Prevalence of SARS-CoV-2 in human post-mortem ocular tissues

BackgroundSARS-CoV-2 is found in conjunctival swabs and tears of COVID-19 patients. However, the presence of SARS-CoV-2 has not been detected in the human eye to date. We undertook this study to analyze the prevalence of SARS-CoV-2 in human post-mortem ocular tissues.MethodsThe expression of SARS-CoV-2 RNA was assessed by RT-PCR in corneal and scleral tissues from 33 surgical-intended donors who were eliminated from a surgical use per Eye Bank Association of America (EBAA) donor screening guidelines or medical director review or positive COVID-19 test. Ocular levels of SARS-CoV-2 RNA (RT-PCR), Envelope and Spike proteins (immunohistochemistry) and anti-SARS-CoV-2 IgG and IgM antibodies (ELISA) in blood were evaluated in additional 10 research-intent COVID-19 positive donors.FindingsOf 132 ocular tissues from 33 surgical-intended donors, the positivity rate for SARS-CoV-2 RNA was ~13% (17/132). Of 10 COVID-19 donors, six had PCR positive post-mortem nasopharyngeal swabs whereas eight exhibited positive post-mortem anti-SARS-CoV-2 IgG levels. Among 20 eyes recovered from 10 COVID-19 donors: three conjunctival, one anterior corneal, five posterior corneal, and three vitreous swabs tested positive for SARS-CoV-2 RNA. SARS-CoV-2 spike and envelope proteins were detected in epithelial layer of the corneas that were procured without Povidone-Iodine (PVP–I) disinfection.InterpretationsOur study showed a small but noteworthy prevalence of SARS-CoV-2 in ocular tissues from COVID-19 donors. These findings underscore the criticality of donor screening guidelines, post-mortem nasopharyngeal PCR testing and PVP-I disinfection protocol to eliminate any tissue harboring SARS-CoV-2 being used for corneal transplantation.     

The evidence of SARS-CoV-2 infection on ocular surface

This is a cross-sectional study of patients who received a COVID-19 diagnosis between December 30, 2019 and February 7, 2020 at Tongji Hospital. A total of 102 patients (48 Male [47%] and 54 Female [53%]) with clinical symptoms, Rt, and chest Computed Tomography (CT) abnormalities were identified with a clinical diagnosis of COVID-19. Patients had a mean [SD] gestational age of 57.63 [14.90] years. Of a total of 102 patients identified, 72 patients (36 men [50%] and 36 women [50%]; mean [SD] age, 58.68 [14.81] years) were confirmed to have COVID-19 by laboratory diagnosis with a SARS-CoV-2 RT-PCR assay. Only two patients (2.78%) with conjunctivitis were identified from 72 patients with a laboratory confirmed COVID-19. Of those two patients, SARS-CoV-2 RNA fragments were found in ocular discharges by SARS-CoV-2 RT-PCR in only one patient. Our findings suspect the incidence of SARS-CoV-2 infection through the ocular surface is extremely low, while the nosocomial infection of SARS-CoV-2 through the eyes after occupational exposure is a potential route. To lower the SARS-CoV-2 nosocomial infection, all health care professionals should wear protective goggles. The inefficient diagnostic method and the sampling time lag may contribute to the lower positive rate of conjunctival swab samples of SARS-CoV-2.     

SARS-CoV-2 and its beta variant of concern infect human conjunctival epithelial cells and induce differential antiviral innate immune response

