Volume 26, Number 11—November 2020 Research Let
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Volume 26, Number 11—November 2020
Research Letter
Saliva Alternative to Upper Respiratory Swabs for SARS-CoV-2 Diagnosis
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Rachel L. Byrne, Grant A. Kay, Konstantina Kontogianni, Ghaith Aljayyoussi, Lottie Brown, Andrea M. Collins, Luis E. Cuevas, Daniela M. Ferreira, Alice J. Fraser, Gala Garrod, Helen Hill, Grant L. Hughes, Stefanie Menzies, Elena Mitsi, Sophie I. Owen, Edward I. Patterson, Christopher T. Williams, Angela Hyder-Wright, Emily R. AdamsComments to Author , and Ana I. Cubas-Atienzar
Author affiliations: Liverpool School of Tropical Medicine, Liverpool, UK (R.L. Byrne, G.A. Kay, K. Kontogianni, G. Aljayyoussi, L. Brown, A.M. Collins, L.E. Cuevas, D.M. Ferreira, A.J. Fraser, G. Garrod, H. Hill, G.L. Hughes, S. Menzies, E. Mitsi, S.I. Owen, E.I. Patterson, C.T. Williams, A. Hyder-Wright, E.R. Adams, A.I. Cubas-Atienzar); National Institute for Health Research, Leeds, UK (A.M. Collins, H. Hill, A. Hyder-Wright); Liverpool University Hospitals National Health Services Foundation Trust, Liverpool (A.M. Collins, H. Hill, A. Hyder-Wright)
Cite This Article
Abstract
PCR of upper respiratory specimens is the diagnostic standard for severe acute respiratory syndrome coronavirus 2 infection. However, saliva sampling is an easy alternative to nasal and throat swabbing. We found similar viral loads in saliva samples and in nasal and throat swab samples from 110 patients with coronavirus disease.
Quantitative reverse transcription PCR (qRT-PCR) is the diagnostic standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease (COVID-19) (1). Testing usually is conducted on upper respiratory specimens collected using swabs (1–3). However, this method requires multiple samples and has a low sensitivity (4). Swab sampling can cause patients to cough or sneeze, uncomfortable reactions that might also increase transmission risks to healthcare workers (A. Wyllie et al., unpub. data, https://doi.org/10.1101/2020.04.16.20067835External Link). Sampling technique proficiency also varies, especially during self-sampling (A. Wyllie et al., unpub. data, https://doi.org/10.1101/2020.04.16.20067835External Link), which can result in false negatives. Furthermore, shortages of swabs, transport media, and personal protective equipment limit healthcare capacity to conduct SARS-CoV-2 tests that rely on swab sampling.
Saliva sampling is a noninvasive alternative to upper respiratory swabbing. We compared paired self-collected saliva samples with healthcare worker–collected nasal and throat swab specimens from 110 patients with suspected SARS-CoV-2 infection. This analysis was part of a prospective study (Facilitating a SARS CoV-2 Test for Rapid Triage) at the Royal Liverpool University and Aintree University Hospitals (Liverpool, UK). We recruited participants who had provided written informed consent and had COVID-19 symptoms. The National Health Service Research Ethics Committee (20/SC/0169) approved the study under Integrated Research Application System no. 282147.
Within 24 hours after patient consent, we collected nasal and throat swab specimens containing 1.0 mL of Amies transport medium (COPAN Diagnostics, https://www.copanusa.comExternal Link). We also asked participants to funnel their saliva into a sterile cryotube (SARSTEDT, https://www.sarstedt.comExternal Link). We immediately extracted RNA from the swab samples; we stored saliva samples at –80°C until processing. We extracted viral RNA using the QIAamp Viral RNA Mini Kit (QIAGEN, https://www.qiagen.comExternal Link) and tested 8 μL of extracted RNA using the genesig Real-Time Coronavirus COVID-19 PCR (genesig, https://www.genesig.comExternal Link). We quantified viral loads using the manufacturer’s positive control (1.67 × 105 copies/µL) as reference.
