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In Vivo Confocal Microscopy Analysis of the Corneal Layers in Adenoviral Epidemic Keratoconjunctivitis.

Introduction

The most common cause of viral conjunctivitis is adenoviruses. Adenoviral conjunctivitis can manifest clinically as acute follicular conjunctivitis, pharyngoconjunctival fever, epidemic keratoconjunctivitis (EKC), or chronic conjunctivitis. EKC caused by adenovirus serotypes 8, 19, and 37 occurs in epidemics, particularly in the summer months, presents with keratitis in 80% of cases, and shows the most severe clinical course. (1)

EKC is one of the viral diseases that cause severe ocular surface inflammation. After a prodromal period of 7-10 days, unilateral or bilateral follicular conjunctivitis develops; within 2-4 days after onset of conjunctivitis, diffuse epithelial keratitis appears, followed by focal epithelial keratitis. A subepithelial infiltration period begins in the third week, and this clinical presentation may last for weeks or even months. (2,3,4)

In vivo confocal microscopy (iVCM) is a non-contact imaging method that enables evaluation of the cornea at the cellular level. (5) in addition to having a well established place in the diagnosis and follow-up of many corneal diseases, studies including iVCM findings have also shown corneal changes in the various stages of EKC. These studies described changes starting at the basal epithelium level and extending into the midstroma, while images targeting the subepithelial infiltration period showed focal inflammatory foci. (6,7) in this study we sought to use iVCM to elucidate corneal alterations that begin in the prodromal period of EKC, evaluate findings seen in the clinical course of the disease, and discuss our results within the context of the literature.

Materials and Methods

The study included 20 eyes of 12 patients (6 males, 6 females) who presented with complaints of burning, watering, and discharge from the eyes and were clinically diagnosed with EKC in the ophthalmology outpatient clinic of the Kocaeli University School of Medicine. Ethical approval was obtained from the university ethics committee, and informed consent was obtained from all participants prior to examination.

Following clinical assessment with biomicroscopy, patients underwent iVCM (Rostock Cornea Module/Heidelberg Retina Tomography 3, Heidelberg Engineering GmBH, Germany) examination under topical anesthesia (0.5% proparacaine Hydrochloride; Alcaine[R]; Alcon Laboratories, Fort Worth, TX, USA). A new sterile polymethylmethacrylate cap (Tomocap[R]; Heidelberg Engineering GmBH, Germany) was placed over the objective lens for each patient. Gel (Viscotears[R]; Carbomer 980, 0.2%; Novartis, North Ryde, Australia) was applied to the cap at the start of imaging. The distance between the cornea and objective was monitored on the camera display as imaging was initiated. After visualizing the surface epithelium on the screen, the objective lens was manually focused to acquire images of the corneal layers sequentially until reaching the endothelium. (8)

At initial examination, patients underwent iVCM both in the eye diagnosed with EKC and the eye with no clinical signs. iVCM imaging was done in the patients' healthy, non-EKC eyes at each follow-up visit in order to capture images in the prodromal period. For the patients whose healthy eyes developed clinical EKC during follow-up, eyes imaged by iVCM within the 7-10 days prior to the appearance of EKC signs were evaluated as prodromal (4 eyes), while eyes that did not develop clinical EKC and remained healthy throughout follow-up were evaluated as the control group (4 eyes). Of the imaged eyes with clinical disease, the routine ophthalmologic examination findings, anterior segment photographs, and iVCM findings of 4 eyes with punctate epithelial keratitis, 4 eyes with deep corneal keratitis, and 4 eyes with subepithelial infiltration were evaluated. Slit-lamp microscopy findings and disease stages were recorded. iVCM findings were scored as 0 (same as control), + (slight increase compared to control), ++ (moderate increase compared to control), and +++ (extreme increase compared to control). (6) All assessments were done at different stages in different patients; disease stages in which patients were examined are shown in Table 1. Patients with history of any ocular disease or with any chronic systemic disease were not included in the study. All eyes with active clinical EKC were treated with topical 0.3% tobramycin (Tobrased, Bilim Ilac, Istanbul, Turkey) 6 times a day and preservative-free artificial tears (Tears Naturale Free, Alcon) 8 times a day. None of the patients in the study were treated with steroids. All treatment except preservative-free tears was discontinued when clinical symptoms had resolved, after about 14 days of treatment.

