Detection of coronavirus-2 by real-time reverse transcription polymerase chain reaction in conjunctival swabs from patients with severe form of Coronavirus disease 2019 in São Paulo, Brazil

Misawa, Mariana Akemi Matsura; Tanaka, Tatiana; Minelli, Tomás; Braga, Pedro Gomes Oliveira; Kato, Juliana Mika; Gouvêa, Michele Soares Gomes; Pinho, João Renato Rebello; Yamamoto, Joyce Hisae

doi:10.6061/clinics/2021/e2913

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ORIGINAL ARTICLE • Clinics 76 • 2021 • https://doi.org/10.6061/clinics/2021/e2913 copy

Detection of coronavirus-2 by real-time reverse transcription polymerase chain reaction in conjunctival swabs from patients with severe form of Coronavirus disease 2019 in São Paulo, Brazil

Authorship SCIMAGO INSTITUTIONS RANKINGS

OBJECTIVES:

To test conjunctival swabs from patients with laboratory-confirmed severe forms of coronavirus disease 2019 (COVID-19) for the presence of SARS-CoV-2 on real-time reverse-transcription polymerase chain reaction (rRT-PCR).

METHODS:

Fifty conjunctival swabs were collected from 50 in-patients with laboratory-confirmed severe forms of COVID-19 at the largest teaching hospital and referral center in Brazil (HCFMUSP, São Paulo, SP). The samples were tested for SARS-CoV-2 on rRT-PCR with the primers and probes described in the CDC protocol which amplify the region of the nucleocapsid N gene (2019_nCoV_N1 and 2019_nCoV_N2) of SARS-CoV-2 RNA and compared with naso/oropharyngeal swabs collected within 24 hours of the conjunctival swabs.

RESULTS:

Five conjunctival samples (10%) tested positive (amplification of the N1 and N2 primer/probe sets) while two conjunctival samples (4%) yielded inconclusive results (amplification of the N1 primer/probe set only). The naso/oropharyngeal swabs were positive for SARS-CoV-2 on rRT-PCR in 34 patients (68%), negative in 14 (28%) and inconclusive in 2 (4%). The 5 patients with positive conjunctival swabs had positive (n=2), negative (n=2) or inconclusive (n=1) naso/oropharyngeal swabs on rRT-PCR. Patients with negative or inconclusive naso/oropharyngeal swabs had the diagnosis of COVID-19 confirmed by previous positive rRT-PCR results or by serology.

CONCLUSION:

This is the first study to present conjunctival swab rRT-PCR results for SARS-CoV-2 in a Brazilian population. In our sample of 50 patients with severe forms of COVID-19, 10% had positive conjunctival swabs, most of which were correlated with positive naso/oropharyngeal rRT-PCR results.

