Estimation of conjunctival swab and nasopharyngeal swab specimens for viral nucleic acid detection in Coronavirus disease 2019 patients to compare the viral load

INTRODUCTION

Coronavirus disease 2019 (COVID-19); the ongoing global major health hazard is caused by the highly transmissible novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) which originated in China in December 2019 and rapidly became a global pandemic posing serious health threat, recognized by the World Health Organization, with the imminent potential of taking a toll with accelerated overburdening of the health-care systems and causing substantial mortality worldwide.^[1] The vicious cycle of close contact human transmission occurs mainly through respiratory droplets, but other routes are under investigation, because SARS-CoV-2 has also been detected in several other body fluids.^[2]

The COVID-19 patients mostly present with signs and symptoms of fever, cough, myalgia, fatigue, sputum production, headache, hemoptysis, diarrhea, and conjunctivitis^[3] Although, the fact that the clinical presentation can lead to a provisional diagnosis, but the confirmatory diagnosis of COVID-19 patients can be made after confirmation with reverse transcription-polymerase chain reaction (RT-PCR) which is a simple and reliable molecular test done on respiratory samples (throat swab/ nasopharyngeal swab/sputum/endotracheal aspirates, and bronchoalveolar lavage).^[4]

The respiratory viruses such as adenovirus, respiratory syncytial virus, influenzae virus, and rhino virus are a well-known entity for ocular tropism^[5] The conjunctiva may be a potential site of direct viral inoculation as a result of SARSCoV droplet from the close contact of an infected patients through aerosol route or migration from upper respiratory tract or through hematogenous spread.^[6-8]

So far, only limited data are available on ocular sampling from patients with COVID-19. Earlier, during the SARS epidemic, the exposure of ocular surface to infectious fluids was associated with an increased risk for SARS-CoV transmission to healthcare workers prompting a grave concern that respiratory illness could be transmitted through ocular secretions.^[6,9]

Similar concerns have been raised with SARS-CoV-2, especially among eye care providers and those on the front lines triaging what could be initial symptoms of COVID-19.

During the ongoing pandemic surge, SARS-CoV-2 ribonucleic acid (RNA) was detected in conjunctival secretions collected from the patient who presented with conjunctivitis from a hospital in China.^[10] However, there is a requirement of further studies to evaluate the infectious potential of the SARSCoV-2 RNA detected in the ocular specimens in comparison to standard nasopharyngeal swab specimen and to determine whether transmission may occur through ocular secretions. The aim of the present study is to estimate the conjunctival swab along with nasopharyngeal swab specimens for viral nucleic acid detection in cases of COVID-19 Patients.

MATERIAL AND METHODS

RESULTS

Total 30 confirmed positive cases of COVID-19 by at least one nasopharyngeal swab in adherence to the WHO standards with or without ocular symptoms were included in the study. Out of 30 patients, 21 patients (70%) were males and the remaining nine patients (30%) were females. Mean age of the patients in the study was 44.80 ± 15.37 years with age ranging between 18 years and 80 years. Mostly, the patients were in the age group of 41–60 years [Table 1]. The mean time of first collection of tear and conjunctival secretions, from the onset of symptoms was 2.30 ± 0.79 days. Twelve patients (40%) had a history of contact to COVID positive patients whereas six patients (20%) gave history of travel to endemic areas. There was no correlation between exposure and travel history with conjunctival swab positivity (P > 0.05 using Chi-square test).

The patients presented commonly with the symptoms of fever, sore throat, cough, and breathlessness, as shown in Table 2. There was an overlap of symptoms in certain patients. However, the correlation between clinical symptoms and conjunctival swab positivity was not statistically significant (P > 0.05 using Fisher’s exact test) [Table 3].

Only one patient (3.3%) in our study was diagnosed with acute conjunctivitis as ocular manifestation after 1 week of hospitalization. Two patients (6.7%) out of 30 patients were positive for real-time RT-PCR SARS-CoV-2 in conjunctival swab with the Ct value of <35. However, three patients had weakly positive Ct value and the result was inconclusive which on repeat testing came out to be negative. There was no statistically significant correlation between nasopharyngeal and conjunctival swab positivity using Pearson’s correlation coefficient (r) = 0.010; P = 0.995 (> 0.05) [Graph 1]. The mean body temperature of the patients was 35.81 ± 1.32°C and average SpO2 was 95.63 ± 2.82% at the time of sampling. The statistical correlation between oxygen saturation level of the patient at the time of admission and conjunctival swab positivity (P = 0.418 using Fisher’s extract test) [Graph 2].

