491

restriction endonucleases. Viral DNA was detected by in situ hybridization using HSV-spe­cific probes and by amplification using the polymerase chain reaction (PCR). The synthesis of various classes of viral proteins was analyzed by immunoblots of infected celllysates. In con­trol cells, Vero 3.3, a cell line stably transformed to express HSV-I ICP27, the replication-de­ficient virus replicated to titers of}07 pfu/ml, expressed a full compliment of viral proteins, and replicated DNA, generating mature, linear 150 kb genome as well as replicative interme­diate forms. Based on digestion with the endonuclease, Spe I, the mature genome consisted of the usual four isoforms. In wild type Vero cells virus replication was reduced by ~ 106• Viral DNA persisted in the form of a replicative intermediate. In the absence of the production of detectable virus, viral DNA was detectable after 20 days and 5 passages by PCR. A dominate viral protein, ICP8, was detectable on immunoblots for 19 days. In corneal stromal cells the virus and viral DNA persisted in a manner similar to Vero cells. Viral DNA remained as a rep­licative intermediate after entering the cell. Surprisingly, a second eye-derived cell, the retinal pigment epithelium did support replication of the virus deletion mutant as evidenced by pro­duction of infectious progeny and production of viral proteins and unit length viral genome. These studies demonstrate several issues regarding gene delivery: (i) it cannot be assumed that the replication incompetence of a particular mutant virus is universal, (ii) viral DNA may persist in cells in the form of a replicative intermediate for extended periods, (iii) selected vi­ral genes may be expressed despite the inability of virus to replicate, and (iv) HSV that is rep­lication-deficient in corneal cells can deliver viral genes to corneal cells.

EFFECT OF GLYCOPROTEIN D2 VACCINATION ON ACUTE AND RECURRENT OCULAR HSV-l INFECTION

J.S. Pepose,! K.A. Laycock,! lK. Miller,! K.K. Hook,! E.D. Fenoglio,! M. Francotte,z M. Slaoui,z P.M. Stuart,! and T.L. Keadle!

!Department ofOphthalmQlogy and Visual Sciences, Washington University, S1. Louis, MO and 2SmithKline Beecham Biologicals, Rixensart Belgium

Purpose: To compare the prophylactic and therapeutic efficacy of herpes simpler virus type 2 (HSV-2) glycoprotein D (gD2) vaccines in a mouse model of recurrent ocular HSV-l infection. Methods: Vaccinations consisted of gD2 in alum, gD2 in aluml3DMPL, adjuvant or saline. In prophylactic studies, the corneas of vaccinated and control mice were inoculated with HSV-l McKrae. In therapeutic studies, mice with preexisting latent ocular HSV-I infec­tion and mock infected controls were infected with gD2 vaccines, adjuvant or saline. Corneal opacity, mortality, virus titers, latency, and reactivation rates were determined at time points before and after vaccination/infection and ultraviolet-B (UV-B)- induced recurrent ocular vi­rus shedding. Results: Prophylactically, gD2 alum and gD2 aluml3DMPL vaccines decreased acute disease severity and mortality Only gD2 aluml3DMPL vaccination resulted in signifi­cant reductions in latency, UV-B induced virus reactivation, and post reactivation disease. Therapeutically, gD2 vaccination decreased the number of days recurrent viral shedding was detected. Conclusions: Whereas gD2 vaccination was protective when administered prior to infection, its effects on reactivated latent ocular infections in mice was limited to reduced length of viral shedding, with no effect on subsequent ocular disease. These disparate findings underlie the need to evaluate herpes vaccines in models of both primary and recurrent ocular herpetic stromal keratitis, so that valid independent conclusions about their prophylactic and therapeutic value can be reached.

492 Abstracts

PENETRATING KERATOPLASTY FOR ACUTE PERFORATIONS IN HERPETIC KERATITIS

Ramon Naranjo-Tackman

Cornea Department, Asociacion Para Evitar la Ceguera en Mexico, Mexico, D.F.

Purpose: The evaluation of clinical outcome of cases that underwent Penetrating Keratoplasty (PK) for recently perforated herpetic keratitis.

Methods: The retrospective evaluation of cases with active Herpetic Keratitis (HK), that evolved to corneal perforation, was done. Visual outcome, graft evolution as well as postoperative complications, were evaluated.

Results: The files of 13 cases: 13 eyes of 13 patients, were reviewed. Males were predominant in 62% of cases, with a mean age of 23.6 years of age (Range: 9 to 52 years). Right eyes were more affected than the left eye in 61 % of cases. In 11 cases there was a history of recurrent herpetic keratitis, the average time of evolution of the herpetic disease since the first attack to the perforation, was 7.5 years, with a mean rate of recurrences of 3 episodes. Only 2 cases (15.38%) were reported as the initial episode, an both cases were associated with the use of topical steroids. In 11 cases (84.62%),perforation was central, for an average of 11 days before PK. Stains and cultures routinely practiced showed only saprophitic flora in all 13 cases.

