key: cord-0840522-bj1idnf6
authors: Akgun Dogan, O.; Kose, B.; Agaoglu, N. B.; Yildiz, J.; Alkurt, G.; Kendir Demirkol, Y.; Irvem, A.; Dinler-Doganay, G.; Doganay, L.
title: Does sampling saliva increase detection of SARS-CoV-2 by RT-PCR? Comparing saliva with oro-nasopharyngeal swabs
date: 2020-07-28
journal: nan
DOI: 10.1101/2020.07.26.20158618
sha: 7a8ea9018434f00d1e9d72c5fcbb6eb96da6a50a
doc_id: 840522
cord_uid: bj1idnf6

The gold standard method in the diagnosis of SARS-CoV-2 infection is the detection of viral RNA in nasopharyngeal sample by RT-PCR. Recently, saliva samples has been suggested as an alternative due to being fast, reliable and non-invasive, rather than nasopharyngeal samples. We compared RT-PCR results in nasopharyngeal, oro-nasopharyngeal and saliva samples of COVID-19 patients. 98 of 200 patients were positive in RT-PCR analysis performed before the hospitalization. In day 0, at least one sample was positive in 67% of 98 patients. Positivity rate was 83% for both oro-nasopharyngeal and nasopharyngeal samples, while it was 63% for saliva samples (p<0.001). On day 5, RT-PCR was performed in 59 patients, 34% had at least one positive result. The positivity rate was 55% for saliva and nasopharyngeal samples, while it was 60% for oro-nasopharyngeal samples. Our study shows that the sampling saliva does not increase the sensitivity of RT-PCR tests at early stages of infection. However, on 5th day, viral RNA detection rates in saliva were similar to nasopharyngeal and oro-nasopharyngeal samples. In conclusion, we suggest that, in patients receiving treatment, virus presence in saliva, in addition to the standard samples, is important to determine the isolation period and to control the transmission.

3

The SARS CoV-2 infection, which has influenced the world since the end of 2019, causes serious problems in both health and socio-economic fields. 1, 2 Currently, it has affected approximately 6 million people over more than 200 countries. 3 SARS-CoV-2 infection can cause serious lower respiratory tract infections that can be fatal in some patients, however, many individuals remain asymptomatic during the infection. 4, 5 Especially asymptomatic individuals have been a major factor in increasing the transmission rate of the disease and evolving it into a pandemic.

COVID-19 diagnosis is based on clinical findings, besides, the detection of the virus in patients' specimens is of great importance in terms of monitoring the disease, guidance of treatment, and infection control, 6 Quarantine, which is launched all over the world urging people to stay home, is considered as the only way to reduce transmission, but it started to cause serious social and economic problems due to loss of labor. 7, 8 This situation reveals the need for a fast, reliable, easily applicable, and non-invasive test that quickly identifies infected individuals to be isolated. Currently, gold standard method in the diagnosis of SARS-CoV-2 infection is the detection of viral RNA in the nasopharyngeal swab sample by Real Time Polymerase Chain Reaction (RT-PCR) analysis. 9 The most important disadvantage of this method is the presence of limited trained personnel available in sampling during the outbreak. High risk of nosocomial infections that such personnel are exposed to is an additional obstacle. Moreover, in terms of patients, discomfort experienced, especially, in repeated tests is the most frequently reported problem.

The abovementioned disadvantages have led the researchers to study on non-invasive, easy to selfapplicable sampling methods for massive screening. [10] [11] [12] [13] [14] As the main source of transmission of SARS-CoV-2 infection is salivary droplets, viral RNA RT-PCR in saliva samples have been suggested as possible alternative testing for diagnosis. 15 The main advantages of saliva sampling are self-availability, no need for specialized staff and the comfort of the procedure. However, although turnaround time and self-applicability of the tests are important, the reliability of the sampling method to be chosen should also be tested very well. It is obvious that the use of tests with a high false negativity rate will adversely affect the course of the pandemic.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 28, 2020. Here, we compared the results of RT-PCR in nasopharyngeal, oro-nasopharyngeal, and saliva samples in patients diagnosed with COVID-19 to investigate their possible relationships with clinical findings.

