key: cord-0702043-pnowmv6b authors: Gunduz, Ayten; Firat, Murat; Turkoglu, Gamze title: Comparison of the simultaneous conjunctiva and oropharynx–nasopharynx swab results in patients applying to the SARS‐CoV‐2 outpatient clinic for the first time date: 2021-05-03 journal: J Med Virol DOI: 10.1002/jmv.26981 sha: 39639f7ecd90ea91c2dffc0e98aff9ef2ba633d4 doc_id: 702043 cord_uid: pnowmv6b The aim is to comparatively evaluate the results of simultaneous conjunctiva and oropharynx–nasopharynx (ONP) swabs in patients who had presented to the outpatient department with a suspicion of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). An ONP sample was obtained following bilateral conjunctiva swabs in 85 subjects with a contact history or symptoms but unknown SARS‐CoV‐2 status and with no ocular symptoms or findings. The results were evaluated according to the patient's symptoms and how the swab was taken. The conjunctiva swab was positive in 29 (34.1%) cases and the ONP swab in 20 (23.5%) cases. Both methods produced positive results in 11 (14.1%) cases. The mean cycle threshold (C (t)) value was 30.15 ± 3.41 in symptomatic cases and 33.62 ± 1.76 in asymptomatic cases (p = .008). The mean C (t) value was 24.37 ± 3.48 when only the ONP swab was positive and 31.22 ± 1.99 when only the conjunctiva swab was positive. In cases that were positive by both methods, the mean C (t) value was 25.21 ± 4.94 for the ONP swab and 30.29 ± 5.05 for the conjunctiva swab. We found higher SARS‐CoV‐2 detection rates with the conjunctiva swab than the ONP swab in cases with unknown SARS‐CoV‐2 status in the early period. In addition, the conjunctival viral load seemed to be higher in symptomatic cases than in asymptomatic cases. We, therefore, believe a conjunctiva swab could be an alternative method to detect SARS‐CoV‐2 at the time of the first presentation to the outpatient department. The conjunctiva is a mucosal surface directly exposed to air and contains angiotensin-converting enzyme 2 receptors; it is also connected to the nasal cavity through the nasolacrimal canal, making it an attractive candidate as a route for the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 1, 2 There are several reports of SARS-CoV-2 detection in conjunctiva swabs. [3] [4] [5] However, the relationship between the conjunctival presence of the virus and the pathogenesis is controversial. 6, 7 Previous studies have been conducted by taking a conjunctiva swab from subjects after the positive status had already been confirmed. This prevents determining the rate at which a positive conjunctiva swab result indicates a new case. Note: 1st and 3rd places are affiliated hospitals. The aim of this study was to evaluate the comparative results of simultaneously obtained conjunctiva and oropharynx-nasopharynx (ONP) swabs in patients presenting to the outpatient department for the first time with unknown SARS-CoV-2 status and determine the relationship between the results and the symptoms. This prospective study was conducted on 85 adult patients who had a contact history or were symptomatic and presented to the Turgut Ozal University Malatya Training and Research Hospital's outpatient department for the first time in December 2020. Simultaneous conjunctiva and ONP swabs were obtained from the patients with unknown SARS-CoV-2 infection status who had been randomly selected. All subjects were queried regarding symptoms related to SARS-CoV-2 and a detailed SARS-CoV-2 contact history was taken. The medical history was also evaluated regarding ocular surgery or medication use. All subjects were examined with a pen-light to detect conjunctivitis. Patients with conjunctivitis or conjunctival hyperemia on the eye examination were excluded from the study. Other exclusion criteria were the use of any ophthalmic medication, ocular surgery within the last 3 months, chronic eye disease, and any anatomical deformity of the conjunctiva or ONP that would present when performing an optimum swab procedure. Patients who could not tolerate the ONP swab procedure were also excluded. Permission for the study was obtained from the local ethics committee. Written informed consent was obtained from all study subjects. The Helsinki declaration principles were adhered to at every stage of the study. When the patient was looking up with both eyes, the swab for the reverse-transcription polymerase chain reaction (RT-PCR) test was obtained from the lower lid conjunctiva after drawing the lower lids slightly downward. The same swab was used to obtain swabs from both eyes in sequence. A topical anesthetic was not administered before the procedure. The technician changed gloves after each patient to avoid cross contamination. Another swab was then used to