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ORIGINAL ARTICLE
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Presence of SARS-CoV-2 in human tears detected by quantitative real time PCR (qRT-PCR)


1 Optometry, Dr. D. Y. Patil Institute of Optometry and Visual Sciences, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune, Maharastra, India
2 Central research facility, Physiology, community medicine, microbiology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune, Maharastra, India
3 Center for online Learning, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune, Maharastra, India

Date of Submission15-Dec-2020
Date of Decision28-Dec-2021
Date of Acceptance27-Apr-2022

Correspondence Address:
Neeta Mishra,
Dr. D. Y. Patil Institute of Optometry and Visual Sciences, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune - 411 018, Maharastra
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mjdrdypu.mjdrdypu_708_20

  Abstract 


Background: COVID-19 has caused a pandemic since the end of the year 2019. Controversy regarding the presence of the SARS-CoV-2 in tears and conjunctival sac has created suspense throughout. Moderate to severe dry eye has been observed in the patients infected with COVID-19. Material and Method: We conducted a descriptive, prospective observational study. We enrolled 16 patients fulfilling the inclusion criteria for our study. Patients affected with acute COVID-19 from 28-9-2020 to 9-10-2020 registered at our hospital and willing to participate were included. Tears and conjunctival swabs were taken at the interval of 24 h; consecutively three samples were taken from one COVID-19 infected patient. In total, 48 samples were analyzed by qRT-PCR. Result: We had 16 participants, 47% were females and 53% were males; 2/16 [12.5%] patients showed the presence of SARS-CoV-2 in tears and conjunctival swabs. Both the infected patients were females, and they did not have any obvious abnormal ocular conditions. Schirmer strip reading, however, showed a severe dry eye in both the patients. Conclusion: We identified dry eye ranging from mild to moderate to severe [Schirmer 1 reading ranged from 2 to 8 mm] in all our patients admitted to the COVID-19 ward. The SARS-CoV-2 gene seems to be mutating. More work needs to be done to find the viral load in the tears and any abnormalities in the eyes accordingly.

Keywords: COVID -19, qRT-PCR, Schirmer strip



How to cite this URL:
Mishra N, Raut C, Prasad N, Bhawalkar J S, Farooqui S, Mirza S, Madan VV. Presence of SARS-CoV-2 in human tears detected by quantitative real time PCR (qRT-PCR). Med J DY Patil Vidyapeeth [Epub ahead of print] [cited 2022 Dec 1]. Available from: https://www.mjdrdypv.org/preprintarticle.asp?id=345911




  Introduction/Background Top


COVID -19 has caused a pandemic since the end of the year 2019, causing many causalities all around the world. The virus that causes COVID-19 is mainly transmitted through droplets generated when an infected person coughs, sneezes, or exhales. Anybody near someone who has encountered COVID-19 may get infected by droplet transmission if proper precautions like respiratory cough etiquette are not followed judicially. Isolation of the virus has been done from various body samples up till now but there are few studies related to the presence of SARS-CoV-2 in tears. The knowledge about the presence of SARS-CoV-2 in the tears is very important for the public and health professionals if it is a potential source of infection. There have been studies done in the past for the presence of SARS-CoV-2 in the tears.[1],[2] The tears samples collected early during infection have been found to have viral shedding more in the early days than in the latter days.[1] There has been a prevalence of conjunctivitis in mild COVID-19 patients.[3] These data are confusing as one study contradicts the other. The study is designed in a manner to see if there is a presence of the virus in tears.


  Method Top


Ethical approval was taken before the study from the institutional review board and university ethical committee ref no.- DYPV/EC/588/2020. Informed consent was duly signed by all the subjects who were included in the study. It was a longitudinal, prospective, observational study done at the Dr. D.Y. Patil Medical College, Hospital and Research Centre, Pimpri, Pune, India. A total of 48 tear samples were collected from 16 patients clinically and laboratory-confirmed cases of COVID -19 patients admitted to the hospital. The patients who tested negative for the test were excluded from further collection of samples. Collection of samples was carried on for the patients with a positive report at the interval of 24 h. The samples were collected at the time of admission, 24 and 48 h later. We included 16 acute cases admitted to our hospital from 28-9-2020 to 9-10-2020. We included asymptomatic patients infected with COVID-19, mild, moderate, and severe.