A cross-sectional study was conducted in repurposed Genomic Laboratory (GLAB), Umraniye

Teaching and Research Hospital, in Istanbul, with a total of 200 consecutive patients who met the possible case definition for COVID-19 and hospitalized with moderate-severe disease. 16 According to is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 28, 2020. . https://doi.org/10.1101/2020.07.26.20158618 doi: medRxiv preprint Patients admitted to ICU, not giving consent to study, incapable of providing saliva sample and patients under age of 18 were excluded.

In all patients, an oro-nasopharyngeal sample was taken with a cotton swab used for the viral RT-PCR test before hospitalization as a standard diagnostic approach. In the scope of our study, oronasopharyngeal samples were taken with cotton swab and nasopharyngeal samples with dacron swab.

Details of the sampling processes are given in Figure 1 . Before the saliva collection, participants were given brief explanations about the difference between saliva and sputum, then they were asked to give saliva samples prior to other samples by a drooling technique. They spit approximately 1 mL into the falcon tubes containing the viral transport medium (VTM, Innomed VTM001) used in standard sampling. Single trained healthcare professional took samples to avoid possible variations in the collection technique. All samples were transferred to our laboratory within 1 hour of sampling stored in the refrigerator, and RT-PCR was performed on the day they were collected.

ORF1ab and N gene of SARS-CoV-2 were targeted for the diagnosis of the infected patients. For this purpose, The Direct Detection of SARS-CoV-2 Detection Kit was used (Coyote Bioscience Co., Ltd).

The kit procedure was based on the detection of the conserved region of ORF1ab coated with a pair of specific primers and a fluorescently labelled probe, and the N gene of SARS-CoV-2 by the RT-PCR method. Since this kit did not need a separate RNA extraction, the samples in the VTM medium had briefly vortexed and taken directly into the study. Biorad CFX 96 Real-Time PCR systems were used.

FAM channel for ORFlab gene, ROX channel for N gene, and HEX channel for internal RNase P gene of human control were selected. These channels should have the logarithmic growth period with the Ct value ≤29, for a positive result.

In the RT-PCR results of the samples, the internal RNase P gene was positive for all samples. If both the ORFlab and the N gene were positive, the result was considered as presumptive positive. If both the . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 28, 2020. . https://doi.org/10.1101/2020.07.26.20158618 doi: medRxiv preprint 6 ORFlab and the N gene were negative, the result was considered as presumptive negative. If one of them was positive and the other was negative, the test for this sample was repeated. If the same result was achieved again, a new nasopharyngeal and oropharyngeal swab was requested. In the RT-PCR analysis on day 0, of 102 patients with a previous negative result, 95 remained as negative, while in 7 (6·9%) patients positive RT-PCR results obtained in at least one sample type ( Figure   . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 28, 2020. . https://doi.org/10.1101/2020.07.26.20158618 doi: medRxiv preprint 2). While the oro-nasopharyngeal sample was positive in all 7 patients, nasopharyngeal samples were positive in 3. There was only one patient whose saliva sample was positive. Oro-nasopharyngeal and nasopharyngeal samples in this patient were also positive.

On day 5, we were able to collect samples in 59 of 200 (30%) patients. Of 59 patients, 20 (34%) had at least one positive result in saliva or in one of the swab samples. The sensitivity rate was determined as 11/20 (55%) for both saliva and nasopharyngeal samples, while it was 12/20 (60%) for oronasopharyngeal samples (Figure 3b ). In the statistical analysis, there was no difference between the sample types in terms of the sensitivity (p=0.368). The mean Ct values determined for FAM and ROX were shown in Figure 4 . On day 5, there were 5 patients who showed RT-PCR positivity only on the saliva sample. These patients had longer prior history with positive RT-PCR results in oronasopharyngeal samples, home-treated with hydroxychloroquine for five days, clinically deteriorated with radiological findings, tested positive again in oro-nasopharyngeal samples and hospitalized.