In all cases a penetrating keratoplasty was performed, the size of grafts, varied from 6.5 to 9.0 mm in diameter. Only interrupted sutures were used. No associated surgical pro­cedure other than anterior iris synechia liberation were performed. Postop treatment con­sisted in topical steroids and topical antiviral ointment (Ophthalmic acyclovir ointment), for a mean 11 months period. Post-op follow-up, ranged from 14 months to 4 years. Preop vision ranged from Light perception to count fingers, after surgery it improved In 10 eyes (BCVA:20/40 or better in 3 eyes)

Main complications were: Glaucoma 30.7%, Uncontrolled graft rejection: 23%, Cataract: 23%, Vascularization 38%. Recurrent herpetic ulcers in the graft: 2 eyes (15.38%).

Conclusions: Most of cases evolved after multiple recurrences. The main predispos­ing factor was topical steroids, specially in new cases. Main complications of the Grafts were the same as reported in other series, however results are encouraging, specially after long follow-up, with sustained combined steroid, antiviral therapy.

DE NOVO HERPES SIMPLEX VIRUS KERATITIS AFTER PENETRATING KERATOPLASTY

L. Remeijer, P. Doornenbal, AJ.M. Geerards, WJ. Rijneveld, and W.H. Beekhuis

Cornea and External Eye Disease Service; Eye Hospital Rotterdam; Rotterdam, The Netherlands

Background: In corneal transplantation for diagnoses not related to Herpes Simplex Virus(HSV) keratitis there is not always suspicion of an HSV infection in the case of non-

Section VII: Microbial Keratitis-Viral 493

specific epithelial defects. The purpose of this study was to determine the incidence of "De Novo Herpetic Keratitis" after penetrating keratoplasty and to analyse possible fac­tors contributing to this clinical entity.

Methods: We retrospectively studied the medical records of our group of patients transplanted in the period from January 1980 to January 1995.Factors related to the opera­tion and possible reactivating stimuli were recorded.

Results: From 1980 to 1995 a group of 18 patients presented with epithelial herpetic keratitis in their corneal graft whereas the primary diagnosis was not related to HSV kera­titis. The incidence of "De Novo Herpetic Keratitis" is 1,2 per 1000 person years. In most cases the infection occurs in the first two years after the operation and in the majority of cases known reactivating stimuli could have caused the HSV infection.

Conclusions: Herpes simplex virus keratitis can occur in penetrating keratoplasty even in the absence of a clinical history of HSV in the host. The high incidence of this clinical picture and the frequent occurrence of the infection in the first two years after the operation strongly indicates a relation between the corneal transplantation and the presen­tation of the infection.

DETECTION OF HERPES SIMPLEX VIRUS GENOMES IN TEARS FROM PATIENTS AFTER PENETRATING KERATOPLASTY USING POLYMERASE CHAIN REACTION

M. Tei, K. Nishida, T. Sasagawa, W. Adachi, and S. Kinoshita

Department of Ophthalmology; Kyoto Prefectural University of Medicine, Kyoto, Japan

Purpose: Diagnosis of recurrent herpetic epithelial keratitis after penetrating kerato­plasty (PKP) is considered to be difficult, since many factors such as steroid administra­tion, insufficient innervation in the graft modify ocular surface appearance. In diagnosing the herpetic epithelial keratitis mentioned above, polymerase chain reaction (peR) method may be effective and swift, in comparison with viral culture, immunofluorescence, and se­rum antiboby. In the present study, we investigated the efficacy of peR method in diag­nosing HSV -suspected epithelial keratitis in patients after PKP for herpetic stromal keratitis.

Methods: Three eyes of 3 patients showed epithelial keratitis characteristic of large geographic-shaped ulcer located at host-graft border, weak ciliary injection and a little epithelial infiltration at the edge of the ulcer. Tears were collected by Schirmer's strips from these 3 patients and 3 eyes of 3 patients without recurrent HSV keratitis after PKP served as control. DNA was extracted and submitted to peR. Subsequently, Southern blot hybridization was performed to determine herpes simplex genomes.

Results: Using peR, we identified HSV DNA in 3 eyes (100%) of HSV-suspected epithelial keratitis, but not in control eyes.

Conclusions: The results indicate that peR is an effective and swift method to diag­nose possible herpetic epithelial keratitis after PKP, which is difficult to judge from clini­cal observation.

494 Abstracts

IMMUNOPATHOGENESIS OF HERPES SIMPLEX STROMAL KERATITIS

Corneal blindness secondary to scarring from recurrent herpes simplex keratitis (HSK) still occurs in epidemic proportions, despite 30 years of effective topical antiviral medication. Why some individuals are capable of an adequate immune response which clears the infection without producing excessive, tissue-damaging inflammation, while others are unable to do this is unclear. Our work in a defined murine model of necrotizing HSK has shown that discreet genetic differences produce profound immunologic and clinical differences following corneal encounter with HSV. Underregulation of an overly exuberant inflammatory response is only one of those differences responsible for the blinding keratopathy experienced by some individuals. We now know that in mice with a specific genotype, frank autoimmunity to corneal antigens develops, contributing to the necrotizing stromal keratitis seen in these mice. These data indicate even more profound reasons for major efforts at developing selective immunomodulatory strategies for treating The inflammatory response associated with HSK, with strategies that dissociate The anti­inflammatory effect from the wound healing impairment and infection enhancing effects of steroid therapy.