obtain the ONP swab for the SARS CoV-2 diagnosis. The samples were then placed in a transfer tube (Bio-Speedy vNAT, Bioeksen Ar-Ge Tekn. Ltd. Şti) and the cap closed. The samples were transferred to the laboratory under a temperature of +2°C to 8°C. The ONP and conjunctiva samples from the patients were transferred to the laboratory and simultaneously studied. The tests were conducted within a Level-2 Biosafety Hood inside a Level-2 Biosafety Laboratory. The samples were kept for at least 30 min before the study so that the cells could be degraded and the RNA released. The samples were then vortexed for 15-20 s and 100 µl was transferred to an Eppendorf tube. The reaction setup procedure was started and an RT-PCR kit (Bio-Speedy SARS CoV-2 Double Gene RT-qPCR, Bioeksen Ar-Ge Tekn. Ltd. Şti) was used. As described in the kit leaflet, 10 µl of 2X prime script mix, 5 µl of CVD Di Oligo Mix, and 5 µl of template nucleic acid were mixed to form a mixture of 20 µl. The ONP and conjunctival samples were then simultaneously placed in the device (Rotor-Gene Q, Qiagen). Again according to the kit leaflet, the procedure consisted of 1 cycle at 52°C for 5 min, 1 cycle at 95°C for 10 s, and 40 cycles at 95°C for 1 s and at 55°C for 1 s. The reaction curves were interpreted according to the rules of the kit procedure once the test study was completed. Values with a cycle threshold (C t ) of less than 38 (C t < 38) were accepted as positive. Internal and external controls were performed to evaluate false positivity and false negativity in our study. There was no adverse situation in these controls. All the stages of the sample analysis were conducted by two microbiologists (Gunduz and Turkoglu) experienced in RT-PCR. The SPSS version 25.0 (IBM Corp.) software was used for statistical analysis. The Kolmogorov-Smirnov test was used to determine whether the measurable variables conformed to a normal distribution. The measurable data are presented as mean ± standard deviation (SD) (minimummaximum). The Mann-Whitney U test was used for the independent groups and the Wilcoxon signed rank test for the dependent groups when evaluating measurable data. Categorical data were evaluated with Pearson's χ 2 test or Fisher's exact test. Spearman test was used for correlation analysis. A p value of less than .05 was used to indicate statistical significance. The mean age of the study subjects was 38.74 ± 14.36 (18- For cases with a positive result from both samples, the C t value for the ONP swab was lower than that for the conjunctiva swab. In addition, there was no relationship between the number of symptoms of the patients and the conjunctiva or ONP swab C t values. Table 2 Note: There was no statistically significant difference between the mean C t values of the conjunctival swabs of C(+)/OP(−) and C(+)/OP(+) and between the mean C t values of the oropharynx-nasopharynx swabs of C(+)/OP(+) and C(−)/OP(+) (p = 1.000 and p = .603, respectively; Mann-Whitney U). Abbreviations: C, conjunctiva, C t , cycle threshold; n, sample size; OP, oropharynx-nasopharynx; SD, standard deviation. F I G U R E 1 Amplification curves of three cases whose conjunctival and/or oro-nasopharynx samples were positive by RT-PCR test. The left amplification curve shows the conjunctiva sample and the right one shows the amplification curve of the oro-nasopharynx sample of the same patient. Respectively from top to bottom: (A) conjunctiva positive and (B) oro-nasopharynx negative patient (1st patient in Table 2 ), (C) conjunctiva positive and (D) oro-nasopharynx positive patient (19th patient in Table 2 ) and (E) conjunctiva negative and (F) oro-nasopharynx positive patient (34th patient in Table 2 ) The cycle threshold values of the SARS-CoV-2 positive subjects by gender, age, number of symptoms and the conjunctiva and oro-nasopharynx swab results Table 3 ). The demonstration of SARS CoV-2 virus receptors on the conjunctiva and cornea has shown that virus spread through the ocular surface could be possible. 1 In addition, the detection of the virus in conjunctiva swabs and tears has resulted in speculation that the virus could spread with ocular secretions. 6 Another view on such spread via the conjunctiva is that the cause is viruses passing to the nasal mucosa from the conjunctiva through the nasolacrimal canal. 8, 9 The fact that the virus itself can cause keratoconjunctivitis 10 We were able to detect a higher percentage of cases with the conjunctiva swab than the ONP swab in this study. However, the percentage of cases that both the conjunctiva sample and the ONP sample could detect was relatively low. This indicates that different factors are at work for the detection of SARS CoV-2 by the two methods. One of these factors could be the difference in the period when the virus is present on the ONP and the conjunctiva. There are reports that the conjunctiva samples are more frequently positive in the early period. 