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.     

Ascorbic acid levels in aqueous and vitreous humors of the rabbit: effects of inflammation and ceruloplasmin

A HPLC method for determination of ascorbic acid and dehydroascorbic acid in plasma and aqueous and vitreous humors of rabbits is described. Values for total ascorbic acid concentration found in this study are in agreement with those of previous investigators. Endotoxin-induced ocular inflammation caused a decrease in the concentration of ascorbic acid in the aqueous humor and an increase in the vitreous humor. The additional lack of correlation between levels of ascorbic acid in normal aqueous and vitreous humors from the same uninflamed eye indicates that the aqueous humor is not the source of vitreal ascorbic acid. The copper concentration of the aqueous humor is increased during ocular inflammation, most likely due to the influx of ceruloplasmin from plasma when the blood-aqueous barrier is disrupted. Ceruloplasmin caused a decrease in the amount of ascorbic acid in the aqueous humor in vitro and the vitreous humor in vivo. The presence of ceruloplasmin in the aqueous humor during inflammation thus may contribute to the decreased concentration of ascorbic acid in this fluid.     

Eubacterial PCR for bacterial detection and identification in 100 acute postcataract surgery endophthalmitis

PURPOSE: To evaluate eubacterial PCR compared with conventional cultures for detection and identification of bacterial agents in ocular samples from patients with acute postcataract endophthalmitis. METHODS: Broad-range eubacterial PCR amplification was used, followed by direct DNA sequencing in ocular samples (aqueous humor, vitreous samples from tap or vitrectomy) from 100 consecutive patients presenting with acute postcataract endophthalmitis. Bacterial cultures were performed on the same ocular samples by using traditional methods (brain-heart infusion broth). RESULTS: At the time of admission, the detection rate was not significantly different between cultures and PCR (38.2% for cultures versus 34.6% for PCR in aqueous humor samples; 54% versus 57% in vitreous from a vitreous tap). In contrast, in the vitreous obtained from vitrectomy, after intravitreous injection of antibiotics, PCR detected bacteria in 70% of the cases, compared with 9% in cultures. By combining PCR and cultures, bacterial identification was obtained in 47% of aqueous humor samples at admission, in 68% of vitreous samples from a vitreous tap at admission, and in 72% of vitreous samples from pars plana vitrectomy. Gram-positive bacteria predominated (94.3%). The concordance between cultures and PCR was 100%. The contamination rate was 2%. CONCLUSIONS: Cultures and eubacterial PCR are complementary techniques for bacterial identification in eyes with acute postcataract endophthalmitis. PCR technique was needed for identification of the involved microbial pathogen in 25% of all the cases. Eubacterial PCR is more effective than cultures in detecting bacteria in vitreous samples from patients with previous intravitreous administration of antibiotics.     

葛根素经全身用药在兔眼房水和玻璃体中的药代动力学

目的:测定腹腔注射单次剂量的葛根素后不同时间点新西兰白兔眼房水、玻璃体中葛根素的浓度变化,探讨葛根素在兔眼房水和玻璃体中的药动学变化。方法:新西兰白兎随机分组,每只白兔腹腔注射葛根素80mg/kg,在用药前(0h)和用药后0.5、1、2、3、4、6、8、12、16、24h取房水液、玻璃体液,采用反相高效液相色谱法(RP-HPLC)进行测定。3P87软件拟合药动学参数。结果:腹腔注射葛根素后,其浓度在正常新西兰白兔房水、玻璃体呈开放式二房室模型。理论值:高峰浓度(Cmax)分别为1.61、0.09μg/ml,达峰时间(tmax)为1.68、1.81h,半衰期t1/2α为1.36、1.05h,t1/2β为19.72、15.18h,清除率(CL)分别为2.17、12.43L/h。实测值30min分别为(0.78±0.29)μg/ml、(0.06±0.02)μg/ml,2h达高峰,分别为(2.32±0.15)μg/ml、(0.12±0.04)μg/ml,随后逐渐下降,6h后房水中葛根素含量降至0.57μg/ml,玻璃体为0.05μg/ml,16h后,葛根素在房水和玻璃体中的浓度降至0.03μg/ml或以下。结论:本方法灵敏、特异、准确和快速,可用于房水、玻璃体中葛根素浓度的测定;葛根素通过腹腔注射能透过血-眼屏障进入房水、玻璃体,进入房水的葛根素药量较大,进入玻璃体的药量有限。     

