COVID-19 Tests: a Systematic Comparison of Viral RNA Test Sample Taking for RT-qPCR, in This Case With a Nasopharyngeal Swab or Saliva Collected With Salivette® Cortisol, to Diagnose SARS-CoV-2 Virus Infection
Phase: NA
Conditions: Covid-19
Sponsor: Boehringer Ingelheim

 
COVID-19 Tests Project @ BI 
Comparison of swab vs Salivette® Cortisol 
Programme to identify SARS-CoV-2/COVID-19 infection with RT-
qPCR using a nasopharyngeal swab vs saliva collected with 
Salivette® Cortisol  
 
 
Test protocol 
T 
Document date: 
28 July 2020 
BI Project No  
0352-2150 
Title 
COVID-19 tests: a systematic comparison of viral RNA test sample 
taking for RT-qPCR, in this case with a nasopharyngeal swab or 
saliva collected with Salivette® Cortisol, to diagnose SARS-CoV-2 
virus infection 
Short title 
COVID-19 tests: nasopharyngeal swab vs saliva collected with 
Salivette® Cortisol for viral RNA sampling for RT-qPCR  
Area of investigation 
COVID-19 
In vitro diagnosis 
compared in the test 
programme 
• 
Kylt® for detection/confirmation of SARS-CoV-2 (PCR) 
• 
cobas® SARS-CoV-2 (PCR) test  
• 
TaqPathTM COVID-19 CE-IVD RT-PCR kit 
• 
Salivette® Cortisol 
Sampling methods 
1. Swab – by the medical staff 
2. Salivette® Cortisol – by the participant 
Global Programme 
Telephone: 
-
 
Email:
Local Programme 
Telephone: 
 ext 
 / 
 
Email:

 
Coordinating 
Investigator 
 
 ext 
 
24-hour telephone 
 
Email:
 
 Laboratory 
Analyst (internal) 
Telephone: 
 
Email:
 
 
Laboratory (external) 
 
Telephone: 
 
Email: 
 
 Statistician  
Telephone: 
 
Email:
 
 
 
Status 
Protocol description document 
Version and date 
Version: 1.0  
Date: 28 July 2020 
Page 2 of 18 
Protected confidential information 
© 2020 Boehringer Ingelheim International GmbH or one or more of its affiliated companies. All rights reserved. 
This document may not be transferred, reproduced, published or used in any way - in full or in part - without prior written permission  
 

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PROTOCOL SUMMARY 
Company name 
Boehringer Ingelheim (BI) 
Protocol date 
28 July 2020 
Revision date 
Not applicable 
BI project number 
0352-2150 
Programme title 
COVID-19 tests @ BI: a systematic comparison of viral RNA test 
sample taking for RT-qPCR, in this case with a nasopharyngeal swab 
or saliva collected with Salivette® Cortisol, to diagnose SARS-CoV-2 
virus infection 
Coordinating 
Investigator:  
 
 ext 
 
24-hour telephone 
 
Email:
 
Test sites 
Laboratory sites 
 
Area of investigation 
COVID-19 
Justification for the 
programme 
In order to have a correct diagnosis for individuals infected with SARS-CoV-2, reliable 
diagnostic tools must be identified, selected and regularly used. All results must be 
interpreted in relation to medical history and clinical evidence. 
RT-qPCR is the standard for identifying SARS-CoV-2 infection using viral RNA 
detection, generally with medical staff taking nasopharyngeal samples. This is 
considered to be an established procedure and is expected to provide reliable results; 
however, it has been found that the procedure depends on participant cooperation 
and staff experience. Compared to the previous technique, self-sampling is not 
considered optimal and may produce false-negative results due to the complex 
collection techniques and confounding human factors. In some cases it is difficult or 
impossible to obtain suitable samples. Self-sampling can lead to a lower risk of 
spreading the virus, as no visits to special centres are required. Furthermore, the 
biosafety level and risk of infections of medical staff when taking swab samples are 
reduced significantly. 
Objective of the 
programme 
The objective of the comparison is to demonstrate that saliva collection with 
Salivette® Cortisol is as reliable as the use of throat swabs in sampling viral RNA 
for RT-qPCR and identifying SARS-COV-2 infections.  
PCR for RNA detection of SARS-CoV-2 in throat swab samples and saliva samples 
collected with Salivette® Cortisol 
BI internal laboratories 
Certified external laboratories  

