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   Table of Contents      
ORIGINAL ARTICLE
Year : 2020  |  Volume : 68  |  Issue : 6  |  Page : 1015-1017

Presence of viral RNA of SARS-CoV-2 in conjunctival swab specimens of COVID-19 patients


1 Department of Ophthalmology, Minto Ophthalmic Hospital, Bangalore Medical College and Research Institute, Bengaluru, Karnataka, India
2 Department of Medicine, Bangalore Medical College and Research Institute, Bengaluru, Karnataka, India
3 Department of Microbiology, Bangalore Medical College and Research Institute, Bengaluru, Karnataka, India
4 Department of Pulmonary Medicine, Bangalore Medical College and Research Institute, Bengaluru, Karnataka, India
5 Department of Pharmacology, Bangalore Medical College and Research Institute, Bengaluru, Karnataka, India

Date of Submission05-May-2020
Date of Acceptance16-May-2020
Date of Web Publication25-May-2020

Correspondence Address:
Dr. Kiran Kumar
Cataract, Cornea and Refractive Services, Minto Regional Institute of Ophthalmology, Bangalore Medical College and Research Institute, Bengaluru - 560 002, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijo.IJO_1287_20

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  Abstract 


Purpose: To detect the presence of viral RNA of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in conjunctival swab specimens of coronavirus disease-19 (COVID-19) patients. Methods: Forty-five COVID-19 patients positive for real-time reverse transcription-polymerase chain reaction (RT-PCR) for SARS-CoV-2 in nasopharyngeal swab with or without ocular manifestations were included in the study. The conjunctival swab of each patient was collected by an ophthalmologist posted for COVID duty. Results: Out of 45 patients, 35 (77.77%) were males and the rest were females. The mean age was 31.26 ± 12.81 years. None of the patients had any ocular manifestations. One (2.23%) out of 45 patients was positive for RT-PCR SARS-CoV-2 in the conjunctival swab. Conclusion: This study shows that SARS-CoV-2 can be detected in conjunctival swabs of confirmed cases of COVID-19 patients. Though the positivity rate of detecting SARS-CoV-2 in conjunctival swabs is very less, care should be exercised during the ocular examination of patients of COVID-19.

Keywords: Conjunctival swab, coronavirus disease-19, nasopharyngeal swab, SARS-CoV-2


How to cite this article:
Kumar K, Prakash AA, Gangasagara SB, Rathod SB, Ravi K, Rangaiah A, Shankar SM, Basawarajappa SG, Bhushan S, Neeraja T G, Khandenahalli S, Swetha M, Gupta P, Sampritha U C, Prasad GN, Jayanthi CR. Presence of viral RNA of SARS-CoV-2 in conjunctival swab specimens of COVID-19 patients. Indian J Ophthalmol 2020;68:1015-7

How to cite this URL:
Kumar K, Prakash AA, Gangasagara SB, Rathod SB, Ravi K, Rangaiah A, Shankar SM, Basawarajappa SG, Bhushan S, Neeraja T G, Khandenahalli S, Swetha M, Gupta P, Sampritha U C, Prasad GN, Jayanthi CR. Presence of viral RNA of SARS-CoV-2 in conjunctival swab specimens of COVID-19 patients. Indian J Ophthalmol [serial online] 2020 [cited 2020 Oct 21];68:1015-7. Available from: https://www.ijo.in/text.asp?2020/68/6/1015/284769



Since December 2019, there have been cases of coronavirus disease 2019 (COVID-19) reported in China, which soon spread to other parts of the world. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen which is a member of the Coronaviridae family. A few researchers have reported keratoconjunctivitis as an ocular abnormality of SARS-CoV-2.[1] Signs and symptoms of COVID-19 have been described as fever, cough, myalgia, fatigue, sputum production, headache, hemoptysis, diarrhea, and conjunctivitis.[2] Since the diagnosis of COVID-19 patients cannot be only based on symptoms, reverse transcription-polymerase chain reaction (RT-PCR) is a simple and reliable molecular test on respiratory samples (throat swab/nasopharyngeal swab/sputum/endotracheal aspirates, and bronchoalveolar lavage).[3]

Close examination of patients by ophthalmologists and physical contact with patients' eyes is inevitable. This could be a potential source of the spread of SARS-CoV-2. Other viruses like adenovirus and influenza viruses are known for ocular tropism.[4] Therefore, this study was designed to understand whether SARS-CoV-2 can be detected in the conjunctival swab of patients of COVID-19.