Viral load (copies/mL) of SARS-CoV-2 RNA recovered from paired saliva samples and nasal and throat swab specimens from 14 patients with coronavirus disease, United Kingdom, 2020. Viral loads are shown on a logarithmic scale. NS, not significant; NT, nasal and throat; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
Figure. Viral load (copies/mL) of SARS-CoV-2 RNA recovered from paired saliva samples and nasal and throat swab specimens from 14 patients with coronavirus disease, United Kingdom, 2020. Viral loads are shown...
Of the 110 adults recruited from April through June 2020, a total of 61 (55.5%) were women. Most participants were hospitalized; 21 (19.1%) were discharged to home directly from the emergency department. Overall, 12 (10.9%) saliva and 14 (12.7%) nasal and throat swab specimens of 110 paired samples tested positive for SARS-CoV-2 RNA. Viral loads for all samples ranged from 36 to 3.3 × 106 copies/mL. Overall viral loads were similar among all positive samples (Figure).
Insignificant viral load discrepancies existed among all positive samples (p = 0.1955 by Wilcoxon signed-rank paired test). Two patients tested positive (<10 copies/mL) on nasal and throat swab samples and negative on saliva samples; the discrepancies might have resulted from the different processing times of the 2 specimens because freeze-thawing can reduce the stability of RNA (5,6).
Saliva sampling can improve SARS-CoV-2 diagnostic techniques. Saliva samples are easier to collect than nasal and throat samples; the technique is noninvasive, presumably preferred by the participant, and does not require sampling proficiency. In addition, saliva sampling does not require swabs and transport media, which have limited availability during the pandemic. Our technique uses a funnel which, although helpful, might not be necessary for sample collection. Our study focused on symptomatic hospitalized participants; further research is needed on saliva sampling for patients with mild and asymptomatic SARS-CoV-2 infection.
Further studies should document the effects of storage and transport on RNA and viral loads. Rapid processing of saliva samples might benefit patients in low- and middle-income countries, where the pandemic is still accelerating and swab availability is limited (7). Furthermore, high-income countries can establish a cold chain for sample transportation. A cold chain could enable home sampling and screening of children who have rejected swabbing. It could also streamline research studies that require repeat sampling.
As rates of SARS-CoV-2 infection increase, we must continue to investigate efficient diagnostic strategies. Easy and effective diagnostic techniques, such as saliva sampling, should be evaluated in certified clinical laboratories.
Ms. Byrne is a doctoral candidate at the Liverpool School of Tropical Medicine, Liverpool. Her primary research interest is the application of new molecular diagnostics for the detection of emerging infectious diseases.
Top
Acknowledgments
We are grateful to the participants in the Facilitating A SARS CoV-2 Test for Rapid Triage study for their involvement in this research. We also thank the research nurses from Liverpool University Hospitals National Health Services Foundation Trust and National Institute for Health Research and the Liverpool School of Tropical Medicine team that assisted with the sample collection and processing. We acknowledge the advice, guidance, and community of the Clinical Research Network in the North West Coast.
This study was financially supported by the Department for International Development/Wellcome Trust Epidemic Preparedness coronavirus grant no. 220764/Z/20/Z and Pfizer grant no. WI255862 (D.M.F., E.M., A.C.). E.R.A. and L.E.C. are funded by the National Institute for Health Research Health Protection Research Unit in Emerging and Zoonotic Infections, the Centre of Excellence in Infectious Diseases Research, and the Alder Hey Charity. We also acknowledge the financial support of Liverpool Health Partners and the Liverpool Malawi COVID-19 consortium.
This article was preprinted at https://www.medrxiv.org/content/10.1101/2020....20149534v1.
