Molecular Detection of SARS-CoV-2 From Oropharyngeal Swabs Performed With or Without Specimen Collection From the Palatine Tonsils - a Multicenter Randomized Controlled Trial
Phase: NA
Conditions: SARS-CoV Infection, Coronavirus, Corona Virus Infection
Sponsor: Rigshospitalet, Denmark

 
1 
Molecular detection of SARS-CoV-2 from oropharyngeal 
swabs performed with or without specimen collection 
from the palatine tonsils – a research proposal for a 
multicenter randomized controlled trial 
 
Benedikte Hartvigsen1,2, Kathrine Kronberg Jakobsen1,3, Thomas Benfield3,4, Niels Tobias Gredal, BSN5, Annette 
Kjær Ersbøll5,6, Mathias Peter Grønlund1, Nina Steenhard7, Christian von Buchwald1,3 and Tobias Todsen1,2,3 
 
 
1Department of Otorhinolaryngology, Head and Neck Surgery and Audiology, Rigshospitalet – Copenhagen 
University Hospital, Copenhagen, Denmark.   
2Copenhagen Academy for Medical Education and Simulation, Capital Region, Copenhagen, Denmark.   
3Department of Clinical Medicine, University of Copenhagen, Denmark.   
4Department of Infectious Diseases, Copenhagen University Hospital, Amager and Hvidovre, Hvidovre, 
Denmark.   
5Copenhagen Emergency Medical Services. University of Copenhagen. Copenhagen. Denmark   
6National Institute of Public Health, University of Southern Denmark, Copenhagen, Denmark.   
7TestCenter Danmark, Statens Serum Institut, Copenhagen, Denmark. 
 
Contact information: 
 
Tobias Todsen, MD, PhD, Associate Professor 
 
Department of Otorhinolaryngology, Head and Neck Surgery and Audiology, Rigshospitalet, 
Copenhagen University Hospital, DK-2100 Copenhagen, Denmark. 
Tobias.todsen@regionh.dk 
 
 
 
Clinical trial number: NCT05611203 
 
Date for latest revision: 21/12 2022 
 
 

 
2 
Table of content 
 
INTRODUCTION ........................................................................................................................................................ 3 
BACKGROUND AND RATIONALE ............................................................................................................................................. 3 
RESEARCH QUESTION .......................................................................................................................................................... 4 
METHODS ................................................................................................................................................................. 5 
STUDY SETTING .................................................................................................................................................................. 5 
ELIGIBILITY CRITERIA ........................................................................................................................................................... 5 
RANDOMIZATION TO INTERVENTION AND CONTROL GROUP ........................................................................................................ 6 
OUTCOMES ....................................................................................................................................................................... 7 
SAMPLE SIZE ...................................................................................................................................................................... 7 
STATISTICAL ANALYSIS ......................................................................................................................................................... 8 
QUALITY ASSURANCE .......................................................................................................................................................... 9 
FOLLOW-UP ...................................................................................................................................................................... 9 
ETHICS AND SAFETY CONSIDERATIONS ................................................................................................................................... 10 
DATA PLAN ..................................................................................................................................................................... 10 
DECLARATION OF INTERESTS ............................................................................................................................................... 10 
ACCESS TO DATA .............................................................................................................................................................. 10 
APPENDICES ............................................................................................................................................................ 11 
APPENDIX 1: QUALITY ASSURANCE CHECKLISTS ...................................................................................................................... 11 
APPENDIX 2: SURVEY FOR PARTICIPANTS ............................................................................................................................... 13 
APPENDIX 3: REDCAP DATABASE ........................................................................................................................................ 14 
APPENDIX 4: PARTICIPANT INFORMATION ............................................................................................................................. 18 
APPENDIX 5: MALLAMPATI SCORE ....................................................................................................................................... 21 
APPENDIX 6: STATISTICAL ANALYSIS PLAN (SAP) ................................................................................................................... 22 
REFERENCES ............................................................................................................................................................ 28 
 
 
 