COVID-19; Conjunctiva; SARS-CoV-2

Patient	Age range (years)	Sex	Result of PCR for SARS-CoV-2 rRT in naso/oropharyngeal and conjunctival swabs	Initial systemic symptoms	Systemic comorbidities	Duration of disease (days)^a
Positive conjunctival swab
1	60s	M	Negative^b	Sore throat, cough, asthenia, SOB	CVD, previous alcoholism, smoking	9
2	40s	M	Negative^b	Cough, fever, SOB	No previous comorbidities	11
3	60s	F	Inconclusive	Cough, nasal discharge, asthenia	CVD, DM, HTN, RA	8
4	50s	M	Positive	Cough, fever, myalgia, SOB	DM	7
5	60s	F	Positive	Chest pain, SOB	Cancer, COPD, DM, HTN, pulmonary thromboembolism	1
Inconclusive conjunctival swab
6	60s	F	Positive	Fever, SOB	Asthma, HTN, obesity	10
7	70s	M	Positive	Cough, fever, SOB	Cancer, CKD, COPD, CVD, DM, smoking, OSA	5
Negative conjunctival swab
8	50s	F	Negative^b	Cough, fever, SOB	Obesity, previous smoking, schizophrenia	3
9	60s	F	Negative^b	SOB	COPD, CVD, dyslipidemia, HTN, kidney transplant, previous smoking, overweight	8
10	50s	M	Negative^b	Cough	DM, HTN	8
11	60s	M	Negative^b	Anosmia, ageusia, fever, cough, myalgia	No previous comorbidities	10
12	60s	M	Negative^b	Cough, SOB	DM, dyslipidemia, HTN, previous	9
13	40s	M	Negative^b	Cough, inappetence, fever, abdominal pain, nausea and vomiting, SOB	CKD, DM, HTN	14
14	50s	F	Negative^b	Asthenia, headache, inappetence, SOB	DM, fibromyalgia, HTN, obesity	12
15	60s	M	Negative^b	Anosmia, ageusia, cough	Cancer, HTN, previous smoking	6
16	70s	M	Negative^b	Cough, nasal discharge, wheezing, headache, myalgia	CVD, DM, HTN, obesity	9
17	50s	F	Negative^b	Ageusia, hyposmia, headache, cough	No previous comorbidities	9
18	40s	M	Negative^b	Anosmia, dysgeusia, fever, chest pain, SOB	Obesity	11
19	50s	F	Negative^b	Anosmia, SOB	DM, HTN	5
20	60s	M	Inconclusive^b	Headache, fever, myalgia, SOB	Benign prostatic hyperplasia, HTN, obesity	4
21	30s	F	Positive	Cough	CVD, depression, dyslipidemia, DM, HTN, obesity	4
22	20s	F	Positive	Sore throat, headache, fever	Sickle cell anemia	2
23	60s	M	Positive	Asymptomatic	DM, hepatic cirrhosis, HTN, smoking	1
24	80s	F	Positive	Inappetence, wheezing	Actinic pulmonary fibrosis, cancer, Parkinson disease	4
25	30s	F	Positive	Cough, fever, SOB	CKD, HTN, lupus	7
26	40s	M	Positive	Headache, SOB	DM, myasthenia gravis	6
27	60s	F	Positive	Dysgeusia, fever, cough, myalgia	Dyslipidemia, previous smoking	7
28	30s	M	Positive	Cough, fever, diarrhea, SOB	DM	9
29	70s	F	Positive	Inappetence, cough, diarrhea, SOB	CVD, DM, HTN	12
30	50s	M	Positive	Asthenia, cough, chest pain, fever	DM, HTN, kidney transplant	13
31	70s	F	Positive	Hyposmia, headache, cough, nausea, vomiting, inappetence, diarrhea, myalgia, SOB	DM, dyslipidemia, HTN, obesity, previous smoking	8
32	50s	F	Positive	Cough, SOB	Bariatric surgery, cancer	4
33	50s	F	Positive	Fever, diarrhea	Cancer	6
34	70s	M	Positive	SOB	CKD, CVD, DM, HTN, previous smoking	9
35	20s	F	Positive	Cough	A1AD, CKD, granulomatosis with polyangiitis	1
36	70s	F	Positive	Cough, myalgia, SOB	No previous comorbidities	8
37	40s	M	Positive	Anosmia, ageusia, headache, cough, fever, SOB	Cancer	4
38	70s	F	Positive	Cough, nasal discharge, sneezing, headache	CKD, CVD, HTN, previous smoking	8
39	30s	M	Positive	Anosmia, headache, cough, fever, inappetence	Obesity	6
40	60s	M	Positive	Asthenia, nausea, vomiting, SOB	Cancer	3
41	40s	F	Positive	Cough, sore throat, backache, SOB	DM, HTN, obesity	8
42	60s	M	Positive	Ageusia, headache, fever, myalgia	DM, dyslipidemia	12
43	80s	F	Positive	Anosmia, dysgeusia, asthenia, SOB	DM, HTN, sarcoidosis	15
44	60s	M	Positive	Fever, diarrhea, vomiting, SOB	Cancer, HTN, kidney transplant	2
45	50s	M	Positive	Cough, myalgia, SOB	Alcoholism, HTN, previous smoking	12
46	60s	F	Positive	Cough, asthenia, fever, SOB	Cancer, HTN	9
47	80s	F	Positive	Dysgeusia, asthenia, headache, fever, SOB	Asthma, COPD, DM, HTN, obesity, depression, OSA, previous smoking	10
48	30s	M	Positive	Cough, headache, fever, myalgia, SOB	No previous comorbidities	9
49	70s	M	Positive	Cough, fever	CVD, HTN, previous smoking and alcoholism	8
50	60s	M	Positive	Cough, myalgia, SOB	Liver transplant	15

