严重急性呼吸系统综合症冠状病毒2(SARS-CoV-2)通过角膜移植物传播的风险正在进行辩论,并导致角膜采购受到严格限制,导致眼银行活动大幅减少。这项研究的目的是专门评估人角膜感染SARS-CoV-2的能力,并促进其体外复制。并通过检测诊断为2019年冠状病毒病(COVID-19)的供体和未受影响的供体的角膜中的SARS-CoV-2RNA来评估角膜污染的现实生活风险。
为了评估人角膜被SARS-CoV-2感染的能力,SARS-CoV-2受体血管紧张素转换酶2(ACE-2)和激活剂TMPRSS2和组织蛋白酶B和L在未受影响的供体的眼表组织中的表达模式通过免疫组织化学(角膜=10,78±11岁,40%女性)和qPCR(n=5角膜,80±12岁,40%女性)。此外,5个新鲜切除的角膜(80±12年,40%的女性)用高浓度的SARS-CoV-2溶液(106中位数组织培养感染剂量(TCID50)/mL)离体感染。从组织和培养基中提取病毒RNA,并在感染后30分钟(H0)和24小时(H24)通过逆转录定量PCR(RT-qPCR)(病毒RNA拷贝)定量。为了评估SARS-CoV-2角膜污染的风险,通过RT-qPCR(Ct值)在来自14名诊断为COVID-19(74±10年,29%的女性)和26名健康捐赠者(79±13岁,57%女性),并且仅在来自2个眼库的133个连续未受影响的供体的器官培养基中(73±13年,29%女性)。受体和激活剂的表达在蛋白质和mRNA水平上在样品之间是可变的。根据免疫组织化学结果,ACE-2主要位于周围角膜最浅表上皮细胞,角膜缘,和结膜,而TMPRSS2主要在球结膜的所有层中表达。从感染后30分钟到24小时,在中央角膜中观察到IP4RNA序列(RdRp病毒基因)的总链和正链显着增加(1.1×108[95%CI:6.4×107至2.4×108]至3.0×109[1.4×109至5.3×109],p=0.0039和2.2×107[1.4×107到3.6×107]到5.1×107[2.9×107到7.5×107],p=0.0117,分别)和角膜巩膜边缘(4.5×109[2.7×109至9.6×109]至3.9×1010[2.6×1010至4.4×1010],p=0.0039和3.1×108[1.2×108到5.3×108]到7.8×108[3.9×108到9.9×108],分别为p=0.0391)。离体角膜中的病毒RNA拷贝在一个供体与另一个供体之间高度可变。最后,在28个被诊断为COVID-19的供体角膜中,有3个(11%)检测到病毒RNA。来自159个未受影响的供体的所有样品对于SARS-CoV-2RNA是阴性的。这项研究的主要局限性在于样本量有限,由于获得被诊断为COVID-19的供体的机会有限,并且伴随着从未受影响的供体获得角膜的减少。
在这项研究中,我们观察到SARS-CoV-2受体和激活剂在人眼表的表达,以及在实验感染新鲜切除的人角膜后24小时病毒RNA拷贝的可变增加。我们还发现病毒RNA仅在非常有限百分比的鼻咽PCR阳性的供体中。诊断为COVID-19的供体阳性率低,这让人们质疑供体选择算法的实用性。
AgencedelaBiomedecine,PFS-20-011https://www。agence-biomedecine。fr/.
The risk of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission through corneal graft is an ongoing debate and leads to strict restrictions in corneas procurement, leading to a major decrease in eye banking activity. The aims of this
study are to specifically assess the capacity of human cornea to be infected by SARS-CoV-2 and promote its replication ex vivo, and to evaluate the real-life risk of corneal contamination by detecting SARS-CoV-2 RNA in corneas retrieved in donors diagnosed with Coronavirus Disease 2019 (COVID-19) and nonaffected donors.
To assess the capacity of human cornea to be infected by SARS-CoV-2, the expression pattern of SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE-2) and activators TMPRSS2 and Cathepsins B and L in ocular surface tissues from nonaffected donors was explored by immunohistochemistry (n = 10 corneas, 78 ± 11 years, 40% female) and qPCR (n = 5 corneas, 80 ± 12 years, 40% female). Additionally, 5 freshly excised corneas (80 ± 12 years, 40% female) were infected ex vivo with highly concentrated SARS-CoV-2 solution (106 median tissue culture infectious dose (TCID50)/mL). Viral RNA was extracted from tissues and culture media and quantified by reverse transcription quantitative PCR (RT-qPCR) (viral RNA copies) 30 minutes (H0) and 24 hours (H24) after infection. To assess the risk of corneal contamination by SARS-CoV-2, viral RNA was tested by RT-qPCR (Ct value) in both corneas and organ culture media from 14 donors diagnosed with COVID-19 (74 ± 10 years, 29% female) and 26 healthy donors (79 ± 13 years, 57% female), and in organ culture media only from 133 consecutive nonaffected donors from 2 eye banks (73 ± 13 years, 29% female). The expression of receptor and activators was variable among samples at both protein and mRNA level. Based on immunohistochemistry findings, ACE-2 was localized mainly in the most superficial epithelial cells of peripheral cornea, limbus, and conjunctiva, whereas TMPRSS2 was mostly expressed in all layers of bulbar conjunctiva. A significant increase in total and positive strands of IP4 RNA sequence (RdRp viral gene) was observed from 30 minutes to 24 hours postinfection in central cornea (1.1 × 108 [95% CI: 6.4 × 107 to 2.4 × 108] to 3.0 × 109 [1.4 × 109 to 5.3 × 109], p = 0.0039 and 2.2 × 107 [1.4 × 107 to 3.6 × 107] to 5.1 × 107 [2.9 × 107 to 7.5 × 107], p = 0.0117, respectively) and in corneoscleral rim (4.5 × 109 [2.7 × 109 to 9.6 × 109] to 3.9 × 1010 [2.6 × 1010 to 4.4 × 1010], p = 0.0039 and 3.1 × 108 [1.2 × 108 to 5.3 × 108] to 7.8 × 108 [3.9 × 108 to 9.9 × 108], p = 0.0391, respectively). Viral RNA copies in ex vivo corneas were highly variable from one donor to another. Finally, viral RNA was detected in 3 out of 28 corneas (11%) from donors diagnosed with COVID-19. All samples from the 159 nonaffected donors were negative for SARS-CoV-2 RNA. The main limitation of this
study relates to the limited sample size, due to limited access to donors diagnosed with COVID-19 and concomitant decrease in the procurement corneas from nonaffected donors.
In this
study, we observed the expression of SARS-CoV-2 receptors and activators at the human ocular surface and a variable increase in viral RNA copies 24 hours after experimental infection of freshly excised human corneas. We also found viral RNA only in a very limited percentage of donors with positive nasopharyngeal PCR. The low rate of positivity in donors diagnosed with COVID-19 calls into question the utility of donor selection algorithms.
Agence de la Biomédecine, PFS-20-011 https://www.agence-biomedecine.fr/.