13, 14 In addition, the different peak times of the viral load in oral and nasal swabs could indicate that the conjunctival peak time could differ from the pharyngeal peak time. 6, 15 Considering that the patients included in our study are early stage patients, the fact that conjunctival samples can detect more cases than the ONP sample may make the conjunctival sample taken in the early period valuable. Another possible cause is the technical difficulties encountered while obtaining an ONP swab. An ONP sample is obtained without seeing the swab's tip and it is possible that the swab tip does not adequately contact the mucosa surface. However, the technician can fully visualize the contact between the swab tip and the mucosa when obtaining the conjunctiva swab. The nasopharyngeal swab is used as the gold standard in detecting the disease 16 but false-negative results are also possible. 17, 18 The test sensitivity can vary depending on the duration since disease onset and the disease severity, necessitating the use of other swab sites. 19 It is obvious that the new site should not make it more difficult to obtain swabs than from an ONP swab. The fact that it is essential to detect asymptomatic cases for the control of the disease could make a conjunctiva swab more desirable in outpatient departments with few resources and in population screening as it is quite easy to take and has a low cost. Obtaining a conjunctiva swab simultaneously with the ONP swab, while using separate swabs, could increase the sensitivity, especially in cases known to have contacted a SARS CoV-2 patient or who have presented to the outpatient department for the first time. Obtaining an ONP swab can be difficult as it commonly causes signs of irritation such as coughing and nausea in the patient and can therefore result in false negatives. 20 The patients could show much less resistance to a conjunctiva swab test as it is localized to the eye and causes relatively less irritation. The large number of droplets that are distributed to the environment when obtaining an ONP sample is also important regarding the spread of the disease. 21 Spread via droplets is much less important with a conjunctiva sample. A conjunctiva sample can also be a practical alternative for patients who cannot tolerate the ONP procedure or who have a structural deformity in the airway passage leading to the ONP. The pulmonary functions have been reported to be better in intensive care patients when both the conjunctiva and ONP samples are negative compared to those when both are positive. 22 The mean and a positive conjunctiva swab test to be seen at the early stage. A limitation of our study is that our subjects' symptoms were subjective. However, it is not possible to conduct tests for most symptoms. Another limitation of our study is that the quarantine applied to SARS-CoV-2 patients in our country and the outpatient follow-up of the patients who participated in our study prevented the follow-up of the patients and the detection of false positivity and false negativity status. In conclusion, it is possible that the SARS CoV-2 virus becomes positive in the conjunctiva sample at an earlier stage than the pharynx sample. A conjunctiva swab test can therefore be an The distribution of the gender, age, and symptoms at presentation of the subjects according to the oro-nasopharyngeal and conjunctival swab results alternative method for patients who present to the outpatient department for the first time or in population screening (early stage patients). In addition, it can be an easy, inexpensive, and low-risk alternative in patients with low tolerance to the ONP swab procedure and whose upper respiratory pathway is not anatomically suitable. We also believe preferring conjunctiva swabs is important as regards decreasing the risk of spread to the health care worker taking the sample. The tests used in this study will need to be performed on subjects of various races and using various devices and test kits to support these results. Studies conducted on larger study populations are also needed. We would like to acknowledge the Biyoeksen Ar-Ge Tekn. Ltd. company due to their RT-PCR kits donated for our use utilized in this study. The authors declare that there are no conflict of interests. The peer review history for this article is available at https://publons. com/publon/10.1002/jmv.26981 The data that support the findings of this study are available from the corresponding author upon reasonable request. 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