Top
References
World Health Organization. Laboratory testing strategy recommendations for COVID-19. 2020 [cited 2020 July 1]. https://www.who.int/publications/i/item/labor...ceExternal Link
Centers for Disease Control and Prevention. Interim guidelines for collecting, handling, and testing clinical specimens from persons for coronavirus disease 2019 (COVID-19). 2020 [cited 2020 Jun 22]. https://www.cdc.gov/coronavirus/2019-ncov/lab...imens.html
To KKW, Tsang OTY, Leung WS, Tam AR, Wu TC, Lung DC, et al. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study. Lancet Infect Dis. 2020;20:565–74. DOIExternal LinkPubMedExternal Link
Zhang W, Du RH, Li B, Zheng XS, Yang XL, Hu B, et al. Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes. Emerg Microbes Infect. 2020;9:386–9. DOIExternal LinkPubMedExternal Link
Ji X, Wang M, Li L, Chen F, Zhang Y, Li Q, et al. The impact of repeated freeze–thaw cycles on the quality of biomolecules in four different tissues. Biopreserv Biobank. 2017;15:475–83. DOIExternal LinkPubMedExternal Link
Kuang J, Yan X, Genders AJ, Granata C, Bishop DJ. An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research. PLoS One. 2018;13:e0196438. DOIExternal LinkPubMedExternal Link
Gilbert M, Pullano G, Pinotti F, Valdano E, Poletto C, Boëlle PY, et al. Preparedness and vulnerability of African countries against importations of COVID-19: a modelling study. Lancet. 2020;395:871–7. DOIExternal LinkPubMedExternal Link
Top
Figure
Figure. Viral load (copies/mL) of SARS-CoV-2 RNA recovered from paired saliva samples and nasal and throat swab specimens from 14 patients with coronavirus disease, United Kingdom, 2020. Viral loads are shown......
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Cite This Article
DOI: 10.3201/eid2611.203283
Original Publication Date: September 11, 2020
Table of Contents – Volume 26, Number 11—November 2020
Comments
Please use the form below to submit correspondence to the authors or contact them at the following address:
Emily R. Adams, Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK
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Page created: August 10, 2020
Page updated: October 19, 2020 1:43 PM EDT
Page reviewed: October 19, 2020 1:43 PM EDT
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
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Research Letter
Saliva Alternative to Upper Respiratory Swabs for SARS-CoV-2 Diagnosis
On This Page
Research Letter
Cite This Article
Figures
Figure
Downloads
Article
RIS [TXT - 2 KB]
Altmetric
Article has an altmetric score of 158
Metric Details
Related Articles
TB and COVID-19 Transmission and Control
COVID-19 Infection Control Measures in Long-Term Care Facility, Pennsylvania, USA
Intrauterine Transmission of SARS-CoV-2
More articles on Coronavirus, COVID-19
Rachel L. Byrne, Grant A. Kay, Konstantina Kontogianni, Ghaith Aljayyoussi, Lottie Brown, Andrea M. Collins, Luis E. Cuevas, Daniela M. Ferreira, Alice J. Fraser, Gala Garrod, Helen Hill, Grant L. Hughes, Stefanie Menzies, Elena Mitsi, Sophie I. Owen, Edward I. Patterson, Christopher T. Williams, Angela Hyder-Wright, Emily R. AdamsComments to Author , and Ana I. Cubas-Atienzar
Author affiliations: Liverpool School of Tropical Medicine, Liverpool, UK (R.L. Byrne, G.A. Kay, K. Kontogianni, G. Aljayyoussi, L. Brown, A.M. Collins, L.E. Cuevas, D.M. Ferreira, A.J. Fraser, G. Garrod, H. Hill, G.L. Hughes, S. Menzies, E. Mitsi, S.I. Owen, E.I. Patterson, C.T. Williams, A. Hyder-Wright, E.R. Adams, A.I. Cubas-Atienzar); National Institute for Health Research, Leeds, UK (A.M. Collins, H. Hill, A. Hyder-Wright); Liverpool University Hospitals National Health Services Foundation Trust, Liverpool (A.M. Collins, H. Hill, A. Hyder-Wright)
Cite This Article
Abstract
PCR of upper respiratory specimens is the diagnostic standard for severe acute respiratory syndrome coronavirus 2 infection. However, saliva sampling is an easy alternative to nasal and throat swabbing. We found similar viral loads in saliva samples and in nasal and throat swab samples from 110 patients with coronavirus disease.