Results

Clinical features, disease stages, slit-lamp examination findings, and iVCM findings of the patients are given in Table 1. in eyes examined in the prodromal period before the onset of clinical EKC, the epithelial, Bowman's, and stromal layers appeared normal in iVCM, while the subbasal plexus showed an increased number of Langerhans cells (Figure 1).

Clinical EKC eyes evaluated during the punctate epithelial keratitis stage showed cell clusters surrounded by inflammatory cell infiltration in the basal epithelium. An increased number of branching dendritic cells were observed in Bowman's layer. Hyperreflective cells were noted in the anterior stroma (Figure 2).

Eyes in the deep epithelial keratitis stage showed basal epithelial cells with peripheral hyperreflectivity in keratitis foci, inflammatory cells in the form of punctate hyperreflectivity, and the hyperreflective areas in the anterior stroma had acquired round focal borders. The increase in Langerhans cells in the subbasal plexus continued (Figure 3).

In the subepithelial infiltrate period, the basal epithelium still exhibited hyperreflective foci and inflammatory cells, but the areas of anterior stromal hyperreflectivity formed more distinct round hyperreflective plaques. The eyes exhibited no changes in the deep stromal layers or endothelium during the course of EKC (Figure 4).

Discussion

The cornea is the most densely innervated tissue in the body, and this innervation provides corneal sensitivity. Many diseases disrupt corneal sensitivity, including ocular infections, herpetic eye disease, dry eye syndrome, and diabetes. (9,10,11,12,13,14) Animal studies have shown a correlation between corneal inflammation and innervation. (15,16) Hamrah et al. (17) and Liu et al. (18) demonstrated that immature dendritic cells in the cornea had matured after inflammation and transplantations. in noninflammatory, quiet conditions, dendritic cells are found in the central corneal epithelium and anterior stroma, whereas during inflammation they infiltrate the entire cornea, thus preparing it to respond to pathogens. (19) With iVCM enabling in vivo visualization of these cells, it has become possible to document their increase in immune active situations.

Corneal involvement occurs during the course of EKC, and various corneal findings can be observed in the different disease stages. Corneal involvement leads to symptoms such as dry eye, glare, blurry or low vision, and irregular astigmatism. (20) No corneal and conjunctival findings occur in the prodromal period, but clinical signs of conjunctivitis appear within 7-10 days after this period. Despite apparently normal biomicroscopic and clinical findings during the prodromal period, iVCM revealed a marked increase in Langerhans cells in the subbasal plexus in our study, indicating that inflammation has already started. These findings suggest an active prodromal process in the healthy eye that precedes clinical disease.

The active follicular conjunctivitis phase is characterized by the formation of corneal punctate epithelial keratitis, followed by a long-term inflammatory process with subepithelial infiltration, believed to be a result of type 4 hypersensitivity reaction. in EKC, inflammatory cell infiltration in the basal epithelium and anterior stromal surfaces has been demonstrated by the higher concentration of dendritic cells observed in iVCM. (6,7,21)

The increase in dendritic cells in the subbasal plexus is considered an important iVCM finding in EKC and herpes simplex keratitis. ozturk et al. (22) reported that herpetic keratoconjunctivitis can often be confused with adenoviral EKC due to similarities in their clinical course and common iVCM findings. in addition, a temporary reduction in corneal sensitivity has been observed following inflammatory cell activation in 74% of patients with EKC. (22)

In the subepithelial infiltration phase, an increase in inflammatory cells is observed in addition to inflammatory foci in the stroma. Dosso and Rungger-Brandle (6) reported that Langerhans cells were reduced in more advanced disease stages. However, our study encompassed the earlier subepithelial infiltrate stage and showed an increase in Langerhans cells, consistent with the literature.

Conclusion

Our study demonstrates based on iVCM findings that corneal involvement in EKC begins not in the clinical disease stage but in the prodromal phase, with an increase of Langerhans cells. in clinical disease stages, findings such as increased dendritic cells accompanying the development of epithelial keratitis, and hyperreflective plaques in the basal epithelial layer and anterior stromal surface are seen on iVCM. in the subepithelial infiltration phase, lesions become more focal and persist without extension to the posterior stromal surface. Based on our findings, we suggest that corneal findings in iVCM signal the development of clinical EKC starting in the prodromal period.

Ethics

Ethics Committee Approval: Obtained (Project number: KU GOKAEK 2016/237).

Informed Consent: Obtained.

Peer-review: internally peer-reviewed.