PurposeSARS-CoV-2 RNA has been detected in ocular tissues, but their susceptibility to SARS-CoV-2 infection is unclear. Here, we tested whether SARS-CoV-2 can infect human conjunctival epithelial cells (hCECs) and induce innate immune response.MethodsConjunctival tissue from COVID-19 donors was used to detect SARS-CoV-2 spike and envelope proteins. Primary hCECs isolated from cadaver eyes were infected with the parental SARS-CoV-2 and its beta variant of concern (VOC). Viral genome copy number, and expression of viral entry receptors, TLRs, interferons, and innate immune response genes were determined by qPCR. Viral entry receptors were examined in hCECs and tissue sections by immunostaining. Spike protein was detected in the cell culture supernatant by dot blot.ResultsSpike and envelope proteins were found in conjunctiva from COVID-19 patients. SARS-CoV-2 infected hCECs showed high viral copy numbers at 24–72h post-infection; spike protein levels were the highest at 24hpi. Viral entry receptors ACE2, TMPRSS2, CD147, Axl, and NRP1 were detected in conjunctival tissue and hCECs. SARS-CoV-2 infection-induced receptor gene expression peaked at early time points post-infection, but gene expression of most TLRs peaked at 48 or 72hpi. SARS-CoV-2 infected hCECs showed higher expression of genes regulating antiviral response, RIG-I, interferons (α, β, & λ), ISG15 & OAS2, cytokines (IL6, IL1β, TNFα), and chemokines (CXCL10, CCL5). Compared to the parental strain, beta VOC induced increased viral copy number and innate response in hCECs.ConclusionsConjunctival epithelial cells are susceptible to SARS-CoV-2 infection. Beta VOC is more infectious than the parental strain and evokes a higher antiviral and inflammatory response.     

Expression of the SARS-CoV-2 Receptor ACE2 in Human Retina and Diabetes—Implications for Retinopathy

PurposeTo investigate the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 in human retina.MethodsHuman post-mortem eyes from 13 non-diabetic control cases and 11 diabetic retinopathy cases were analyzed for the expression of ACE2. To compare the vascular ACE2 expression between different organs that involve in diabetes, the expression of ACE2 was investigated in renal specimens from nondiabetic and diabetic nephropathy patients. Expression of TMPRSS2, a cell-surface protease that facilitates SARS-CoV-2 entry, was also investigated in human nondiabetic retinas. Primary human retinal endothelial cells (HRECs) and primary human retinal pericytes (HRPCs) were further used to confirm the vascular ACE2 expression in human retina.ResultsWe found that ACE2 was expressed in multiple nonvascular neuroretinal cells, including the retinal ganglion cell layer, inner plexiform layer, inner nuclear layer, and photoreceptor outer segments in both nondiabetic and diabetic retinopathy specimens. Strikingly, we observed significantly more ACE2 positive vessels in the diabetic retinopathy specimens. By contrast, in another end-stage organ affected by diabetes, the kidney, ACE2 in nondiabetic and diabetic nephropathy showed apical expression of ACE2 tubular epithelial cells, but no endothelial expression in glomerular or peritubular capillaries. Western blot analysis of protein lysates from HRECs and HRPCs confirmed expression of ACE2. TMPRSS2 expression was present in multiple retinal neuronal cells, vascular and perivascular cells, and Müller glia.ConclusionsTogether, these results indicate that retina expresses ACE2 and TMPRSS2. Moreover, there are increased vascular ACE2 expression in diabetic retinopathy retinas.     

Aqueous humor induces lymphatic regression on the ocular surface

PurposeThis study is to investigate the potential effect of aqueous humor on already formed lymphatic vessels of the ocular surface including the conjunctiva and the cornea.MethodsAqueous humor harvested from fresh bovine or murine eyeballs were used in the study. It was injected into the subconjunctival space of Prox-1-GFP (green fluorescent protein) transgenic mice. Pre-existing conjunctival lymphatics were observed in vivo using our advanced live imaging system. Additionally, ex vivo tissue cultures were performed in aqueous humor with normal conjunctival tissues or inflamed corneas with newly formed lymphatic vessels. Time lapse images were taken by an advanced live cell imaging system with an incubator. Moreover, human primary microdermal lymphatic endothelial cell culture system was employed to evaluate the effect of aqueous humor on lymphatic tube regression in vitro.ResultsAqueous humor induced lymphatic regression in both normal conjunctiva and inflamed corneas. It also led to the regression of formed lymphatic tubes by the lymphatic endothelial cells in vitro.ConclusionsThis study provides the first direct and real time live imaging evidence showing that aqueous humor induces lymphatic regression. Further investigation promises for divulging new mechanisms and therapeutic strategies to treat lymphatic diseases that occur both inside and outside the eye.     