 
3 
Introduction 
Background and rationale 
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) testing strategy is essential for 
the diagnostic work up and screening during the coronavirus disease 19 (COVID-19) pandemic. 
Proper upper respiratory specimen collection is the most critical step in the diagnosis of SARS-
CoV-2 virus in public settings and standard sample guidelines are recommended(1).  An 
oropharyngeal swab (OPS) specimen is the preferred sample in many countries, either alone or 
combined with nasal swab(2, 3). However, there is a great variability in the diagnostic accuracy for 
OPS with a 95% confidence interval from 52-100% reported in systematic reviews(4, 5) and The 
Infectious Diseases Society of America therefore do not recommend OPS for SARS-CoV-2 testing(6). 
Many of the previous studies performing OPS also lack detailed description of the sample 
technique, which makes it difficult to compare the different diagnostic accuracy results. Currently, 
there are no standardized oropharyngeal sample guidelines, and some only collect specimen from 
the posterior oropharyngeal wall while other recommend also to include a swab of the palatine 
tonsils for SARS-CoV-2 testing (Figure A). The Danish Health Authority advised to avoid the 
palatine tonsils in the oropharyngeal swab in the beginning of the COVID-19 pandemic, but later 
changed their recommendation so it was up to the individual testing center to decide(7). However, 
studies suggest that the palatine tonsils could have a tissue tropism for SARS-CoV-2 which may 
improve the SARS-CoV-2 detection during sampling(8). This may explain the variation of sensitivity 
reported, but no clinical studies have explored the differences between the two sample 
techniques. We therefore plan to conduct a randomized clinical trial to compare an oropharyngeal 
swab with or without the palatine tonsils. 

 
4 
 
FIGURE A. Image A. Anatomic visualization of the posterior oropharyngeal wall (green) and 
palatine tonsils (pink). Image B. Oropharyngeal swab including the posterior oropharyngeal wall 
and palatine tonsils. Image C. Oropharyngeal swab including the posterior oropharyngeal wall, but 
avoiding the palatine tonsils. 
Research question 
In a cohort of individuals tested for COVID-19 at a public test center, what is the sensitivity of an 
OPS sample, including the palatine tonsils, compared to only swabbing the posterior 
oropharyngeal wall in molecular detection of SARS-CoV-2? 
 
 
 

 
5 
Methods 
We will conduct a randomized, controlled study to compare the molecular detection rate of SARS-
CoV-2 by an OPS performed from the posterior oropharyngeal wall and the palatine tonsils 
(intervention group) or the posterior oropharyngeal wall only (control group). This trial is 
approved by the Danish Data Protection Agency (Protocol No. P-2022-803) and reported to the 
Regional Ethics Committee of the Capital Region of Denmark who considered it exempted from 
further processing (Protocol No. H-22022937). The protocol was registered with the 
ClinicalTrials.gov database (NCT05611203). A grant was received from the Novo Nordisk 
Foundation (Grant number NNF21SA0069151) without influence on study design, data collection, 
data analysis, writing of the report, and the decision to submit the results for publication. 
Study setting 
Participants will be enrolled in a free public Covid-19 Testcenter in Valby and Hillerød, Capital 
Region, Denmark starting from November 2022. All samples will be sent for molecular testing at 
Statens Serum Institut and data analysis will be performed at Rigshospitalet.  
Only special trained healthcare workers at Valby and Hillerød Testcenter will collect specimens 
from the participants enrolled in the study. Before enrollment, all healthcare workers will receive 
training and a competency-based assessment of their skills (appendix 1). 
 
Eligibility criteria 
Individuals with or without symptoms of upper respiratory tract infection age 18 or above who 
visit Valby or Hillerød Testcenter for RT-PCR-testing for SARS-CoV-2 will be offered participation in 
the study. Participation is voluntary, and participants are required to provide oral and written 
consent to participate prior to entering the study (appendix 2).  
The exclusion criteria are individuals with a tracheostomy, laryngectomy, or prior oropharyngeal 
cancer surgery that would make the OPS difficult. Further, individuals without a Danish civil 

 
6 
registration number (CPR) will be excluded to participate. Individuals who are not included in the 
study will have the standard oropharyngeal swab performed in the test center.  
 
The same individual will only be allowed to participate in the study once. If a participant is 
enrolled in the study more than once, only the first enrollment (or the first positive test case) will 
be included, while the following test results will be excluded from the statistical analysis. 
Randomization to intervention and control group 
After enrollment, the participants will be randomized in a 1:1 ratio having either oropharyngeal 
swab performed including the posterior oropharyngeal wall and both palatine tonsils (intervention 
group) or only including the posterior oropharyngeal wall and avoiding the palatine tonsils (control 
group), see Figure B.  
A block randomization list was generated by T.T. using an online randomization software(7) 
afterward uploaded to REDCap. The randomization group will be disclosed in connection with trial 
registration in REDCap using the REDCap “randomize” function. The oropharyngeal sample will 
afterward be performed by the trained healthcare workers with or without the palatine tonsils 
depending on the randomization to either the intervention or control group. 
 