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Programme design 
Parallel tests of different sample matrices  
Number of participants  
Total number: minimum of 20 and up to 80 participants who are positive for SARS-
CoV-2 
Main inclusion criteria 
for the pilot phase 
COVID-19 diagnosis confirmed by PCR (within the past 5 days)  
In vitro diagnosis 
• 
Kylt® for detection/confirmation of SARS-CoV-2 (PCR)
• 
cobas® SARS-CoV-2 (PCR) test  
 
• 
TaqPathTM COVID-19 CE-IVD RT-PCR kit 
 
 
Day of the test 
One single sample is planned  
Statistical methods 
Descriptive statistics directly comparing the test results 

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TABLE OF CONTENS 
 
TEST PROTOCOL  ................................................................................................ 1 
PROTOCOL SUMMARY ........................................................................................ 3 
TABLE OF CONTENTS ......................................................................................... 5 
ABBREVIATIONS .................................................................................................. 6 
1. 
INTRODUCTION ............................................................................................ 7 
1.1 
MEDICAL BACKGROUND ................................................................... 7 
1.2 
DIAGNOSTIC PROFILE........................................................................ 8 
1.2.1 Quantitative reverse transcription polymerase chain 
reaction (RT-qPCR) ......................................................................... 8 
1.3 
JUSTIFICATION ................................................................................... 8 
1.4 
RISK-BENEFIT ASSESSMENT ............................................................ 9 
2. 
OBJECTIVES AND GOALS ......................................................................... 13 
2.1 
MAIN OBJECTIVES AND PRIMARY GOALS .................................... 13 
2.1.1 Main objectives .............................................................................. 13 
2.1.2 Main diagnosis for admission .................................................... 13 
2.1.3 Inclusion criteria ............................................................................ 13 
2.1.4 Withdrawal of consent or decision by BI to discontinue 
the test programme ....................................................................... 13 
2.2 TEST METHODS .................................................................................... 13 
2.2.1 Identity of the in vitro diagnosis at BI ........................................ 14 
2.2.2 Storage and transportation conditions – Mexico to 
Germany ........................................................................................... 15 
2.2.3 Responsibility for the in vitro diagnosis ................................... 15 
2.3 REFERENCE MEASURES ..................................................................... 16 
2.3.1 Restrictions ....................................................................................... 16 
2.3.2 Handling of swabs and saliva samples ..................................... 16 
3. 
SAMPLE SIZE .............................................................................................. 17 
4. 
ADVERSE EVENT REPORTING………………………………………………..17 
5. 
REFERENCES ............................................................................................. 18 
 

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ABBREVIATIONS 
BI 
Boehringer Ingelheim 
CA 
Competent authority 
COVID-19 
Coronavirus disease 2019 
GCP 
Good Clinical Practice 
ICF 
Informed consent form 
Ig 
Immunoglobulin 
IPC 
Internal positive control 
IU 
International units 
K 
Dispersion factor 
LLOQ 
Lower limit of quantification 
MERS 
Middle East Respiratory Syndrome 
OD 
On-demand 
PI 
Package insert 
PoC 
Point of care 
RNA 
Ribonucleic acid 
RT-qPCR 
Quantitative reverse transcription polymerase chain reaction 
SARS  
Severe Acute Respiratory Syndrome 
SARS-CoV-2 
Severe Acute Respiratory Syndrome Coronavirus 2 
ULN 
Upper limit of normal 
ULOQ 
Upper limit of quantification 
UTM 
Universal Transport Medium 
WFH 
Work from home 
WHO 
World Health Organization 
 