严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)眼表感染研究进展

严重急性呼吸综合征冠状病毒2型(SARS-CoV-2,又称COVID-19)经眼表传播远较经口鼻传播困难,然而现有证据认为眼表传播仍是一条可能的途径,尤其对于医护人员.本文描述了SARS-CoV-2相关的眼表症状,阐释了眼表SARS-CoV-2入胞的可能机制和与眼表相关的SARS-CoV-2传播机制,并对现有的和未来可...     

Conjunctival goblet cells: Ocular surface functions,disorders that affect them,and the potential for their regeneration

Conjunctival goblet cells (CGCs) are specialized cells that produce and secrete soluble mucins to the tear film that bathes the ocular surface. CGC numbers and functions are affected in various ocular surface diseases including dry eye disease with diverse etiologies. In this review we will (i) summarize the important functions of CGCs in ocular surface health, (ii) describe the ocular surface diseases that affect CGC numbers and function, (iii) provide an update on recent research outcomes that elucidate CGC differentiation, gene expression and functions, and (iv) present evidence in support of the prediction that restoring CGC numbers and/or functions is a viable strategy for alleviating ocular surface disorders that impact the CGCs.     

2型糖尿病患者眼表改变的临床分析

目的观察2型糖尿病患者角膜知觉、泪液分泌等眼表改变,及激光共焦显微镜下角膜上皮、神经纤维组织形态学变化,探讨2型糖尿病患者角膜病变的可能机制。方法前瞻性病例对照研究。选取2型糖尿病患者108例,依据眼底情况分为无眼底改变（NDR）组[42例（59眼））和增殖性糖尿病性视网膜病变（PDR）组[66例（86眼）1;选取无全身及眼部疾病的健康体检者（性别、年龄匹配）33例作为对照组。对所有研究对象均进行角膜敏感度测定、基础泪液分泌试验,泪膜破裂时间（BUT）、泪膜分析、角膜荧光素染色及激光共焦显微镜检查。分别对相关数据进行单因素方差分析、卡方检验、Spearman等级相关分析。结果NDR组除泪膜分析结果（大于等于Ⅲ级者占32％）与对照组（14％）的差异有统计学意义外（p,0．01）,其他观察项目均无统计学意义。PDR患者角膜敏感度为（33．0±12．4）mm,低于对照组的（47．2±9．7）mm（P〈0．01）;泪液分泌量为（11．8±4．2）mm,少于对照组的（15．2±4．3）mm（P〈0．011;BUT值为（7．3±2．5）s,低于对照组的（13．7±4．0）s（P〈0．01）;泪膜脂质层光干涉图像为Ⅲ级及以上者占50％,高于对照组的14％（P〈0．01）;角膜荧光染色阳性者占74％,高于对照组的8％（P〈0．01）;角膜上皮细胞密度为（4407±480）个／mm^2,小于对照组的（4736±313）个／mm^2（P〈0．01）;角膜神经纤维密度为（898±153）Ixm／视野。低于对照组的（1231＋176）txm／视野（P〈0．05）。且随糖尿病患病时间的延长,角膜敏感度下降fr=0．657,P=0．020）、角膜荧光素染色增多（rm=-0．460,P=0．012）、角膜神经纤维密度减少（r=-0．473,P=-0．020）。结论NDR患者角膜会出现轻度改变,应引起注意,需定期检查;PDR患者角膜已发生明显改变．应积极诊治。     

Risk of SARS-CoV-2 virus transmission from donor corneal tissue: A review

Since the outbreak of respiratory coronavirus disease (COVID-19) caused by the coronavirus SARS-CoV-2, there is an ongoing discussion about whether the virus could be transmitted through corneal transplantation from donor to recipient. The purpose of this review was to summarize the current knowledge in the scientific community to provide aid in risk evaluation for potential virus transfer by corneal transplants. Literature was searched in PubMed.gov for relevant articles on coronavirus in conjunction with cornea processing, cornea transplantation and eye banking. Further, guidelines of health authorities and eye banking associations were reviewed. Studies have shown that SARS-CoV-2 RNA can be detected in ocular swabs and/or fluid of patients with COVID-19. However, the risk of SARS-CoV-2 virus transmission through these ocular tissues or fluid of patients is judged differently. To date, per literature and official guidelines, no evidence of viable virus in ocular tissue and no cases of transmission of SARS-CoV-2 via tissue preparations have been reported.     