Figure B. Study flowchart. 
 
 
The swabs will be placed in separate tubes dry and sent to the clinical microbiology laboratory for 
detection of SARS-CoV-2 by single-target RT-PCR at Statens Serum Institute. Samples with viral 
cycle threshold (Ct) values below 38 are considered positive, 38-40 inconclusive and above 40 
negative(8). 

 
7 
All participants are required to fill out a survey on-site about their reason for being tested, prior 
Covid-19 infection(s), vaccinations status, symptoms, and if any prior tonsillectomy has been 
performed (appendix 2).  
All healthcare workers participating in the data collection have been given a unique ID which will 
be registered for each OPS performed in the study. Further, the healthcare worker will also be 
asked to rate the Mallampati score of the participants including in the study (appendix 5).  
All data will be documented on-site in a secure web database (REDCap, appendix 3). 
 
Outcomes 
The primary outcome will be reported as: 
• SARS-CoV-2 RNA by RT-PCR test result (positive, negative, inconclusive)  
 
The secondary outcome will be reported as: 
• SARS-CoV-2 RT-PCR cycle threshold (Ct) value 
• Test discomfort on a 11-point NRS-scale  
• Development of COVID-19 disease after testing  
• SARS-CoV-2 detection rate for each healthcare worker  
• Mallampati Score of participants being tested 
 
Sample size 
Based on a SARS-CoV-2 test positive proportion on 13% in the Danish capital region the last week 
of October 2022, we expected proportion of positive to increase to about 20% during the 
following study period(9). The power calculation was based on an expected improvement in 
diagnostic accuracy of 25% by including the palatine tonsils in the oropharyngeal specimen 
collection compared to a sample without the tonsils. We therefore estimated that a sample of 
2,188 participants (with about 438 with SARS-CoV-2 infection) would provide the trial with 80% 
power at a 5% significance level with an expected test positive proportion of 20% for SARS-CoV-2 

 
8 
among the participants tested (10). As we expect about 10% missing due to dropout and missing 
data, we aim to include 2.407 participants in the study. All individuals entering the testing facility 
who meet the eligibility criteria are offered to participate in the study. We expect about 300-700 
tests performed at both COVID-19 test centers each day and an assumed participation at 10-30%. 
We therefore expect to complete the study within two-three months.   
 
Statistical analysis 
A study participant is considered to have a SARS-CoV-2 infection if the oropharyngeal sample is 
RT–PCR positive (reference standard). Participants with an inconclusive RT-PCR test result will be 
included in the analyses as a negative test result following an intention-to-diagnose approach(11). 
Differences in the proportion of SARS-CoV-2 positive tests between the intervention and the 
control group will be compared using binary logistic regression using test center as fixed effect and 
a generalized estimating equation to adjust for clustering of data within the healthcare workers 
performing the sample. The difference in SARS-CoV-2 detection rate between healthcare workers 
will also be reported separately to estimate the inter-person variance. The Ct values from positive 
RT–PCR samples and the NRS discomfort scores will be compared using a general linear model 
with mixed effects (Ct) and GEE models (NRS discomfort). The 95% confidence intervals (CI) will be 
presented. Categorical data will be summarized with number and percentage while continuous 
data will be summarized by mean and standard deviation. We will not perform tests of statistical 
significance for baseline characteristics. 5% significance level was applied. Statistical Analysis 
Software ver. 9.4 (SAS Institute, North Carolina, U.S.) was used for the statistical analyses. 
 
Planned subgroup analyses  
We planned to do a sensitivity analysis using a lower cycle threshold (Ct) < 25 for positive SARS-
CoV-2 definition to explore the consequences of a higher test specificity for the SARS-CoV-2 
detection rate between specimen types. We also plan to estimate the sensitivity and specificity 
using Bayesian latent class analysis for accounting for an imperfect reference standard. Further, 
we planned to do subgroup analyses exploring the distribution of positive test results for 
participants stratified by symptoms, previous COVID-19 infection, vaccinations status, prior 

 
9 
tonsillectomy and Mallampati score. To explore a potential bias from the distribution of the 
inconclusive test results, we excluded the inconclusive results in a subgroup analyses. 
 