Our study demonstrates that saliva sampling did not improve diagnostic sensitivity in patients who had a negative result in initial testing before hospitalization. Our results also revealed that at the early stages of the infection, saliva sampling had a lower sensitivity to detect viral RNA compared to other sampling methods. In contrast to our findings, three recent studies suggest that the results of RT-PCR analysis in saliva samples are compatible with the results of the nasopharyngeal and oro-nasopharyngeal samples 11, 13, 14 . These studies had either limited number of patients (23 and 12 patients) or limited number of RT-PCR positive patients (21 RT-PCR positive patients). In this study we consecutively recruited 200 inpatients who presented with clinical signs compatible with COVID-19 and at initial testing 98 of them revealed SARS-COV-2 RT-PCR positivity. In this study the sensitivity rate for saliva samples was 63% and this was significantly lower than nasopharyngeal and oro-nasopharyngeal swabs. However, our data on the 5th day showed that the viral RNA detection rates in saliva samples were similar to those of nasopharyngeal and oro-nasopharyngeal samples. Two recent studies supported our results and showed that SARS-CoV-2 detection from saliva was more consistent during extended hospitalization and recovery 11, 14 . Thus, we suggest that RT-PCR analysis in saliva samples may be beneficial when a . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 28, 2020. . https://doi.org/10.1101/2020.07.26.20158618 doi: medRxiv preprint 8 follow up test beyond day 5 is needed especially in hospitalized patients (e.g. before discharge).

Compared to nasopharyngeal swabs, sampling saliva causes less discomfort in patients and reduces risk for nosocomial infection among healthcare workers as patients can give saliva samples by themselves.

On day five, out of 59 patients, 5 (8%) had detectable viral RNA only in saliva sample. The fact that all of these patients were using hydroxychloroquine prior to hospitalization, this may have hindered the detection of the virus in nasopharyngeal and oro-nasopharyngeal samples. With this result, we suggest that taking saliva samples along with the standard method before ending the isolation in individuals treated with hydroxychloroquine can be effective in minimizing contamination in the community by reducing the false negativity rate. In addition, we recommend saliva sampling in patients who show progression despite at-home treatment. Such an approach would increase the detection of positive cases, thereby enabling more accurate planning of treatment, follow-up, and subsequent discharge.

One of the most important issues affecting false negative rates in standard nasopharyngeal sampling is the use of inappropriate techniques. In our study, taking all samples by single trained healthcare staff is the strength of our study and enabled us to achieve a safer result. The most important limitation of our study is that no study has been performed in asymptomatic individuals and mild cases that do not require hospitalization. Therefore, it was not possible to make a comment about whether saliva samples can be used in screening.

In conclusion, our study shows that the saliva sample is not as sensitive as standard nasopharyngeal swabs in determining viral RNA and it does not improve detection rate in PCR negative patient group.

However, in the later stage of the disease, RT-PCR test from saliva samples might help detecting deteriorating patients or determining the isolation period more effectively after treatment.

Funding: None . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 28, 2020. . https://doi.org/10.1101/2020.07.26.20158618 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 28, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 28, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 28, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 28, 2020. . https://doi.org/10.1101/2020.07.26.20158618 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted July 28, 2020. . https://doi.org/10.1101/2020.07.26.20158618 doi: medRxiv preprint Table 2 : RT-PCR study results of saliva, oronasopharyngeal and nasopharyngeal samples on day 0 in 98 patients who were found positive in RT-PCR analysis before hospitalization. Table 3 : RT-PCR study results of saliva, oro-nasopharyngeal and nasopharyngeal samples on day 5 in 59 patients. We would like to remind that of 59 patients, 20 (34%) had at least one positive result in saliva or swab sample.

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