Author contributions

Concept: Nursen Yuksel, Design: Sevgi Subasi, Nursen Yuksel, Data Collection and Processing: Sevgi Subasi, Muge Toprak, Analysis and interpretation: Nursen Yuksel, Sevgi Subasi, Muge Toprak, Busra Yilmaz Tugan, Literature Search: Sevgi Subasi, Writing: Sevgi Subasi.

Conflict of interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.

References

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(2.) Dawson CR, Hanna L, Togni B. Adenovirus type 8 infections in the United States. i V. Observations on the pathogenesis of lesions in severe eye disease. Arch Ophthalmol. 1972;87:258-268.

(3.) Murah WF. Epidemic keratoconjunctivitis. Ann Ophthalmol. 1988;20:36-38.

(4.) Chodosh J, Astley RA, Butler MG, Kennedy RC. Adenovirus keratitis: a role for interleukin-8. Invest Ophthalmol. 2000;41:783-789.

(5.) Niederer RL, McGhee CN. Clinical in vivo confocal microscopy of the human cornea in health and disease. Prog Retin Eye Res. 2010;29:30-58.

(6.) Dosso AA, Rungger-Brandle E. Clinical course of epidemic keratoconjunctivitis. Evaluation by in vivo confocal microscopy. Cornea. 2008;27:263-268.

(7.) Kocabeyoglu S, Mocan MC, Irkec M. In Vivo Confocal Microscopic Findings of Subepithelial Infiltrates Associated with Epidemic Keratoconjunctivitis. Turk J Ophthalmol. 2015;45:119-121.

(8.) Bitirgen G, Ozkagnici A. Saglikli Insan Korneasinda Hucre ve Sinir Liflerinin In Vivo Konfokal Mikroskopi ile Degerlendirilmesi Turkiye Klinikleri J Med Sci. 2014;34:256-261.

(9.) Kurbanyan K, Hoesl LM, Schrems WA, Hamrah P. Corneal nerve alterations in acute Acanthamoeba and fungal keratitis: an in vivo confocal microscopy study. Eye (Lond). 2012;26:126-132.

(10.) Rosenberg ME, Tervo TM, Muller LJ, Moilanen JA, Vesaluoma MH. In vivo confocal microscopy after herpes keratitis. Cornea. 2002;21:265-269.

(11.) Hamrah P, Cruzat A, Dastjerdi MH, Zheng L, Shahatit BM, Bayhan HA, Dana R, Pavan-Langston D. Corneal sensation and subbasal nerve alterations in patients with herpes simplex keratitis: an in vivo confocal microscopy study. Ophthalmology. 2010;117:1930-1936.

(12.) Tuominen IS, Konttinen YT, Vesaluoma MH, Moilanen JA, Helinto M, Tervo TM. Corneal innervation and morphology in primary Sjogren's syndrome. Invest Ophthalmol Vis Sci. 2003;44:2545-2549.

(13.) Villani E, Galimberti D, Viola F, Mapelli C, Ratiglia R. The cornea in Sjogren's syndrome: an in vivo confocal study. Invest Ophthalmol Vis Sci. 2007;48:2017-2022.

(14.) De Cilla S, Ranno S, Carini E, Fogagnolo P, Ceresara G, Orzalesi N, Rossetti LM. Corneal subbasal nerves changes in patients with diabetic retinopathy: an in vivo confocal study. Invest Ophthalmol Vis Sci. 2009;50:5155-5158.

(15.) Ferrari G, Chauhan SK, Ueno H, Nallasamy N, Gandolfi S, Borges L, Dana R. A novel mouse model for neurotrophic keratopathy: trigeminal nerve stereotactic electrolysis through the brain. Invest Ophthalmol Vis Sci. 2011;52:2532-2539.

(16.) Ueno H, Ferrari G, Hattori T, Saban DR, Katikireddy KR, Chauhan SK, Dana R. Dependence of corneal stem/progenitor cells on ocular surface innervation. Invest Ophthalmol Vis Sci. 2012;53:867-872.

(17.) Hamrah P, Liu Y, Zhang Q, Dana MR. The corneal stroma is endowed with a significant number of resident dendritic cells. Invest Ophthalmol Vis Sci. 2003;44:581-589.

(18.) Liu Y, Hamrah P, Zhang Q, Taylor A, Dana MR. Draining lymph nodes of corneal transplant hosts exhibit evidence for donor major histocompatibility complex (MHC) class II-positive dendritic cells derived from MHC class II-negative grafts. J Exp Med. 2002;195:259-268.