新型冠状病毒感染及其疫苗接种相关的眼科疾病

由新型冠状病毒(严重呼吸综合征-冠状病毒-2型,SARS-CoV-2)引起的新型冠状病毒肺炎(COVID-19),因其极高的传染致病性,严重威胁人类的生命健康,同时也给目前的医疗模式带来全新挑战。已有众多文献资料显示SARS-CoV-2感染不仅可以造成肺脏、肾脏、肠道等全身损害,而且也能累及眼组织,从较为常见的眼表疾病如角膜、结膜、巩膜炎症,到相对少见的旁中心急性中层黄斑病变和急性黄斑神经视网膜病变。对以眼部症状为首发或伴随症状的SARS-CoV-2感染患者而言,眼科医生如何甄别眼部表现与SARS-CoV-2感染之间的相关性无疑面临严峻的挑战。本文将结合近年相关文献,探讨由SARS-CoV-2感染和接种新型冠状病毒肺炎疫苗所引起的相关眼部病变,涵盖了眼表、葡萄膜、视网膜和黄斑以及颅神经等病理改变。     

Ocular Features and Associated Systemic Findings in SARS-CoV-2 Infection

ABSTRACT

Purposes

To describe the prevalence of ocular features among COVID-19 patients and their relationship with clinical data, inflammatory markers and respiratory support therapy (including CPAP); to investigate SARS-CoV-2 in ocular secretions of symptomatic patients.     

Fulminant fungal endogenous endophthalmitis following SARS-CoV-2 infection: A case report

Systemic corticosteroids and immunocompromised state following SARS-CoV-2 infection can predispose individuals to endogenous endophthalmitis. A 66-year-old gentleman presented with complaints of diminution of vision and redness one week post discharge after hospitalization for COVID-19 infection. Clinical examination suggested fulminant endogenous endophthalmitis which responded poorly even after aggressive treatment requiring evisceration. Culture and gene sequenced analysis confirmed Aspergillus fumigatus to be the causative organism. A high degree of suspicion is warranted in the presence of recent onset of floaters in COVID-19-infected individuals to facilitate early diagnosis and outcomes.     

SARS-CoV-2经结膜感染与结膜炎研究述评

当前因新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)引起的新型冠状病毒感染的肺炎(corona virus disease 2019,COVID-19)在我国传播乃至全球爆发,快速蔓延至世界各国,严重威胁世界人民的生命安全与健康。由于特效药物的缺失,积极预防仍是目前抵御COVID-19的有效方法。临床观察发现COVID-19患者可并发结膜炎,动物实验也证实结膜是SARS-CoV-2传播的途径之一。但已有的研究结果存在明显差异性,SARS-CoV-2感染与结膜炎发生的关系也未阐明。所以本文结合目前最新的研究和报道,通过各项分析研究结果,进一步阐述SARS-CoV-2感染与结膜及结膜炎的关系,探讨结膜作为SARS-CoV-2传播途径之一对于该病防护的意义,为该病与眼表关系的研究和临床判读提供帮助。     

Time course of ocular surface and lacrimal gland changes in a new scopolamine-induced dry eye model