			Conjunctival swab
Patient	Duration (days)	Naso/Oropharyngeal swab	CT N1	CT N2
1	9	Negative	30.8	33.7	Positive
2	11	Negative	32	31.4	Positive
3	8	Inconclusive	31.6	31.6	Positive
4	7	Positive	37	35.9	Positive
5	1	Positive	36.2	40	Positive
6	10	Positive	36.9	Undetermined	Inconclusive
7	5	Positive	36	Undetermined	Inconclusive

Author	Ocular symptoms (n)	Patients (n)	Positive rate (%)	Form of disease
Xia et al. (¹³13. 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(6):589-94. https://doi.org/10.1002/jmv.25725 https://doi.org/10.1002/jmv.25725... )	1	30	3	Common type (n=21)
				Severe (n=9)
Liang et al. (⁴4. Loffredo L, Pacella F, Pacella E, Tiscione G, Oliva A, Violi F. Conjunctivitis and COVID-19: A meta-analysis. J Med Virol. 2020;92(9):1413-4. https://doi.org/10.1002/jmv.25938 https://doi.org/10.1002/jmv.25938... )	3	37	2.7	Mild (n=25)
				Severe (n=12)
Zhang et al. (¹²12. 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;18(3):360-2. https://doi.org/10.1016/j.jtos.2020.03.010 https://doi.org/10.1016/j.jtos.2020.03.0... )	2	72	1.4	Not reported
Seah et al. (⁹9. Shemer A, Einan-Lifshitz A, Itah A, Dubinsky-Pertzov B, Pras E, Hecht I. Ocular involvement in coronavirus disease 2019 (COVID-19): a clinical and molecular analysis. Int Ophthalmol. 2021;41(2):433-40. https://doi.org/10.1007/s10792-020-01592-1 https://doi.org/10.1007/s10792-020-01592... )	1	17	0	Not reported
Wu et al. (²2. Li JO, Lam DSC, Chen Y, Ting DSW. Novel Coronavirus disease 2019 (COVID-19): The importance of recognising possible early ocular manifestation and using protective eyewear. Br J Ophthalmol. 2020;104(3):297-8. https://doi.org/10.1136/bjophthalmol-2020-315994 https://doi.org/10.1136/bjophthalmol-202... )	12	38	5.3	Moderate (n=4)
				Severe (n=2)
				Critical (n=6)
Xie et al. (³⁰30. Fang Z, Zhang Y, Hang C, Ai J, Li S, Zhang W. Comparisons of viral shedding time of SARS-CoV-2 of different samples in ICU and non-ICU patients. J Infect. 2020;81(1):147-78. https://doi.org/10.1016/j.jinf.2020.03.013 https://doi.org/10.1016/j.jinf.2020.03.0... )	0	33	6.1	Not reported
Fang et al. (³¹31. Xie HT, Jiang SY, Xu KK, Liu X, Xu B, Wang L, et al. SARS-CoV-2 in the ocular surface of COVID-19 patients. Eye Vis (Lond). 2020;7:23. https://doi.org/10.1186/s40662-020-00189-0 https://doi.org/10.1186/s40662-020-00189... )	Not reported	32	15.6	In ICU (n=8)
Karimi et al. (²⁴24. Karimi S, Arabi A, Shahraki T, Safi S. Detection of severe acute respiratory syndrome Coronavirus-2 in the tears of patients with Coronavirus disease 2019. Eye (Lond). 2020;34(7):1220-3. https://doi.org/10.1038/s41433-020-0965-2 https://doi.org/10.1038/s41433-020-0965-... )	1	43	7	Severe (n=43)
Atum et al. (²¹21. Atum M, Boz AAE, Çakir B, Karabay O, Köroglu M, Ögütlü A, et al. Evaluation of Conjunctival Swab PCR Results in Patients with SARS-CoV-2 Infection. Ocul Immunol Inflamm. 2020;28(5):745-8. https://doi.org/10.1080/09273948.2020.1775261 https://doi.org/10.1080/09273948.2020.17... )	10	40	7.5	Not reported
Kumar et al. (²⁵25. Kumar K, Prakash AA, Gangasagara SB, Rathod SBL, Ravi K, Rangaiah A, et al. Presence of viral RNA of SARS-CoV-2 in conjunctival swab specimens of COVID-19 patients. Indian J Ophthalmol. 2020;68(6):1015-7. https://doi.org/10.4103/ijo.IJO_1287_20 https://doi.org/10.4103/ijo.IJO_1287_20... )	0	45	2.2	Not reported