(19.) Hamrah P, Zhang Q, Liu Y, Dana MR. Novel characterization of MHC class II-negative population of resident corneal Langerhans cell-type dendritic cells. Invest Ophthalmol Vis Sci. 2002;43:639-646.

(20.) Sundmacher R, Hillenkamp J, Reinhard T. Prospects for therapy and prevention of adenovirus keratoconjunctivitis. Ophthalmologe. 2001;98:991-1007.

(21.) Alsuhaibani AH, Sutphin J, Wagoner MD. Confocal microscopy of subepithelial infiltrates occuring after epidemic keratoconjunctivitis. Cornea. 2006;25:1102-1104.

(22.) Ozturk HE, Sonmez B, Beden U. Corneal sensitivity may decrease in adenoviral epidemic keratoconjunctivitis-a confocal microscopic study. Eye Contact Lens. 2013;39:264-268.

Sevgi Subasi (*), [iD] Nursen Yuksel (**), [iD] Muge Toprak (***), [iD] Busra Yilmaz Tugan (****)

(*) Korfez State Hospital, Ophthalmology Clinic, Kocaeli, Turkey

(**) Kocaeli University Faculty of Medicine, Department of Ophthalmology, Kocaeli, Turkey

(***) Gebze Fatih State Hospital, Ophthalmology Clinic, Kocaeli, Turkey

(****) Agri Patnos State Hospital, Ophthalmology Clinic, Agri, Turkey

Address for Correspondence: Sevgi Subasi MD, Korfez State Hospital, Ophthalmology Clinic, Kocaeli, Turkey

Phone: +90 544 915 58 93 E-mail: sevgiozel_5@hotmail.com ORCiD-iD: orcid.org/0000-0002-1099-9626

Received: 15.08.2017 Accepted: 05.05.2018

DOI: 10.4274/tjo.59251
Table 1. The patients' clinical, slit-lamp microscopy, and in vivo
confocal microscopy evaluations

                                              Deep
Patient/  Age/  Examination        Punctate               Subepithelial
Eye       Sex   time         BCVA  keratitis  epithelial  infiltrate
                                              keratitis

 1/OD     28/M  Day 7        0.9   +
 1/OS           P            1.0
 2/OD     40/F  Day 5        0.9   +
 3/OS     37/M  Day 6        0.8   +
 4/OS     44/F  Day 4        1.0   +
 5/OD     29/M  Day 10       1.0              +
 5/OS           P            1.0
 6/OD     65/E  P            1.0
 6/OS           Day 12       0.8              +
 7/OD     53/F  Day 14       0.9              +
 7/OS           P            1.0
 8/OD     42/M  Day 10       1.0   +          +
 9/OD     44/M  Day 20       0.9                          +
 9/OS           C            1.0
10/OD     38/F  Day 22       0.9                          +
10/OS           C            1.0
11/OD     28/M  C            1.0
11/OS           Day 21       0.8                          +
12/OD     50/F  C            1.0
12/OS           Day 20       0.9                          +

            Deep
Patient/    Dendritic  Basal epithelial   Anterior stromal
Eye         cells      hyperreflectivity  hyperreflectivity


 1/OD       ++         ++                 +
 1/OS       +          0                  0
 2/OD       +          ++                 ++
 3/OS       ++         +++                +
 4/OS       ++         +++                +
 5/OD       ++         ++                 ++
 5/OS       +          0                  0
 6/OD       +          0                  0
 6/OS       +++        ++                 ++
 7/OD       ++         ++                 ++
 7/OS       +          0                  0
 8/OD       ++         ++                 +++
 9/OD       ++         +                  ++
 9/OS       0          0                  0
10/OD       +++        +                  ++
10/OS       0          0                  0
11/OD       0          0                  0
11/OS       +++        ++                 +++
12/OD       0          0                  0
12/OS       ++         ++                 +++

BCVA: Best corrected visual acuity, OD: Right eye, OS: Left eye, P:
Prodromal, C: Control, Scoring is described in the Materials and
Methods section. The fellow eyes of patients 2, 3, 4, and 8 were not
included in the study
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Title Annotation:Original Article
Author:Subasi, Sevgi; Yuksel, Nursen; Toprak, Muge; Tugan, Busra Yilmaz
Publication:Turkish Journal of Ophthalmology
Date:Nov 1, 2018
Words:2475
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