Quality assurance 
All the healthcare workers who will be including participants for this study will be trained and have 
at least three months prior experience in oropharyngeal swabs. Further, they will complete a 
special standardized training- and certification program taught by a board-certified 
otorhinolaryngologist to ensure all healthcare workers perform the same technique on the 
participants in the intervention and control group. A two-hour long training session will cover the 
theoretical and practical part of oropharyngeal sample techniques and the study design with 
following elements: 
• A walk-through of the practical study setup and how participants are enrolled, and 
diagnostic interventions are performed 
• Theory on oropharyngeal swabbing including upper airway anatomy and anatomical 
variations/Mallampati Score. 
• Practical exercises in oropharyngeal swab technique. 
Checklists outlining the two different sampling techniques for oropharyngeal swabs with and 
without palatine tonsils, respectively, will be used to assess the swab performance of all the 
healthcare workers by a board-certified otolaryngologist or special trained nurse before study 
enrollment (see appendix 1). 
An on-site healthcare professional is in charge of internal daily quality assurance (N.T.G.).  
 
Follow-up 
The participants enrolled in the study will be asked to agree to be contacted in case of a positive 
test result. About one month after a positive test results the participants will receive an online 
questionnaire or phone call to follow up. The follow-up questions can be found in appendix 3. 
 

 
10 
Ethics and safety considerations 
The purpose of the study is to compare the detection rate of the two different sampling 
techniques. Both techniques used in this study are implemented in the Danish healthcare system 
and are thus considered reliable methods for detecting and diagnosing infection with SARS-CoV-2. 
The study protocol is approved by the local ethical committee and the participants of the study 
are not subject to any adverse intervention and are put at no greater risk than the risk associated 
with a regular SARS-CoV-2 test.  
 
Data plan 
Data will be collected on-site in the secure database REDCap. Identifiers such as CPR-number, 
healthcare worker-ID and sample-ID are registered directly in the database for each new 
participant. The survey will be marked with a REDCap-ID allowing the research group to later 
match records with the physical surveys. Surveys are filled out on site by the participants and 
healthcare workers and stored in accordance with GDPR-legislation until data entry. Data entry 
will be done either by on-site healthcare workers continuously throughout the data collection 
period or by the research group once data collection is completed. No data analysis will be 
conducted before data collection is completed.  
 
Declaration of interests 
The authors have no competing interests to declare. 
 
Access to data 
The anonymized data will be made publicly available through an open access database after 
publication.  
 

 
11 
Appendices 
Appendix 1: Quality assurance checklists 
Checklist for oropharyngeal swab (OPS) including palatine tonsils 
Name: 
Correct 
Incorrect 
Not relevant 
1 OPS performed with proper use of 
protection equipment 
 
 
 
2 OPS performed in compliance with infection 
prevention guidelines 
 
 
 
3 OPS performed in alignment with the 
participant ensuring proper visualization of 
the back wall of the oropharynx 
 
 
 
4 The swab is placed correctly between 
thumb, index and middle finger 
 
 
 
5 Participant is instructed to say “aaaah” 
making the soft palate rises and a spatula is 
used 
 
 
 
6 The swab is inserted and retracted without 
making contact with the mucous membrane 
of the oral cavity or the tongue 
 
 
 
7 The swab gathers sufficient material from 
the back wall of the oropharynx including 
both palatine tonsils in a rotating or brushing 
movement 
 
 
 
8 The swab is placed in the sample tube in 
accordance with local guidelines 
 
 
 
Total score 
 
 
Poor 
Unacceptable 
Good 
Acceptable 
Excellent 
General assessment 
 
 
 
 
 

 
12 
 
Checklist for oropharyngeal swab (OPS) excluding palatine tonsils 
Name: 
Correct 
Incorrect 
Not relevant 
1 OPS performed with proper use of 
protection equipment 
 
 
 
2 OPS performed in compliance with infection 
prevention guidelines 
 
 
 
3 OPS performed in alignment with the 
participant ensuring proper visualization of 
the back wall of the oropharynx 
 
 
 
4 The swab is placed correctly between 
thumb, index and middle finger 
 
 
 
5 Participant is instructed to say “aaaah” 
making the soft palate rises and a spatula is 
used 
 
 
 
6 The swab is inserted and retracted without 
making contact with the mucous membrane 
of the oral cavity or the tongue 
 
 
 
7 The swab gathers sufficient material from 
the back wall of the oropharynx in a rotating 
or brushing movement 
 
 
 
8 The swab is placed in the sample tube in 
accordance with local guidelines 
 
 
 
Total score 
 
 
Poor 
Unacceptable 
Good 
Acceptable 
Excellent 
General assessment 
 
 
 
 
 
 
 
 

 
13 
Appendix 2: Survey for participants 
Survey to be filled out by participant 
1. Are you vaccinated against COVID-19? 
□YES 
 
□NO 
2. 
Have you previously been infected with COVID-19? 
 