Guemes-Villahoz et al. (¹⁵15. Zhou Y, Zeng Y, Tong Y, Chen C. Ophthalmologic evidence against the interpersonal transmission of 2019 novel coronavirus through conjunctiva. medRxiv. 2020. https://doi.org/10.1101/2020.02.11.20021956 https://doi.org/10.1101/2020.02.11.20021... )	18	36	5.5	Mild (n=17)
				Moderate (n=12)
				Severe (n=7)
Arora et al. (¹⁶16. Savastano MC, Gambini G, Savastano A, Falsini B, De Vico U, Sanguinetti M, et al. Evidence-based of conjunctival COVID-19 positivity: An Italian experience: Gemelli Against COVID Group. Eur J Ophthalmol. 2020;1120672120976548. https://doi.org/10.1177/1120672120976548 https://doi.org/10.1177/1120672120976548... )	0	75	24	Moderate (n=36)
				Severe (n=39)
Shemer et al. (¹⁰10. Mahmoud H, Ammar H, El Rashidy A, Ali AH, Hefny HM, Mounir A. Assessment of Coronavirus in the Conjunctival Tears and Secretions in Patients with SARS-CoV-2 Infection in Sohag Province, Egypt. Clin Ophthalmol. 2020;14:2701-8. https://doi.org/10.2147/OPTH.S270006 https://doi.org/10.2147/OPTH.S270006... )	3	16	0	Not reported
Kaya et al. (⁶6. Kaya H, Çalışkan A, Okul M, Sarı T, Akbudak İH. Detection of SARS-CoV-2 in the tears and conjunctival secretions of Coronavirus disease 2019 patients. J Infect Dev Ctries. 2020;14(9):977-81. https://doi.org/10.3855/jidc.13224 https://doi.org/10.3855/jidc.13224... )	0	32	15.6	Mild (n=5)
				Severe (n=22)
				Critical (n=5)
Mahmoud et al. (⁸8. Seah IYJ, Anderson DE, Kang AEZ, 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(7):977-9. https://doi.org/10.1016/j.ophtha.2020.03.026 https://doi.org/10.1016/j.ophtha.2020.03... )	10	28	28.6	Mild/moderate (n=25)
				On ventilator (n=3)
Dutescu et al. (⁷7. Dutescu RM, Banasik P, Schildgen O, Schrage N, Uthoff D. Detection of Coronavirus in Tear Samples of Hospitalized Patients With Confirmed SARS-CoV-2 From Oropharyngeal Swabs. Cornea. 2021;40(3):348-50. https://doi.org/10.1097/ICO.0000000000002562 https://doi.org/10.1097/ICO.000000000000... )	0	18	27.7	In ICU (n=5)
Zhou et al.(¹⁴14. Güemes-Villahoz N, Burgos-Blasco B, Arribi-Vilela A, Arriola-Villalobos P, Rico-Luna CM, Cuiãa-Sardiãa R, et al. Detecting SARS-CoV-2 RNA in conjunctival secretions: Is it a valuable diagnostic method of COVID-19? J Med Virol. 2021;93(1):383-8. https://doi.org/10.1002/jmv.26219 https://doi.org/10.1002/jmv.26219... )	1	67	4	Not reported
Savastano et al. (¹⁷17. Arora R, Goel R, Kumar S, Chhabra M, Saxena S, Manchanda V, et al. Evaluation of SARS-CoV-2 in Tears of Patients with Moderate to Severe COVID-19. Ophthalmology. 2020;128(4):494-503. https://doi.org/10.1016/j.ophtha.2020.08.029 https://doi.org/10.1016/j.ophtha.2020.08... )	5	50	8	Hospitalized and ICU patients
Yan et al. (²⁰20. Yan Y, Zeng B, Zhang Z, Hu C, Yan M, Li B, et al. Detection of SARS-CoV-2 in Simultaneously Collected Tear and Throat Swab Samples from the Patients with 2019- new SARS-CoV-2 Infection Disease: A Single Center Cross-sectional Study. Ophthalmic Epidemiol. 2021;1-7. https://doi.org/10.1080/09286586.2021.1875011 https://doi.org/10.1080/09286586.2021.18... )	0	35	8.6	Mild/moderate (n=29)
				Severe (n=6)
Present study	0	50	10	Severe (n=50)

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[1] *Corresponding author. E-mail: marianamisawa@gmail.com