Detection of SARS-CoV-2 RNA in a conjunctival swab sample in real-time-polymerase chain reaction positive COVID-19 patients and its association with comorbidity and severity at a designated COVID-19 hospital in Central India

Purpose:To evaluate the presence of SARS-CoV-2 RNA in the conjunctival swab sample of positive confirmed COVID-19 patients and to find out its association with comorbidity and severity of COVID-19 disease.Methods:We conducted an observational cross-sectional study at a dedicated tertiary COVID-19 hospital in central India for a period of 8 weeks from February 2021to March 2021. We included patients who tested positive for SARS-CoV-2 RNA through nasopharyngeal swab and were above 18 years of age. Swab samples have been collected within 48 h of admission. Conjunctival swab was taken from the lower fornix of both eyes and sent to microbiology laboratory for real-time- polymerase chain reaction (RT-PCR).Results:Out of 150 patients, conjunctival swab RT-PCR was positive in five patients (3.33%). Two patients had conjunctival manifestations in the form of conjunctivitis but conjunctival swab RT-PCR was negative in those patients. Among the RT-PCR positive patients, two (40%) were from mild, one (20%) was from moderate, and two (40%) were from severe category. No association could be established between conjunctival swab RT-PCR positivity and severity of the disease or associated comorbidity.Conclusion:Our study provides evidence that SARS-CoV-2 RNA could be detected in conjunctival secretions, and though the risk is relatively low, the eye may act as source of transmission. Extra caution should be taken by healthcare workers, and use of proper precautions like face shields and goggles should be encouraged.     

新生血管性青光眼患者眼内液血管 内皮生长因子的检测

目的了解新生血管性青光眼(neovascular glaucoma, NVG)患者房水及玻璃体中血管内皮生长因子(vascular endothelial growth factor, VEGF)的含量以及对虹膜新生血管发生的影响。方法应用酶联免疫吸附测定法(enzyme linked immu nosorbent assay, ELISA)分别对11例NVG患者行内眼手术时抽取的房水、玻璃体22个标本以及同期6例黄斑裂孔手术患者(对照组)房水、玻璃体12个标本进行VEGF检测。结果NVG患者房水、玻璃体中VEGF含量分别为(1.451±0.247)、(1.610±0.125) ng/ml,较对照组房水、玻璃体中的 VEGF含量(0.189±0.038)、(0.201±0.055) ng/ml明显增高, 两组间差别有非常显著性意义(t=12.007,P<0.001;t=26.057,P<0.001)。结论NVG患者房水、玻璃体中VEGF浓度显著增高,提示VEGF在NVG患者虹膜新生血管形成过程中可能具有一定作用。(中华眼底病杂志,2001,17:305-306)     