Quantitative reverse transcription PCR (qRT-PCR) is the diagnostic standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease (COVID-19) (1). Testing usually is conducted on upper respiratory specimens collected using swabs (1–3). However, this method requires multiple samples and has a low sensitivity (4). Swab sampling can cause patients to cough or sneeze, uncomfortable reactions that might also increase transmission risks to healthcare workers (A. Wyllie et al., unpub. data, https://doi.org/10.1101/2020.04.16.20067835External Link). Sampling technique proficiency also varies, especially during self-sampling (A. Wyllie et al., unpub. data, https://doi.org/10.1101/2020.04.16.20067835External Link), which can result in false negatives. Furthermore, shortages of swabs, transport media, and personal protective equipment limit healthcare capacity to conduct SARS-CoV-2 tests that rely on swab sampling.
Saliva sampling is a noninvasive alternative to upper respiratory swabbing. We compared paired self-collected saliva samples with healthcare worker–collected nasal and throat swab specimens from 110 patients with suspected SARS-CoV-2 infection. This analysis was part of a prospective study (Facilitating a SARS CoV-2 Test for Rapid Triage) at the Royal Liverpool University and Aintree University Hospitals (Liverpool, UK). We recruited participants who had provided written informed consent and had COVID-19 symptoms. The National Health Service Research Ethics Committee (20/SC/0169) approved the study under Integrated Research Application System no. 282147.
Within 24 hours after patient consent, we collected nasal and throat swab specimens containing 1.0 mL of Amies transport medium (COPAN Diagnostics, https://www.copanusa.comExternal Link). We also asked participants to funnel their saliva into a sterile cryotube (SARSTEDT, https://www.sarstedt.comExternal Link). We immediately extracted RNA from the swab samples; we stored saliva samples at –80°C until processing. We extracted viral RNA using the QIAamp Viral RNA Mini Kit (QIAGEN, https://www.qiagen.comExternal Link) and tested 8 μL of extracted RNA using the genesig Real-Time Coronavirus COVID-19 PCR (genesig, https://www.genesig.comExternal Link). We quantified viral loads using the manufacturer’s positive control (1.67 × 105 copies/µL) as reference.
Viral load (copies/mL) of SARS-CoV-2 RNA recovered from paired saliva samples and nasal and throat swab specimens from 14 patients with coronavirus disease, United Kingdom, 2020. Viral loads are shown on a logarithmic scale. NS, not significant; NT, nasal and throat; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
Figure. Viral load (copies/mL) of SARS-CoV-2 RNA recovered from paired saliva samples and nasal and throat swab specimens from 14 patients with coronavirus disease, United Kingdom, 2020. Viral loads are shown...
Of the 110 adults recruited from April through June 2020, a total of 61 (55.5%) were women. Most participants were hospitalized; 21 (19.1%) were discharged to home directly from the emergency department. Overall, 12 (10.9%) saliva and 14 (12.7%) nasal and throat swab specimens of 110 paired samples tested positive for SARS-CoV-2 RNA. Viral loads for all samples ranged from 36 to 3.3 × 106 copies/mL. Overall viral loads were similar among all positive samples (Figure).
Insignificant viral load discrepancies existed among all positive samples (p = 0.1955 by Wilcoxon signed-rank paired test). Two patients tested positive (<10 copies/mL) on nasal and throat swab samples and negative on saliva samples; the discrepancies might have resulted from the different processing times of the 2 specimens because freeze-thawing can reduce the stability of RNA (5,6).
Saliva sampling can improve SARS-CoV-2 diagnostic techniques. Saliva samples are easier to collect than nasal and throat samples; the technique is noninvasive, presumably preferred by the participant, and does not require sampling proficiency. In addition, saliva sampling does not require swabs and transport media, which have limited availability during the pandemic. Our technique uses a funnel which, although helpful, might not be necessary for sample collection. Our study focused on symptomatic hospitalized participants; further research is needed on saliva sampling for patients with mild and asymptomatic SARS-CoV-2 infection.
Further studies should document the effects of storage and transport on RNA and viral loads. Rapid processing of saliva samples might benefit patients in low- and middle-income countries, where the pandemic is still accelerating and swab availability is limited (7). Furthermore, high-income countries can establish a cold chain for sample transportation. A cold chain could enable home sampling and screening of children who have rejected swabbing. It could also streamline research studies that require repeat sampling.