Tears samples were collected using conjunctival swabs and Schirmer's paper. The tear was collected 5 min after inserting Schirmer paper [without anesthesia] in the lower eyelid one-third from the fornix. The swab was placed in the cul-de-sac for 10 s for tear collection. The samples [tear and conjunctival swab] were preserved separately in a viral transport medium and transported in a triple container to prevent any leakage till it reached the laboratory. The samples were stored at -80°C deep freezer until use. The samples were collected wearing a personal protective kit. The sample collection method and procedure followed in previous research[4],[5] were for the extraction of nucleic acid for deoxyribonucleic acid [DNA], whereas we needed to extract ribonucleic acid [RNA]. The sample was extracted using an automated nucleic acid extraction machine- Genepure. Indigenous “TRU RT-PCR kit (3B BlackBio)” was used for the detection of E gene, RNase P, and RdRp and N. Cycle Time [CT] cutoff value was 35 for the kit we used for the detection process. Values less than 35 were positive and values more than that indicated negative for SARS-CoV-2. The lesser the CT more vulnerable the sample. Permission and COVID protocols for specimen collection, packaging, transportation, and disposing were followed according to the ICMR/NIV guidelines. Data analysis was done on the percentage change being a longitudinal study.


  Result Top


The demographical distribution is summarized in [Table 1]. Eight males and eight females were involved in our study. The mean age was 29.08 years (SD +/-5 years). The patient presenting the disease at the time and day of admission was 3–5 days. The mean body temperature was 96.98 degrees Fahrenheit at the time of admission, 60% of the patients presented with fever. Shortness of breath and cough was 53.33 and 66.66%, respectively. Ocular involvement of burning sensation was 40% whereas conjunctivitis, foreign body sensation, and watering from the eye contributed 20%, and itching 6.66%, respectively.
Table 1: Demographic and clinical characteristics of the study patients

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Nasopharyngeal, tears, and conjunctival swab samples were analyzed by RT-PCR which resulted in 2 patients being positive of the 16 for the SARS-CoV-2 in tears. One of the two patients tested positive for tears but was negative for the nasopharyngeal swab. No patients had comorbidities. The percent positivity of tears and the conjunctival swab was 12.5% [Table 2].
Table 2: Nasopharyngeal, tear, and conjunctival swab samples RT-PCR results for COVID-19 patients

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  Discussion Top


A [Table 3] below shows a summarized view of the studies done to detect the SARS-CoV-2 in tears and conjunctiva with the ocular signs.
Table 3: Studies done to detect SARS-CoV-2 in tears and conjunctiva with the ocular signs

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The data we have are mostly from the late months of the pandemic for SARS-CoV-2. The percentage of presence of SARS-CoV-2 in tears is slightly higher than earlier reported.[6],[7],[8],[9],[10],[11],[12] Earlier studies, however, confirmed the presence of the virus in association with conjunctivitis, whereas our study did not report positivity of the virus in tears in association with conjunctivitis. It was interesting to observe that two of the patients with SARS-CoV-2 positive for tears and conjunctival swab had no ocular complaints although the Schirmer strip showed severe dry eye averaging 3.5 and 3 mm, respectively, for the right and left eyes.


  Conclusion Top


Though the sample size was less due to the limitation of our study, detection of SARS-CoV-2 in the tears has shown a positive indication. To prevent further transmission, the patient and people around, professionals, and healthcare workers should not only take precautions with clinically confirmed COVID-19 patients but also with asymptomatic positives and NPS RT-PCR-negative optometrists, ophthalmologists, and related healthcare setting workers should be extra cautious while dealing with their patients to maintain biosafety.