□YES                           
□NO 
3.  
Please indicate your reason for getting tested today: 
□Symptoms 
 
□Close contact 
□Positive quick test 
□Screening 
□Test ahead of social gathering or nursing home visit    □Other 
If ’yes’ to symptoms, which symptoms do you have? 
□Sore throat    
□Headache  
□Cough      □Muscle and joint pain 
□Fever 
 
□Fatigue 
 
□Reduced sense of smell or taste 
If “yes” to symptoms, for how many days have you had symptoms? 
Indicate number of days: _____________day(s) 
4. 
Have you had your tonsils removed?? 
□YES 
 
□NO 
5. 
On a scale from 0-10, how uncomfortable was today’s test for COVID-19? 
 No discomfort 
 
 
 
               Worst possible discomfort 
 
 
_______________________________________________________________________________________ 
To be filled out by personnel: 
REDCap ID:________________________ 
Personnel ID:__________________________ 
Participant’s Mallampati score: 
□1 
 
□2 
 
□3 
 
□4 

 
14 
Appendix 3: REDCap database 
REDCap-ID 
 
Date 
DD-MM-YYYY 
Randomization 
Oropharyngeal swab including palatine tonsils 
Oropharyngeal swab excluding palatine tonsils 
CPR-number 
 
Sample ID 
 
Are you vaccinated against COVID-19? 
YES 
NO 
Have you previously been infected with COVID-19? 
YES 
NO 
Please indicate your reason your reason for getting tested today 
Symptoms 
Close contact 
Positive quick test 
Screening 
Test ahead of social gathering / visit to a nursing home 
Other 
If “YES” to symptoms, what symptoms do you have? 
Sore throat 
Headache 
Cough 
Muscle and joint pain 
Fever 
Fatigue 
Reduced sense of smell or taste 
If “YES” to symptoms, for how many days have you had 
symptoms? 
 
Have you had your tonsils removed? 
YES 
NO 
On a scale from 0-10, how uncomfortable was today’s test for 
COVID-19? 
0 
1 

 
15 
2 
3 
4 
5 
6 
7 
8 
9 
10 
Personnel ID 
 
Participant’s Mallampati score 
1 
2 
3 
4 
FOLLOW UP 
Have you since the test and until four weeks later had symptoms 
of COVID-19? 
YES 
NO 
If ‘yes’ to symptoms, when did you develop symptoms? 
I had symptoms when I got tested 
I developed symptoms 1-3 days after the test 
I developed symptoms 3-5 days after the test 
I developed symptoms 5-7 days after the test 
I developed symptoms 1-2 weeks after the test 
I developed symptoms 2-3 weeks after the test 
I developed symptoms 3-4 weeks after the test 
I do not know / I do not wish to disclose 
If ‘yes’ to symptoms, which symptoms did you develop? 
Sore throat 
Headache 
Cough 
Muscle- / joint pain 
Fever 
Fatigue 
Reduced sense of smell / taste 
Other 
If ‘yes’ to fever, what was you highest temperature? 
 

 
16 
If ‘yes’ to fever, how many days did you have a fever for? 
 
If ‘yes’ to symptoms, how many days did you have symptoms in 
total? 
 
Think about period from one week before your test until four 
weeks after. Which of the following statements describes best 
how you felt at your worst? 
I had no symptoms 
I was sick and stayed home 
I was admitted to the hospital due to COVID-19 
Have you taken days off work / from school during your 
infection with COVID-19? 
No, I have not taken any days off 
Yes, I have taken days off between one week before the test 
until four weeks after 
Yes, I have taken days off later than four weeks after 
Not relevant 
If ‘yes’ to having taken days off, how many days have you taken 
off in total? 
 
Do you smoke? 
Yes 
No 
I used to smoke, but I do not smoke anymore 
Not relevant / I do not wish to disclose 
If ‘yes’ to smoking, which of the following statements describes 
you best? 
I smoke sometimes, e.g. at parties 
I smoke daily (less than 10 cigarettes or equivalent to that) 
I smoke daily (10 or more cigarettes or equivalent to that) 
I smoke e-cigarettes 
I do not know / I do not wish to disclose 
If ‘yes’ to having smoked previously, which statement describes 
you best? 
I have not smoked for more than 5 years 
I have smoked within the last 5 years, but I do not smoke 
anymore 
Think about the past half year before the test. Which of the 
following statements describes your level of physical activity 
best? 
I do hard exercise / elite level sports regularly and several 
times per week 
I exercise or perform heavy gardening tasks or similar at 
least four times per week 
I walk, bike or do other lighter exercise at least four times 
per week (incl. walking/biking to work and lighter gardening) 
I read, watch TV or do other still activities 
I do not know / I do not wish to disclose 