增生性玻璃体视网膜疾病眼内液血管内皮生长因子的表达

目的 观察增生性玻璃体视网膜疾病患者眼内液（房水、玻璃体）中血管内皮生长因子（VEGF）含量的变化规律，探讨VEGF在增生性玻璃体视网膜疾病发展变化中的作用。 方法 采用双抗体夹心酶联免疫吸附试验(ELISA)定量检测增生性玻璃体视网膜病变（PVR）、视网膜静脉阻塞（RVO）、增生性糖尿病视网膜病变（PDR）、新生血管性青光眼（NVG）患者组及正常对照组房水、玻璃体VEGF含量。 结果 PVR、RVO、PDR、NVG患者组房水及玻璃体VEGF含量均高于正常对照组，其差异均有统计学意义(P<0.05)；NVG、PDR、RVO、PVR患者组房水及玻璃体VEGF含量依次降低 ，其差异有统计学意义(P<0.05)；PVR、RVO、PDR、NVG患者组和正常对照组玻璃体VEGF含量均高于房水VEGF含量，其差异有统计学意义(P<0.05)；PVR患者病史与房水、玻璃体VEGF含量呈负相关（r分别为－0.819、－0.823，P＜0.05）；RVO患者病史与房水、玻璃体VEGF含量呈正相关（r分别为0.913、0.929，P＜0.05）；PDR患者玻璃体积血时间与房水、玻璃体VEGF含量呈正相关（r分别为0.905、0.920，P＜0.05）。 结论 增生性玻璃体视网膜疾病患者房水及玻璃体VEGF含量明显增高，VEGF可能在增生性玻璃体视网膜疾病发展变化中起着重要的作用。 (中华眼底病杂志， 2006， 22：313-316）     

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.     

Correlation of aqueous humor electrolytes with serum electrolytes in cataract patients

Purpose:To correlate the aqueous humor electrolytes with serum electrolytes in patients with cataract.Methods:In this study, 348 eyes of patients undergoing cataract surgery, who had presented between 1^st January 2019 and 30^th June 2020, were included. Serum electrolytes were obtained and 0.15 ml of aqueous humor was obtained during surgery using an insulin syringe via the side port incision. Mean and standard deviation was used to analyze all the categorical and continuous data including all the electrolytes levels. Pearson’s correlation of coefficient was used to analyze the comparison between serum and aqueous humor electrolytes. Student T-test was used to compare electrolyte values between the serum and aqueous humor.Results:We observed a significant correlation of the aqueous humor electrolytes with their serum counterparts. The coefficient of correlation is 0.155, 0.44, and 0.405 for Na+, K+, and Cl−, respectively. There is also a significant increase in the aqueous humor electrolytes as compared to the serum levels.Conclusion:A significant increase in electrolytes levels in the aqueous humor of cataract patients possibly could have a role in the etiology of cataract.     

眼球穿孔伤患者早期前房房水或玻璃体细菌学培养分析

AlM:To analyze the spectrum and antibiotic sensitivity of bacteria in the aqueous humor and vitreous immediately following the occurrence of penetrating ocular trauma.
METHODS:Aqueous humor or vitreous specimens were collected from 45 eyes of 45 patients with penetrating ocular trauma before operations. The specimens were prepared for bacterial culture and the associated sensitivity tests to several commonly used antibiotics.
RESULTS:Fifteen eyes of all the patients were found to be positive with bacterial infection, and the most frequently identified bacteria was staphylococcus epidermidis, which accounted for 67. 7% ( 10/15 ) of all identified cases. All identified bacteria were sensitive to ciprofloxacin and ofloxacin, and less sensitive to cefoperazone. Most of them were resistant to gentamycin, rifampin and chloramphenicol. Accompanying intraocular foreign body or sclera wound were associated with positive bacterial cultures (χ1 2=6. 544, P1=0.016;χ22=4.5, P2=0.034). Causes of injury(χ2=1.667,P=0. 197), lens rupture (χ2 =1. 125,P=0. 352) or locations where the specimen was collected (χ2=1. 667,P=0. 197) did not contribute to the difference in the positive rates of bacterial cultures.
CONCLUSlON: ln patients with penetrating ocular trauma, the major bacteria entering into the eye was staphylococcus epidermidis. Fluro- Quinolones can be used as the first - line antibiotics for prophylactic treatment. Cefoperazone can be taken as a second choice.     