  Methods Top


A prospective interventional study was conducted at a dedicated tertiary COVID-19 hospital in south India in April 2020. Institute ethical clearance was obtained prior to the onset of the study. Confirmed positive cases of COVID-19 by nasopharyngeal swab according to WHO standards with or without ocular symptoms were included in the study.[5] Suspect and probable cases and those patients who were critically ill were excluded. Signed consent was obtained for each sample collection from patients. The conjunctival swab was collected by an ophthalmologist posted for COVID duty. Eyelids were everted and samples were obtained by sweeping the inferior fornices of either of the two eyes with sterile nylon flocked swabs without topical anesthesia. The tips of the swab sticks were broken off and placed into a viral transport medium—Hi media (HiMedia Laboratories Pvt. Ltd, Nashik, India. Catalog No: 0000431084). Personal protection equipment was used and necessary precautions were used to minimize the risk of spread of infection from one patient to another. Samples were transported to the specified lab facility maintaining the recommended cold chain. In the laboratory, samples were extracted using the magnetic bead extraction method (Thermo scientific viral isolation kit—5X MagMAX, Thermo Fischer scientific Baltics UAB, Vilnius, Lithuania) in Biomek 4000 platform as per the manufacturer's instructions. In eight-strip PCR tubes, 22.5 μL of the mixed reaction solution or master mix (SARS CoV-2 Detection kit; fortitude 2.0) was loaded. To this, 2.5 μL of the extracted nucleic acid was added to each well, covered and the PCR was performed using the CFX96 real-time PCR (Bio-Rad). After the completion of the PCR run, the amplification curves were judged to decide if the results were negative or positive. A cutoff cycle threshold (Ct) value of 40 was considered as positive as per the manufacturer's instructions.


  Results Top


Out of 45 patients, 35 (77.77%) were males and the rest were females. Patients with a minimum age of 6 years and a maximum age of 75 years were present in the study. For our convenience, patients were divided into four age groups: less than 20 years (6); 21–40 years (29); 41–60 years (9), and more than 60 years (1). The mean age (±SD) was 31.26 ± 12.81 years. None of the patients in our study had any ocular symptoms.

Only one (2.23%) out of 45 patients was positive with the Ct value of 33, for real-time RT-PCR SARS-CoV-2 in conjunctival swab as shown in [Table 1].
Table 1: Demography, clinical symptoms, nasopharyngeal swab, and conjunctival swab reports of coronavirus disease-19-positive patients

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  Discussion Top


No reports were suggesting ocular transmission of COVID-19 initially. Xia et al. evaluated the conjunctival secretions of 30 confirmed cases of COVID-19.[6] In one of these patients, both tear and conjunctival secretions tested positive for the virus by RT-PCR. There are numerous anecdotal reports suggesting conjunctivitis as the initial symptom before the onset of pneumonia, including that of Guangfa Wang, a national expert on the panel for pneumonia during the early investigations in Wuhan, China.[7]

Some studies reported the presence of SARS-CoV or Middle-East respiratory syndrome (MERS-CoV) in tears or conjunctival sac.[8] Another study by Chen et al. detected SARS-CoV-2 in the conjunctival sac of only three COVID-19 patients out of 67 positive cases. But these three patients did not have any ocular symptoms.[9] Sun et al. found that among 72 patients confirmed by laboratory diagnosis with SARS-CoV-2 RT-PCR assay, SARS-CoV-2 RNA fragments were found in ocular discharges belonging to one patient.[10] The above studies show that SARS-CoV-2 can be detected in conjunctival sac, in only a small percentage of COVID-19 positive patients. Also, the risk of infection of SARS-CoV-2 through eye secretions remains uncertain.