 
17 
Think about the past half year before the test. How would you 
describe you physical condition? 
Very good 
Good 
Alright 
Below average 
Bad 
I do not know / I do not wish to disclose 
Have you been diagnosed with a chronic illness by a doctor 
previous to your test for COVID-19? 
No, I have not been diagnosed with a chronic illness 
Yes, diabetes 
Yes, asthma 
Yes, COPD or chronic pulmonary disease 
Yes, high blood pressure 
Yes, chronic of frequent headache, including migraine 
Other chronic illnesses 
I do not know / I do not with to disclose 
 
 
 

 
18 
Appendix 4: Participant information 
Deltagerinformation om deltagelse i videnskabeligt forsøg 
Forsøgets titel: Sammenligning af COVID-19 test ved podning i mundsvælget med eller uden 
mandler. 
Vi vil spørge, om du vil deltage i et videnskabeligt forsøg, der udføres af Rigshospitalet i 
samarbejde med akutberedskabet i Region Hovedstaden og Hvidovre Hospital. 
Før du beslutter, om du vil deltage i forsøget, skal du fuldt ud forstå, hvad forsøget går ud på, og 
hvorfor vi gennemfører forsøget. Vi vil derfor bede dig om at læse denne deltagerinformation. Du 
vil også modtage mundtlig deltagerinformation, hvor du kan stille de spørgsmål, du eventuelt har 
til forsøget. 
Hvis du beslutter dig for at deltage i forsøget, vil vi bede dig om at underskrive en 
samtykkeerklæring. Det er frivilligt at deltage i forsøget, og du kan når som helst og uden at give 
en grund trække dit samtykke tilbage. 
Nytte ved forsøget 
Ved at indgå i forsøget hjælper du med at skaffe værdifuld viden om, hvordan man bedst foretager 
COVID-19 test. Det er med til at sikre, at vi kan bruge de testmetoder, der er bedst til at finde de 
smittede og samtidigt er mindst mulig ubehagelig for borgerne. 
  
Formål med forsøget 
Formålet med forsøget er at sammenligne den diagnostiske sikkerhed for to forskellige måder at 
foretage COVID-19 test på. 
Normalt bliver man i Danmark podet med vatpind gennem munden for at samle materiale til PCR-
test for COVID-19. Det varierer fra region til region hvorvidt podningen indbefatter mandlerne. 
Der mangler undersøgelser, der giver os viden om podning af mandlerne øger sikkerheden af 
testen eller ej. Vi vil derfor i dette lodtrækningsforsøg foretage podningen i svælget med eller 

 
19 
uden samtidig podning af mandlerne for at undersøge, hvilken metode er den bedste. For at få 
viden om borger præferencer vil du også blive spurgt ind til testubehag ved undersøgelsen. Når 
studiet er overstået, vil vi således være klogere på hvilken metode der bør anbefales i danske test 
sammenhæng. 
Plan for forsøget 
Alle undersøgelser vil blive udført under samme besøg, og du kan forvente at dette kan tage 
omkring 5 minutter længere. Du vil få taget podning i munden samt besvare et kort spørgeskema. 
Podningen i mundsvælget foregår ved, at man fører en vatpind ind bag ganen (standardpraksis). 
Bliver du randomiseret til at skulle have podet mandlerne også, foregår det ved samme podning. 
Prøven vil blive sendt til PCR-analyse som vanligt og svartiden vil ikke blive forlænget. 
For at undersøge om testen også giver et resultatet som er i overensstemmelse med efterfølgende 
sygdomsudviklingen, vil vi eventuelt efter podningen også lave et opslag i din elektroniske 
patientjournal for at hente information om behov for indlæggelse, behov for medicinsk 
behandling, samt tidligere vaccinationsstatus og COVID-19 infektion. Ved positiv test vil du også 
blive kontaktet nogle uger efter afslutning af forsøget (e-boks eller telefon) for opfølgning på 
udviklingen af mulige alvorlige symptomer på COVID-19. 
Bivirkninger, risici, og ulemper 
Du kan muligvis forvente lidt ekstra ubehag ved podning af mandlerne, men ellers udføres 
podningen ligesom den aktuelle standard praksis. 
  