Background The aim of this study was to set up an animal model of dry eye showing disturbance in several components of the lacrimal functional unit, and to describe the time course of the appearance of clinical signs and inflammatory markers. Methods Dry eye was induced in 6-week-old female Lewis rats by a systemic and continuous delivery of scopolamine via osmotic pumps implanted subcutaneously. We first determined the appropriate dose of scopolamine (6, 12.5, or 25 mg/day) for 28 days. In a second set of experiments, we determined markers after 1, 2, 3, 7, 10, 17, or 28 days of a 12.5-mg/day dose. Clinical signs of corneal dryness were evaluated in vivo using fluorescein staining. MHC II expression and mucin Muc5AC production were detected on the conjunctival epithelium using immunostaining. The level of IL-1β, IL-6, TNF-α, and IFN-γ mRNA was evaluated by real-time polymerase chain reaction in conjunctiva and exorbital lacrimal gland (LG). Lipids were extracted from the exorbital LG for fatty acid analysis. Results Daily scopolamine doses of 12.5 mg and 25 mg applied for a 28-day period induced keratitis, a decrease in Muc5AC immunostaining density in the conjunctival epithelium, and modifications in the fatty acid composition of the exorbital LG. Animals treated with a 12.5-mg/day dose of scopolamine exhibited an increase in corneal fluorescein staining after 2, 10, and 28 days. All animals exhibited unilateral or bilateral keratitis after 17 days. In the conjunctival epithelium, a significant decrease in Muc5AC immunostaining density was observed at early and late time points, and MHC II expression tended to be increased after 1, 7, 10, and 28 days, without reaching statistical significance. The levels of TNF-α, IL-1β and IL-6 mRNA were increased with scopolamine treatment in both conjunctiva and exorbital LG. Arachidonic acid and the Δ5 desaturase index were significantly increased in the exorbital LG of dry eye animals at each time point. Conclusions This systemic and continuous scopolamine-induced model of dry eye in the rat may represent a helpful tool to investigate moderate dry eye, and makes a contribution in the field of dry eye study. Support The PhD fellowship of S. Viau was in part supported by a grant from the Regional Council of Burgundy.     

体外共培养诱导人羊膜间充质干细胞分化为眼表上皮细胞的研究

背景 人羊膜间充质干细胞（hAMSCs）在体外能诱导分化为多种体细胞,但目前有关hAMSCs分化为眼表细胞的研究鲜有报道. 目的 探讨体外共培养诱导hAMSCs向眼表细胞分化的可能性及其分化机制.方法 本研究经广州医科大学第二附属医院伦理委员会批准,在健康产妇的知情同意下从人胎盘中分离hAMSCs并进行培养,采用流式细胞术计数分析CD44、CD45、CD73、CD90在培养细胞中的表达以鉴定细胞,此外通过成骨诱导分化实验和成脂诱导分化实验对培养细胞进行体外分化鉴定.在患者知情同意下获取眼科手术中弃用的人眼球结膜组织,用组织块培养法分离和培养人球结膜成纤维细胞（hBCFs）,将hAMSCs与hBCFs在Transwell培养体系中进行共培养,分为hAMSCs培养组和hAMSCs与hBCFs共培养组,用含质量分数5％胎牛血清（FBS）的DMEM高糖/F12培养基培养7d,采用免疫荧光技术检测hAMSCs中对上皮细胞角蛋白19（CK19）的表达,评价hAMSCs向眼表细胞分化的程度,并检测hAMSCs中α-平滑肌肌动蛋白（α-SMA）的表达,评价其分化过程与间质上皮化转换（MET）过程的关系.结果 传代至3～7代的hAMSCs均为细长形,但随着传代增加,细胞体积增大,细胞中CD44、CD73、CD90表达呈强阳性,但不表达CD45.经成骨或成脂诱导分化后3～4周,细胞中茜素红S（ARS）染色或油红O染色阳性.hAMSCs与hBCFs共培养后1周,hAMSCs形态由细长形变为类上皮细胞型,共培养组的部分细胞中CK19表达阳性,所有细胞α-SMA表达呈微弱阳性,而hAMSCs细胞培养组可见呈绿色荧光的α-SMA阳性细胞,但不表达CK19. 结论 通过体外共培养可诱导hAMSCs向人眼表细胞分化,其分化机制可能与MET过程相关.