Our study showed that only one (2.23%) out of 45 patients had detectable levels of SARS-CoV-2 in conjunctival swabs of COVID-19-positive patients using RT-PCR. Low level of viral detection can be due to various factors like the time for maximum replication of the virus, the timing of performing of sampling, time of presentation of the patient to the hospital and possibly less secretion of the virus through conjunctival secretion, and also low sensitivity of RT-PCR.[11] In a study done by Ziad and colleagues, tracheal aspirates yielded a higher SARS-CoV-2 load than a nasopharyngeal swab.[12] This suggests that viral concentration differs in different sites and secretions. A study was done by de Wit et al. in the rhesus macaque model with MERS-CoV, which is another type of coronavirus.[13] They found out that MERS-CoV RNA could be detected in the conjunctiva well within 6 days of infection beyond which, it failed to detect the virus. Since the majority of people infected with the SARS-CoV-2 virus are asymptomatic, it is difficult to determine the peak of virus load unless multiple samplings are done at varied intervals.

There are several limitations to this study which include relatively small sample size, absence of detailed ocular examinations, sampling done only once from the eyes of each patient, which can increase the prevalence of false-negatives.


  Conclusion Top


This study shows that SARS-CoV-2 can be detected in conjunctival swabs of confirmed cases of COVID-19 patients. Though the positivity rate of detecting SARS-CoV-2 in conjunctival swabs is very less, care should be exercised during the ocular examination of patients of COVID-19.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: Implications for virus origins and receptor binding. Lancet 2020;395:565-74.  Back to cited text no. 1
    
2.
Huang C, Wang Y, Li X. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020;395:497-506.  Back to cited text no. 2
    
3.
Shen M, Zhou Y, Ye J, Abdullah AL-maskri AA, Kang Y, Zeng S, et al. Recent advances and perspectives of nucleic acid detection for coronavirus. J Pharm Anal 2020;10:97-101.  Back to cited text no. 3
    
4.
Nilsson EC, Storm RJ, Bauer J, Johansson SM, Lookene A, Šngström J, et al. The GD1a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis. Nat Med 2011;17:105-9.  Back to cited text no. 4
    
5.
World Health Organization. Case definitions for surveillance of severe acute respiratory syndrome (SARS).  Back to cited text no. 5
    
6.
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. doi: 10.1002/jmv. 25725. [Epub ahead of print].  Back to cited text no. 6
    
7.
Lu CW, Liu XF, Jia ZF. 2019-nCoV transmission through the ocular surface must not be ignored. Lancet 2020;395(10224):e39.  Back to cited text no. 7
    
8.
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. 8
    
9.
Yunyun Zhou, Yuyang Zeng, Yongqing Tong, ChangZheng Chen. Ophthalmologic evidence against the interpersonal transmission of 2019 novel coronavirus through conjunctiva. med Rxiv 2020. doi: https://doi.org/10.1101/2020.02.11.20021956. (Preprint)  Back to cited text no. 9
    
10.
Zhang X, Chen X, Chen L, Deng C, Zou X, Liu W, et al. The evidence of SARS-CoV-2 infection on ocular surface. Ocul Surf. 2020 Apr 11. pii: S1542-0124(20)30065-3. doi: 10.1016/j.jtos.2020.03.010. [Epub ahead of print].  Back to cited text no. 10
    
11.
Yang Y, Yang M, Shen C, Wang F, Yuan J, Li J, et al. Laboratory diagnosis and monitoring the viral shedding of 2019-nCoVinfections. medRxiv preprint. doi: 10.1101/2020.02.11.20021493. (Preprint).  Back to cited text no. 11
    
12.
Memish ZA, Al-Tawfiq JA, Makhdoom HQ, Assiri A, Alhakeem RF, Albarrak A, et al. Respiratory tract samples, viral load, and genome fraction yield in patients with Middle East respiratory syndrome. J Infect Dis 2014;210:1590-4.  Back to cited text no. 12
    
13.
de Wit E, Rasmussen AL, Falzarano D, Bushmaker T, Feldmann F, Brining DL, et al. Middle East respiratory syndrome coronavirus (MERS-CoV) causes transient lower respiratory tract infection in rhesus macaques. Proc Natl Acad Sci U S A 2013;110:16598-603.  Back to cited text no. 13
    



 
 
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