Etisk og databehandling (fortrolighed) 
Forsøget varetages på initiativ af Rigshospitalet, Region Hovedstadens Akutberedskab, og 
Hvidovre Hospital med uafhængig økonomisk støtte af Novo Nordisk Fonden. Projektet er 
godkendt af Videnskabsetisk Komite og Videnscenter for Dataanmeldelser, og håndtering af 
persondata vil blive håndteret med fuld fortrolighed efter gældende retningslinjer. 
  
Adgang til forsøgsresultater 

 
20 
Forsøgets resultater vil blive offentliggjort umiddelbart efter forsøgets afslutning i et 
fagfællebedømt videnskabeligt tidsskrift. 
Vi håber, at du med denne information har fået tilstrækkeligt indblik i, hvad det vil sige at deltage i 
forsøget, og at du føler dig rustet til at tage beslutningen om din eventuelle deltagelse. Hvis du har 
spørgsmål eller vil vide mere om forsøget, er du meget velkommen til at kontakte forsøgsansvarlig 
medicinstuderende Benedikte Hartvigsen (benedikte.hartvigsen@regionh.dk). 
Vi håber du vil have mulighed for at bruge 5 minutter ekstra på at indgå i projektet i forbindelse 
med, at du skal testes for COVID-19. 
På forhånd tak for din interesse i at hjælpe med vores forskningsprojekt! 
På vegne af forskningsgruppen 
Benedikte Hartvigsen, medicinstuderende, Københavns Universitet 
Thomas Benfield, overlæge, professor, Infektionsmedicinsk Afdeling – Hvidovre Hospital 
Annette Kjær Ersbøll, Professor, Region Hovedstadens Akutberedskab 
Nina Steenhard, Ph.d., Testcenter Danmark – Statens Serum Institut 
Tobias Todsen, speciallæge, lektor, Afdeling for Øre-Næse-Halskirurgi og Audiologi – Rigshospitalet 

 
21 
Appendix 5: Mallampati score 
 
 

 
22 
Appendix 6: Statistical Analysis Plan (SAP) 
Title: Molecular detection of SARS-CoV-2 from oropharyngeal swabs performed with or without 
specimen collection from the palatine tonsils – a multicenter randomized controlled trial 
 
SAP version 1, 11122022 
Senior statistician: Professor Annette Kjær Ersbøll, PhD 
Chief investigator: Associate Professor Tobias Todsen, MD, PhD 
 
1. Statistical principles 
 
1.1 Confidence intervals and P values 
The level of statistical significance will be p < 0.05 and 95% confidence interval will be reported.  
 
1.2 Adherence and Protocol deviations 
Definition of adherence to the intervention 
Adherence is defined as participants who has a full registration of identification number (CPR 
number), test center site for collection of specimen and randomization to control or intervention 
group. Further, the participants need to complete the OPS specimen collection send for molecular 
testing.  
 
Compliance is assessed based on the number and percent of subjects who have correct 
registration information and representative samples for RT-PCR.  
 
Description of adherence 
The adherence to the intervention will be summarized in the study flowchart and number and “% 
compliance” will be summarized. 
 
Definition of protocol deviations for the trial 
The participants will be excluded from final analysis if one or more of the following deviations 
from the testing protocol was fund: 

 
23 
 
Missing identification number (CPR number), no registered test center sites or no randomization 
(intervention or control) registered. 
Missing RT-PCR result  
 
Description of which protocol deviations will be summarized 
The number and type of protocol deviation will be registered, and number of participants 
removed will be summarized in figure with study flow.  
 
2. Trial Population 
 
2.1 Screening data 
We aim to invite individuals from Valby and Hillerød Covid-19 test centers to represent citizens 
from two urban areas in Copenhagen, Denmark to participate in the study.  
 
2.2 Eligibility 
All individuals being 18 years or older will be invited to participate in the SAMPLE trial. The same 
individual will only be allowed to participate in the study once.  
 
The exclusion criteria were individuals with  
a tracheostomy, laryngectomy, or prior oropharyngeal cancer surgery 
without a Danish civil registration number (CPR)  
 
2.3 Recruitment 
A trial flow diagram will be used to summarize the number of included participants with 
information about: 
- 
Total number of Covid-19 tested individuals during the study period  
- 
Number of participants lost to identify / registrar  
- 
Number of participants excluded from final analyses due missing test results  
 
2.4 Withdrawal/Follow-up 

 
24 
The level of withdrawal and the missing final RT-PCR test results during the study will be 
tabulated. As they only had a single OPS performed, no dropouts will be expected after the 
intervention – beside missing data.  
 
