key: cord-0784581-dcenh3di
authors: Arora, Ritu; Goel, Ruchi; Kumar, Sumit; Chhabra, Mohit; Saxena, Sonal; Manchanda, Vikas; Pumma, Palak
title: Evaluation of SARS-CoV-2 in tears of moderate to severe COVID-19 patients
date: 2020-08-31
journal: Ophthalmology
DOI: 10.1016/j.ophtha.2020.08.029
sha: c7918d9c6cea44c3ce46d249ad5e7b777f546b1b
doc_id: 784581
cord_uid: dcenh3di

PURPOSE: To investigate the presence of SARS-CoV-2 RNA (Severe Acute Respiratory Syndrome corona virus 2 Ribo nucleic acid) in tears of moderate to severe COVID-19 patients. DESIGN: Cross sectional study SUBJECTS: Laboratory proven moderate to severe COVID-19 patients METHODS: Tears were collected within 48 hours of laboratory confirmation using three methods (group 1) conjunctival swab + Schirmer’s strips, (group 2) conjunctival swab, (group 3) Schirmer’s strips. Samples from both the eyes of each patient were transported in a single viral transport media (VTM) for real time-reverse transcriptase- polymerase chain reaction (real-time RT-PCR). Detailed demographic profile, systemic symptoms, co morbidity and ocular manifestations were noted. MAIN OUTCOME MEASURE: Viral load of a sample was determined using cyclic threshold (Ct) value of E gene. A specimen was positive if the amplification curve for the E gene crossed the threshold line within 35 cycles and positive on RNA-dependent RNA polymerase (RdRp) or Open reading frame 1b (ORF 1b) gene assay. RESULTS: Of the 78 cases enrolled in the study, samples of 3 patients were found inadequate for analysis. Thirty-six (48%) were moderate while 39 (52%) were severe COVID-19 cases with no ocular involvement in any patient. In the 75 patients, RT-PCR of tears was positive in 18 patients (24%) and 29 out of 225 (12.9%) samples were positive. Positivity in group 1, 2 and 3 being 11 (14.7%), 11 (14.7%) and 7 (9.3%) respectively (p=0.3105). Mean Ct values in group 1, 2 and 3 were 28.36±6.15, 29.00±5.58 and 27.86±6.46 (p=0.92) respectively. Five patients had positive RT PCR by all 3 methods (mean Ct values 25.24±6.33) and 12 patients were positive by either of the 3 methods (mean Ct value being 32.16±1.94), the difference in Ct values being statistically significant (p=0.029). The median value of symptomatology in patients with positive RT-PCR in tears was 5 days (4-9 days). CONCLUSION: SARS-CoV-2-RNA was detected in tears of 24% of laboratory proven moderate to severe COVID 19 patients. Conjunctival swab remains the gold standard of tear collection for RT-PCR assay. There is significantly higher possibility of viral transmission through tears in moderate to severe COVID-19 patients.

Coronavirus-19(COVID 19) related morbidity, mortality and economic contraction has taken 2 the world by storm since December 2019. Despite the herculean efforts directed towards 3 curbing its transmission, the disease has continued to spread like fire. 4 5 Coronaviruses (CoVs) are zoonotic pathogens that can infect human beings by undergoing 6 mutations. 1 Air borne respiratory droplet transmission is well recognized, however alternative 7 modes such as ocular secretions and oral-fecal route, though being held responsible for the 8 spread in many studies are yet to be proven conclusively. 2-4 9 Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein binds with 10 the host cellular receptor, human angiotensin-converting enzyme (ACE)2 and gains entry into 11 the cell in presence of transmembrane serine protease2(TMPRSS2), a cell surface associated 12 protease. [5] [6] [7] [8] ACE2 is known to be expressed on epithelial cells in lungs, intestines and 13 kidney. 9 Recent report indicates presence of both ACE2 and TMPRSS2 in human 14 conjunctival as well as corneal cells, making the ocular surface cells as a potential entry point 15 and reservoir for transmission of virus. 10,11 16 The mucous membrane of ocular surface is continuous from puncta via nasolacrimal duct to 17 nasopharynx, resulting in viral transfer in either direction, even to gastrointestinal tract, if 18 swallowed. Blood borne infection of lacrimal gland has also been proposed. 12 

Inspite of the above evidence, the reported prevalence of viral RNA detection in tears varies 20 from 0 to 7%, with higher positivity rates in severe COVID 19 patients. This has been 21 attributed to low sensitivity of real time RT-PCR in picking up small quantities of SARS-

CoV RNA, missing of window of viral shedding at the time of sample collection and a small 23 sample size. The shedding of viral RNA in tears has been observed both in presence as well 24 as in absence of ocular manifestations. 13-19 25 There is inconsistency in study population in different reports, in terms of COVID-19 disease 26 severity and lab confirmation. Also, there is variability in the method of tear sample 27 collection that is, use of conjunctival swabs or Schirmer paper strips with separate viral 28 transport media (VTM) for each eye. To the best of our knowledge different techniques for 29 tear collection for SARS-CoV-2 detection have not been compared. 30 In the present study, to increase the yield of viral RNA in preocular tear film, sampling was 31 done in lab confirmed hospitalized moderate and severe COVID 19 patients, using different 32 methods, within 48 hours of collection of nasal-oropharyngeal swab and samples from both All the samples were transported to the lab as soon as possible maintaining the cold chain. In 81 case of delay the sample was stored at 4 0 C, not beyond 3 days. After reaching the laboratory, 82 the samples were processed immediately or stored at -20 0 C till processing. 83 The real time PCR assay used the TaqMan fluorogenic probe based chemistry that used the 5 ́ The role of ocular surface as a possible portal of entry, reservoir for replication and 158 transmission of SARS-CoV-2 RNA has been extensively explored. 9,23-25 Difference in 159 detection of viral RNA in tears has ranged from 0 to 7 % with some researchers claiming 160 minimal viral shedding in ocular secretions. [13] [14] [15] [16] [17] [18] [19] Recently, live virus has been demonstrated 161 in ocular fluids by demonstrating cytopathic effect in Vero E6 cells. 26 Though initial reports 162 had included asymptomatic, mild and clinically suspected COVID-19 cases, it was noticed 163 that both the patients showing positive RT-PCR in tears were critical COVID-19 patients. 15 164 Sample collection has been described using conjunctival swab and Schirmer paper strip. 3,13- foreign body sensation and the third was asymptomatic. 18 Citing a low viral load in non-209 inflamed tissues, ocular involvement has been suggested as a pre-requisite for viral shedding 210 in tears. 13 The absence of ocular signs and symptoms, in any of the tear positive cases, in 211 our series and also by others implies that viral shedding in tears is not always related to 212 ocular inflammation as proposed previously. 14, 18, 19 Collection of tears and analysis of viral 

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