DIAGNOSTIC IMPRESSION CYTOLOGY FOR VIRAL DISEASES OF THE OUTER EYE

H. Araki, H. Nakagawa, N. Kimata, Y. Nakagawa, and M. Murata

Tokyo Women's Medical College, Tokyo, Japan

Purpose. To evaluate the efficacy of impression cytology (IC) for the diagnosis of viral diseases of the outer eye. Materials and Methods. Three common viral diseases of the oilier eye were studied. (1) Herpes simplex keratoconjunctivitis (n=20): Impression specimens were obtained from eyelid vesicles, bulbar an palpebral conjunctiva, and stel­late, dendritic and geographic epithelial lesions of the cornea or conjunctiva. The speci­mens were stained for herpes simplex virus (HSV) antigens by peroxidase-antiperoxidase method using polyclonal anti-HSV antibodies. (2) Herpes zoster ophthalmicus (n=6): Specimens obtained from dendritic lesions were tested for varicella zoster virus (VZV) an­tigens by Immunofluorsecence with monoclonal anti-VZV antibodies. (3) Adenovirus (Adv) conjunctivitis: Eighty cases of acute follicular conjunctivitis, clinically suspected of Adv conjunctivitis were involved. Impression specimens from bulbar conjunctiva were stained for Adv antigens by modified avidin-biotin method using monoclonal anti-Ad an­tibodies. The patients were also examined for Adv antigens by using Adenoclone®. Re­sults. (1) Twenty cases were all HSV isolation-positive. HSV antigens were identified in 100% of eyelid vesicle samples (9/9) and 91 % of epitheliallesions( 1 0/11) from the cornea and conjunctiva. In contrast, HSV antigens were rarely detected in bulbar (1118) and palpebral (1/15) conjunctiva (2) antigens were detected from dendritic lesions in 3 cases (50%), including one case of zoster sine herpete. (3) Of 42 cases of Adv conjunctivitis proven by positive virus isolation, Ie was positive in 36 cases, sensitivity 85.7% (36/42). In 38 adenovirus-negative cases, 35 cases showed negative in IC, specificity 92.1 %

Section VII: Microbial Keratitis-Viral 495

(35/38). Adenoclone® was 61.9% (26/42) sensitive and 100% (38/38) specific. Conclu­sion. We conclude that IC is an useful technique for the diagnosis of viral diseases of the outer eye because of its simple, rapid, noninvasive procedure and its ability to detect viral antigens even from minute lesions.

DETECTION AND TYPING OF HUMAN PAPILLOMAVIRUS (HPV) IN CORNEAL AND CONJUNCTIVAL TUMORS

S. Kumakura,I,2 T. Iwasaki/ T. Sata,2 R. Muramatsu,l T. Kurata,2 and M. Usui1

Department of Ophthalmology, Tokyo Medical College1; Department of Pathology, NIH2; Tokyo, Japan

Purpose: Human papillomavirus (HPV) has been known to induce corneal and con­junctival tumors. Viral types reported in these tumors are HPV 6, 11 and, 16 which have been considered to be mucosal HPVs. In order to examine and characterize HPV present in corneal/ conjunctival tumors, we analyzed these tumors by polymerase chain reaction (PCR), and compared the sequences of amplified products to those of HPV reported in the literature. Material and methods: PCR using the Li consensus primers (MYI 1 and MY09, Manos MM and Y Ting, 1989) was performed on DNA extracted from formalin­fixed paraffin sections and frozen tissues of 10 cases of squamous cell papilloma, 4 cases of conjunctival dysplasia, and I case of carcinoma in situ. Sequencing of amplified frag­ments was analyzed on 3 recombinants clones from each specimen after T A ligation. Re­sults: Amplified bands, about 450 bases in length, were found in 3 of 10 cases of squamous cell papilloma and in the 1 case of carcinoma in situ. No amplification was ob­served in any of the 4 cases of conjunctival dysplasia. Sequence analysis of DNA frag­ments in squamous cell papillomas showed the sequence of the HPV 11 genome, while the amplified fragment from the case of carcinoma in situ was of HPVI6. Three conserved and 1 non-conserved point mutations and 1 insertional and 1 deletion triplets were found in amplified fragment of HPV 16 in carcinoma in situ, in comparison with the sequence of HPV 16 prototype cloned from invasive cervical carcinoma (Shwartz et al. 1982). In am­plified fragments of HPV 11 in three squamous cell papilloma, only one conserved or non-conserved point mutations were found in comparison to that of HPV 11 prototype cloned from laryngeal papilloma, besides neither deletion or insertional changes were ob­served. B These results suggest that cornea and conjunctiva have a similar susceptibility to HPV that infects genital and laryngeal mucosa. In addition, in cases of squamous cell papilloma and carcinoma in situ, it appears that HPV causes similar histopathology to that seen in genital and laryngeal infections.