2.5 Baseline patient characteristics 
List of baseline characteristics for participants: 
 
Measure 
Outcome 
Description 
Demographic data  
Age and gender 
Data from the Danish civil 
registration number 
Questionnaire 
Test reason, symptom 
description, vaccination 
status, prior SARS-CoV-2 
infection, prior 
tonsillectomy  
Questionnaire registered in 
RedCap 
NRS-scale 
Discomfort score for OPS 
specimen collections 
11-item measure of test-
related discomfort 
answered immediately after 
testing 
Mallampati score 
A visual assessment of the 
distance from the tongue 
base to the roof of the 
mouth of the participants 
4-item measure of amount 
of space in the mouth to 
reach the oropharynx 
assessed by the healthcare 
workers 
 
Categorical data will be summarized with number and percentage while continuous data will be 
summarized by mean and standard deviation. We will not perform tests of statistical significance 
for baseline characteristics.  

 
25 
 
3. Analysis 
3.1 Outcome definitions 
The primary outcome: 
- 
The proportion of positive SARS-CoV-2 RNA samples by RT-PCR for intervention and 
control group 
 
The secondary outcome: 
- 
SARS-CoV-2 RT-PCR cycle threshold (Ct) value 
- 
Test discomfort on a 11-point NRS-scale  
- 
Development of COVID-19 disease after testing  
- 
SARS-CoV-2 detection rate for each healthcare worker  
- 
Mallampati Score of participants being tested 
 
3.1 Analysis methods  
Analysis method and treatment effects 
Differences in the proportion of SARS-CoV-2 positive tests between the intervention and the 
control group will be compared using binary logistic regression using test center as fixed effect and 
a generalized estimating equation to adjust for clustering of data within the healthcare workers 
performing the sample. The difference in SARS-CoV-2 detection rate between healthcare workers 
will also be reported separately to estimate the inter-person variance. The Ct values from positive 
RT–PCR samples and the NRS discomfort scores will be compared using a general linear model 
with mixed effects (Ct) and GEE models (NRS discomfort). 
The 95% confidence intervals (CI) will be presented. The level of statistical significance will be 
defined as p < 0.05. 
 
Adjustment for covariates 
The regression analyses will be adjusted for the effect of the test centers and the individual 
health-care worker performing the sample.  
 
Methods used for assumptions to be checked for statistical methods 

 
26 
Assumptions for the logistic regression analysis included a binary outcome, independent 
observations and linearity in logit for continuous variables. To account for lack of independent 
observations, a generalized estimating equation approach will be applied. No continuous variables 
will be included. 
 
Assumptions for the linear regression analysis included a normal distribution, independent 
observations, equal variation (homoscedasticity) and linearity in logit for continuous variables. 
Normally distributed outcomes and homoscedasticity will be evaluated visually by plots of the 
residuals. To account for lack of independent observations, a generalized estimating equation 
approach and mixed effect models will be applied. No continuous variables will be included. 
 
Details of alternative methods to be used if distributional assumptions do not hold, e.g., 
normality, proportional hazards, etc. 
If the assumption of a normal distribution of the outcome in the linear regression model is not 
fulfilled, a transformation of the outcome will be applied (e.g., logarithmic and rank 
transformations). 
 
Planned subgroup analyses  
We planned to do a sensitivity analysis using a lower cycle threshold (Ct) < 25 for positive SARS-
CoV-2 definition to explore the consequences of a higher test specificity for the SARS-CoV-2 
detection rate between specimen types. We also plan to estimate the sensitivity and specificity 
using Bayesian latent class analysis for accounting for an imperfect reference standard. Further, 
we planned to do subgroup analyses exploring the distribution of positive test results for 
participants stratified by symptoms, previous COVID-19 infection, vaccinations status, prior 
tonsillectomy and Mallampati score. To explore a potential bias from the distribution of the 
inconclusive test results, we excluded the inconclusive results in a subgroup analyses. 
 
 
3.3 Missing data 

 
27 
Participants who will not adhere to the intervention definition (see SAP 1.2) will be reported as 
missing data and excluded from final analysis. Participants with missing data about their baseline 
characteristics from the questionnaire will still be included in the statistical analysis of primary 
outcome and secondary outcome. A table with baseline characteristics will be presented as raw 
data without the participants with missing data from the questionnaire.  
 
3.4 Harms 
Any adverse events during or after the collection of respiratory specimens for the trial will be 
noted and categorized into acute bleeding or foreign body in upper airway.  
 
3.5 Statistical software 
SAS statistical software suite ver. 9.4 (SAS Institute, North Carolina, U.S.)  
 
 

 
28 
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29 
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