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Graph 1:

Correlation between Ct values of nasopharyngeal swab and Conjunctival swab.

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Graph 2:

Correlation between oxygen saturation and conjunctival swab positivity.

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DISCUSSION

Of all the confirmed COVID-19 cases enrolled in our study, viral nucleic acid was detected in the conjunctival secretions of two patients (6.7%). Only one patient reported with the ocular symptoms of redness and discharge from both the eyes and was diagnosed as Acute Conjunctivitis on the 7^th day of admission. The demonstration of the direct association between conjunctivitis and SARS-CoV-2 infection is a diagnostic challenge. In our study, conjunctivitis was seen in conjunctival swab RT-PCR negative patient.^[11] However, based on previous results, the extremely low positive rate of SARS-CoV-2 RNA test by RT-PCR in tears and conjunctival secretions from patients with laboratory confirmed SARSCoV-2 implies that negative test results could be false negative, not excluding the presence of the virus.

During the emergence of the disease, ocular transmission of COVID-19 was not reported initially. However, recent studies have thrown light that SARS-CoV-2 can be detected in conjunctival sac, seen only in smaller percentage of COVID-19 positive patients. Earlier, during the ongoing SARS-CoV infection, studies had reported the presence of virus in the tear and conjunctival fluid^[6,9,12] At present, limited data are available on ocular sampling from patients with COVID-19.

Xia et al. reported one case who tested positive for the virus by RT-PCR in both tear and conjunctival secretions, after evaluating the conjunctival secretions of 30 confirmed cases of COVID-19.^[13]

Study done by Chen et al. also reported the presence of SARS-CoV-2 in the conjunctival sac of three patients out of 67 COVID-19 positive cases confirmed by laboratory diagnosis with SARS-CoV-2 RT-PCR assay.^[14]

This is a prospective interventional study on a small sample size of 30 confirmed COVID-19 patients. We collected their conjunctival swab samples which were subsequently subjected to RT-PCR Assay. Virus detection in conjunctival swab was seen in only two patients possibly due to factors such as sampling time and amount, peak replication time of the virus, time of presentation of the patient to the hospital, and possibly low sensitivity of RT-PCR.^[15]

The current trend of the infected patients with the ongoing SARS-CoV-2 virus shows that mostly the patients are asymptomatic; hence, it is difficult to evaluate the peak virus load in such patients unless multiple sampling at different time frame has been done.

Furthermore, the conjunctival swab and tear film sampling of the convalescent patients should be done to assess the presence and infectivity of the 2019-nCoV in the tear film of such patients.

Although, the current study shows a low risk of coronavirus transmission through the ocular secretion, the need of eye protection in the form of protective goggles, face shields to avoid the eye exposure to disease cannot be overlooked. Especially, the ophthalmologists due to their close proximity to the patients are vulnerable to the risk of acquiring the infections. Hence, safety measures in the form of face shields, slit lamp shields, hand gloves, and protective eye goggles should be encouraged in the routine practice.^[16] This study endeavors to provide insights on the feasibility of using ocular tissue or even tears as a medium of diagnosis.

There were certain unavoidable limitations of this study including a smaller sample size, cursory ocular examination, and chances of increased false-negatives due to single time sampling done.

CONCLUSION

The present study successfully concluded that SARS-CoV-2 can also be detected in conjunctival swabs of the COVID-19 patients. However, definitive studies are required in future to assess the exact infectivity rate. Recently, it has been proposed that angiotensin converting enzyme 2 receptors play a key role in infecting the cells and facilitate in horizontal transmission of the disease. More extensive studies are required in future to prove this hypothesis. Finally, future studies should consider the association between serum viral load and viral shedding in tears. Although the fact, that the rate of detection of SARS-CoV-2 in conjunctival swabs is less, in comparison to standard nasopharyngeal swabs, diligent care and precaution should be practiced while doing the ocular examination of patients of COVID-19 as close contact with the patients by the ophthalmologists is inevitable.