As rates of SARS-CoV-2 infection increase, we must continue to investigate efficient diagnostic strategies. Easy and effective diagnostic techniques, such as saliva sampling, should be evaluated in certified clinical laboratories.
Ms. Byrne is a doctoral candidate at the Liverpool School of Tropical Medicine, Liverpool. Her primary research interest is the application of new molecular diagnostics for the detection of emerging infectious diseases.
Top
Acknowledgments
We are grateful to the participants in the Facilitating A SARS CoV-2 Test for Rapid Triage study for their involvement in this research. We also thank the research nurses from Liverpool University Hospitals National Health Services Foundation Trust and National Institute for Health Research and the Liverpool School of Tropical Medicine team that assisted with the sample collection and processing. We acknowledge the advice, guidance, and community of the Clinical Research Network in the North West Coast.
This study was financially supported by the Department for International Development/Wellcome Trust Epidemic Preparedness coronavirus grant no. 220764/Z/20/Z and Pfizer grant no. WI255862 (D.M.F., E.M., A.C.). E.R.A. and L.E.C. are funded by the National Institute for Health Research Health Protection Research Unit in Emerging and Zoonotic Infections, the Centre of Excellence in Infectious Diseases Research, and the Alder Hey Charity. We also acknowledge the financial support of Liverpool Health Partners and the Liverpool Malawi COVID-19 consortium.
This article was preprinted at https://www.medrxiv.org/content/10.1101/2020....20149534v1.
Top
References
World Health Organization. Laboratory testing strategy recommendations for COVID-19. 2020 [cited 2020 July 1]. https://www.who.int/publications/i/item/labor...ceExternal Link
Centers for Disease Control and Prevention. Interim guidelines for collecting, handling, and testing clinical specimens from persons for coronavirus disease 2019 (COVID-19). 2020 [cited 2020 Jun 22]. https://www.cdc.gov/coronavirus/2019-ncov/lab...imens.html
To KKW, Tsang OTY, Leung WS, Tam AR, Wu TC, Lung DC, et al. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study. Lancet Infect Dis. 2020;20:565–74. DOIExternal LinkPubMedExternal Link
Zhang W, Du RH, Li B, Zheng XS, Yang XL, Hu B, et al. Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes. Emerg Microbes Infect. 2020;9:386–9. DOIExternal LinkPubMedExternal Link
Ji X, Wang M, Li L, Chen F, Zhang Y, Li Q, et al. The impact of repeated freeze–thaw cycles on the quality of biomolecules in four different tissues. Biopreserv Biobank. 2017;15:475–83. DOIExternal LinkPubMedExternal Link
Kuang J, Yan X, Genders AJ, Granata C, Bishop DJ. An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research. PLoS One. 2018;13:e0196438. DOIExternal LinkPubMedExternal Link
Gilbert M, Pullano G, Pinotti F, Valdano E, Poletto C, Boëlle PY, et al. Preparedness and vulnerability of African countries against importations of COVID-19: a modelling study. Lancet. 2020;395:871–7. DOIExternal LinkPubMedExternal Link
Top
Figure
Figure. Viral load (copies/mL) of SARS-CoV-2 RNA recovered from paired saliva samples and nasal and throat swab specimens from 14 patients with coronavirus disease, United Kingdom, 2020. Viral loads are shown......
Top
Cite This Article
DOI: 10.3201/eid2611.203283
Original Publication Date: September 11, 2020
Table of Contents – Volume 26, Number 11—November 2020
Comments
Please use the form below to submit correspondence to the authors or contact them at the following address:
Emily R. Adams, Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK
Return Address
Send To
Send To Authors Editors
Comments
10000 character(s) remaining.
Top
Page created: August 10, 2020
Page updated: October 19, 2020 1:43 PM EDT
Page reviewed: October 19, 2020 1:43 PM EDT
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
About the Journal
Articles
December 2020
Early Release
Past Issues
November 2020
Medscape CME
Search
Subscribe
Author Resource Center
Peer Reviewers
Podcasts
Media Related Content
HAVE QUESTIONS?
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Email CDC-INFO
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