Preoperative COVID-19 testing for elective vitreoretinal surgeries: Experience from a major tertiary care institute in South India

Purpose:To study the prevalence of asymptomatic SARS-CoV-2 virus infection (COVID-19) among patients undergoing elective vitreoretinal surgeries at a tertiary care eye hospital.Methods:This cross-sectional, observational study was performed between July 16, 2020 and August 31, 2020, in the retina clinic of a tertiary care eye hospital in south India. All patients undergoing elective retinal surgical procedures underwent RT-PCR testing for SARS-CoV-2 before being posted for surgery and after obtaining informed consent. Patients planned for surgery under general anesthesia underwent additional computed tomography of the chest. Testing strategies and outcomes were documented.Results:Out of a total of 413 patients who were given appointments for surgery during this period, nine patients (2.2%) were found to have positive RT-PCR for SARS-CoV-2, and their surgeries were postponed. The test positivity (prevalence) rate of asymptomatic COVID-19 infection among all elective vitreoretinal surgical patients in our hospital was 2.2%. None of the patients were symptomatic for COVID-19.Conclusion:Our results showed that among patients visiting high volume ophthalmic centers in the near future, approximately 1 in 45 patients may be asymptomatic, SARS-CoV-2 RT-PCR positive. Asymptomatic COVID-19 patients may lead to chances of transmission of the virus inside healthcare facilities among other visiting patients and healthcare workers.     

Aqueous and vitreous penetration of levofloxacin after topical and/or oral administration

PURPOSE: To investigate the aqueous and vitreous penetration of levofloxacin, the drug was administered topically and/or orally to patients undergoing vitrectomy. METHODS: Thirty-six patients undergoing initial vitrectomy with phacoemulsification and aspiration (PEA) were enrolled, and were divided randomly into three groups. Group 1 was treated with topical application of levofloxacin (three times on the day before surgery and seven times on the day of surgery), Group 2 received oral administration of levofloxacin (200 mg twice on the day before surgery and 200 mg at 3 hours before surgery), and Group 3 received both topical and oral levofloxacin according to the above schedules. The concentration of levofloxacin was measured in aqueous humor and vitreous fluid samples obtained during surgery. RESULTS: In Groups 1, 2, and 3, the mean levofloxacin concentration in aqueous humor was 0.765+/-0.624 micro g/mL, 1.279+/-0.440 micro g/mL, and 1.823+/-0.490 micro g/mL, respectively, while the mean levofloxacin concentration in vitreous fluid was <0.02 micro g/mL, 1.455+/-0.445 micro g/mL, and 1.369+/-0.530 micro g/mL, respectively. CONCLUSIONS: Oral administration of levofloxacin at a dose of 400 mg/day was sufficient for the prophylaxis of ocular infections, because the drug concentrations in both aqueous humor and vitreous fluid were higher than the MIC90 values for major ocular pathogens. Topical application of levofloxacin achieved adequate drug levels in aqueous humor, but not in vitreous fluid, while combined topical and oral administration had an additive effect on the drug concentration in aqueous humor.     

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.     

瘦素在增殖性糖尿病性视网膜病变和增殖性玻璃体视网膜病变中的表达

目的 通过检测瘦素在增殖性糖尿病性视网膜病变（proliferative diabetic retinopathy,PDR）和增殖性玻璃体视网膜病变（proliferative vitreous retinopathy,PVR）中的表达,探讨瘦素在PDR、PVR发生、发展过程中可能的调节机制。方法 分别用免疫组织化学染色的方法和酶联免疫吸附实验检测30例PDR患者、20例PVR病变患者眼内视网膜前膜中瘦素的表达,以及患者的血清、眼前房水、玻璃体液中瘦素的浓度。用Chi－Square Tests统计学方法分析和比较PDR、PVR与对照组之间瘦素表达的差异。结果 免疫组织化学染色结果：30例PDR患者中,有18例患者眼内视网膜前膜的瘦素受体呈阳性表达,阳性率为60%,与对照组比较,差异有统计学意义;20例PVR患者中,有3例患者眼内视网膜前膜的瘦素受体呈阳性表达,其中2例为血管纤维性视网膜前膜,1例为细胞纤维性视网膜前膜,阳性率为15%,与对照组比较,差异无统计学意义。ELISA结果：检测30例PDR患者的血清、眼前房水、玻璃体液中瘦素的浓度,与对照组之间差异有统计学意义（P＜0.05）;检测20例PVR患者的血清、眼前房水、玻璃体液中瘦素的浓度,与对照组之间差异无统计学意义（P＞0.05）。结论 瘦素可能主要是通过促进新生血管的生成参与到增殖性糖尿病性视网膜病变的发生、发展中,与增殖性玻璃体视网膜病变的发生、发展无明显相关性。     