Author contribution

Conception and design: Dr. Neeta Mishra, Dr Chandrashekahar Raut

Analysis and interpretation: Dr Neelam Bala Prasad

Data collection: Dr. Neeta Mishra

Editing: Dr. Safia Farookhi, Dr Neelam Bala Prasad

Obtained funding: DPU/561 (1)/2020

Overall responsibility: Dr. Neeta Mishra

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
DeBroff BM. COVID-19: Ocular manifestations, ocular secretions, and ocular portal of entry. Adv Ophthalmol Vis Syst 2020;10:48-9.  Back to cited text no. 1
    
2.
Lu CW, Liu XF, Jia ZF. 2019-nCoV transmission through the ocular surface must not be ignored. Lancet 2020;22;395:e39.  Back to cited text no. 2
    
3.
Sindhuja K, Lomi N, Asif MI, Tandon R. Clinical profile and prevalence of conjunctivitis in mild COVID19 patients in a tertiary care COVID-19 hospital: A retrospective cross-sectional study. Indian J Ophthalmol 2020;68:1546-50.  Back to cited text no. 3
[PUBMED]  [Full text]  
4.
Satpathy G, Mishra AK, Tandon R, Sharma MK, Sharma A, Nayak N, et al. Evaluation of tear samples for Herpes Simplex Virus 1 (HSV) detection in suspected cases of viral keratitis using PCR assay and conventional laboratory diagnostic tools. Br J Ophthalmol 2011;95:415-8.  Back to cited text no. 4
    
5.
Rentka A, Koroskenyi K, Harsfalvi J, Szekanecz Z, Szucs G, Szodoray P, et al. Evaluation of commonly used tear sampling methods and their relevance in subsequent biochemical analysis. Ann Clin Biochem 2017;54:521-9.  Back to cited text no. 5
    
6.
Wu P, Duan F, Luo C, Liu Q, Qu X, Liang L, et al. Characteristics of ocular findings of patients with coronavirus disease 2019 (COVID-19) in Hubei Province, China. JAMA Ophthalmol 2020;138:575-8.  Back to cited text no. 6
    
7.
Xia J, Tong J, Liu M, Shen Y, Guo D. Evaluation of coronavirus in tears and conjunctival secretions of patients with SARS-CoV-2 infection. J Med Virol 2020;92:589-94.  Back to cited text no. 7
    
8.
Seah I, Anderson D, Kang A, Wang L, Rao P, Young BE, et al. Assessing viral shedding and infectivity of tears in Coronavirus disease 2019 (COVID-19) patients. Ophthalmology 2020;127:977-9.  Back to cited text no. 8
    
9.
Sun C, Wang Y, Liu G, Liu Z. Role of the eye in transmitting human coronavirus: What we know and what we do not know. Front Public Health 2020;8:155.  Back to cited text no. 9
    
10.
Deng C, Yang Y, Chen H, Chen W, Chen Z, Ma K, et al. Ocular Dectection of SARS-CoV-2 in 114 Cases of COVID-19 Pneumonia in Wuhan, China: An Observational Study (2/19/2020). Available at SSRN: https://ssrn.com/abstract=3543587 or http://dx.doi.org/10.2139/ssrn.3543587.  Back to cited text no. 10
    
11.
Zhou Y, Zeng Y, Tong Y, Chen C. Ophthalmologic evidence against the interpersonal transmission of 2019 novel Coronavirus through conjunctiva. medRxi 2020. doi: 10.1101/2020.02.11.20021956.  Back to cited text no. 11
    
12.
Loon SC, Teoh SC, Oon LL, Se-Thoe SY, Ling AE, Leo YS, et al. The severe acute respiratory syndrome coronavirus in tears. Br J Ophthalmol 2004;88:861-3.  Back to cited text no. 12
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

 
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