Isolation of the human T-cell leukemia/lymphotropic virus type III from aqueous humor in two patients with perivasculitis of the retinal vessels

Summary The human T-cell leukemia/lymphotropic virus type III (HTLV-III) has been isolated from aqueous humor in two patients with perivasculitis of the peripheral retinal vessels, an AIDS-related ocular manifestation. Both patients had antibodies to HTLV-III and although they presented with herpes zoster ophthalmicus, they did not present other symptoms known to be associated with HTLV-III infection. The isolation of HTLV-III from aqueous humor in these two patients with retinal perivasculitis suggests that the virus itself may play a role in the etiology of this ocular sign. The presence of infectious HTLV-III in the anterior chamber further emphasises the necessity to discard corneas from HTLV-III infected donors. Address for offprints: Mrs Suzy Sprecher-Goldberger, Institut Pasteur du Brabant, rue Engeland 642, Bruxelles/Uccle Belgium     

Cytokines in the vitreous of patients with proliferative diabetic retinopathy.

Sixteen vitreous and paired serum samples from 13 patients with proliferative diabetic retinopathy, vitreous samples from seven cadaveric control subjects, and aqueous humor samples from 15 normal control subjects were assayed for the cytokines interleukin-1, tumor necrosis factor-alpha, interleukin-6, and interferon-gamma. Interleukin-6 was detected in 15 of 16 vitreous samples (94%) from diabetic patients, but it was not detected in any of the aqueous humor samples. Vitreous interleukin-6 levels positively correlated with ocular disease activity. Interleukin-1 was detected in seven of 16 vitreous samples (44%) and in four of ten aqueous humor samples (40%), whereas tumor necrosis factor-alpha and interferon-gamma were never detected in vitreous or aqueous fluid. Serum samples from diabetic patients and control subjects contained comparable low levels of interleukin-6. Interleukin-1, tumor necrosis factor-alpha, and interferon-gamma were not found in any of the sera. Because interleukin-6 can function as B-cell differentiation factor, this cytokine may have a role in immunoglobulin deposition in the ocular tissues and in the immunopathologic characteristics of proliferative retinopathy.     

Determination and pharmacokinetic profile of liposomal foscarnet in rabbit ocular tissues after intravitreal administration

The treatment of ocular diseases affecting the posterior segment of the eye, such as cytomegalovirus (CMV) retinitis, requires the access of the drugs to the vitreous humor. Foscarnet inhibits replication of herpesviruses, including CMV. The drug's encapsulation in liposomes is meant not only to increase activity and to prolong the effect of the drug, but also to reduce its toxicity. The aims of the present study were to evaluate foscarnet levels and its pharmacokinetic parameters in vitreous humor and retinal tissue of rabbits after the administration of an intravitreal injection of both liposomal foscarnet and foscarnet commercial solution. Liposomes were prepared by the reverse-phase evaporation method. The amount of encapsulated foscarnet (F) was 63% wt. The in vitro diffusion assays showed that F was released more slowly when formulated in liposomes than in the commercial solution. The in vivo studies showed that, as opposed to commercial solution F, liposomal F achieves stable and durable therapeutic levels in retina, going beyond 72 h, reaching the vitreous humor with adequate levels to accomplish the aims of intravitreal therapy. Lyophilization also increased stability and dispersion of liposomes in aqueous medium, although not improving the pharmacokinetic results over those from non-lyophilized liposomes.