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1. 
INTRODUCTION 
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been identified as the cause 
of the pandemic outbreak of the coronavirus disease 2019 (COVID-19). Boehringer Ingelheim (BI) 
has launched a large-scale RT-PCR confirmation programme called COVID-19 Tests to counter the 
pandemic and safeguard public health. This dedicated health care measure was developed to 
complement the actions implemented by public health care to relieve the efforts of the health 
authorities.  
The global pandemic COVID-19 is a reportable disease in most countries and local health authorities 
are monitoring the contacts of infected individuals to detect potential additional infections in order to 
prevent the spread. Contact tests generally depend on the presence of certain respiratory symptoms 
which coincide with the established risk criteria for increasing the probability of a positive nucleosides 
(RNA) result for SARS in swabs or sputum samples [RKI 2020]. It is therefore assumed that tests 
must be performed on as many individuals as possible in order to contain the pandemic situation. As 
such, the sampling method has become a critical factor, as it is more difficult to take swab samples 
than to collect saliva, and sampling must be optimised to allow more flexibility in obtaining samples 
for virus RNA identification, regardless of the availability of medical staff and testing centres. 
Furthermore, a method that could be performed at home and during quarantine will prevent the 
spread and reduce the biosafety risk in general.  
1.1 
MEDICAL BACKGROUND 
SARS-CoV-2 belongs to the Coronaviridae family, which also includes members that cause the 
common cold, Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome 
(MERS) [Liu et al. 2020]. 
It has been confirmed that the novel coronavirus is transmitted from person to person. Close contact 
between individuals (< 1.5 m) promotes the spread of the virus and, as a result, the disease. It is 
spread predominantly through droplets expelled when coughing or sneezing, but also through 
atomised particles when speaking. It must be assumed that transmission takes place when kissing, 
shaking hands or touching contaminated surfaces in public spaces (public transport, door handles, 
buttons, etc.), particularly when touching the mouth, nose or eyes afterwards. Given the many 
uncertainties regarding SARS-CoV-2 infection to date, including the dispersion factor (K), it is 
important to closely monitor the social setting and conduct extensive tests to control the pandemic 
situation. 
COVID-19 symptoms can vary from very mild to severe and may include fever, cough, breathing 
difficulty and fatigue, which commonly occur after an incubation period of 2 to 14 days [Huang et al. 
2020]. These symptoms can become serious and include fatal outcomes. It is thought that 
approximately 20% of infected patients show non-specific symptoms or no symptoms at all, but can 
still spread the virus [He et al. 2020]. Unless they are quickly identified by means of sufficient, 
potentially regular off-site detection activities, these individuals have a high risk of unwittingly 
transmitting SARS-CoV-2, which is a significant threat to public health against a backdrop of acute 
infection driven by an on-demand test scheme. It is understood that the burden of regular tests cannot 
be applied to all, but rather to selected groups. 
There is not yet a vaccine available for SARS-CoV-2, nor are there any specific antiviral drugs for 
COVID-19. The standard treatment is targeted primarily at relieving the symptoms, while 
compassionate use of medications is limited mainly to clinical studies, as well as invasive measures 
to save lives in severe cases, which often require long-term intensive care. The long-term impact of 

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COVID-19 on health cannot be evaluated at this early stage of the disease, but emerging evidence 
suggests that the infections can cause serious harm and potentially cause sequelae with a negative 
impact on overall health [RKI 2020]. As such, a test strategy with the diagnostic tools available for 
prevention or early diagnosis of COVID-19 is essential as an additional measure to the rules in place, 
such as social distancing and hygiene to prevent uncontrolled spread, to prevent the health services 
from collapsing and to reduce the long-term impact on public health. 
1.2 
DIAGNOSTIC PROFILE 
The standard laboratory test for diagnosing SARS-CoV-2 is an RT-qPCR certified protocol to identify 
the RNA of the virus in swab samples (nasopharyngeal/oropharyngeal) or sputum samples 
[Udugama et al. 2020].  
1.2.1 
Quantitative reverse transcription polymerase chain reaction (RT-qPCR) 
RT-qPCR is a bioanalytical method for detecting nucleosides (DNA or RNA) in a wide range of test 
samples (e.g. blood, saliva and swabs). The technique is based on the in vitro amplification of genetic 
material using complementary nucleoside fragments (primers) and specific enzymes (polymerases). 
While the original material is undetectable due to its low concentration, the amplification can easily 
be detected, e.g. by means of gel electrophoresis or, in the case of RT-qPCR, automatically by 
digitally measuring a fluorescent signal in the device. There is currently a wide range of modified RT-
qPCR techniques available for various situations. For RNA detection (SARS-CoV-2 genome), another 
specific enzyme is required: reverse transcriptase (RT). There is a RT-PCR protocol available from 
Charité 
Virology 
for 
diagnosing 
COVID-19 
[Corman 
et 
al. 
2020]. 
The 
Kylt® 
for 
detection/confirmation of SARS-CoV-2 (PCR) kit is based on the PCR method published by 
Corman et al., which detects specific E gene sequences, and specific sequences in the second PCR 
of the RdRP gene and the S gene of SARS-CoV2. 
A second test kit with RT-PCR is the TaqPathTM COVID-19 CE-IVD RT-PCR kit, provided by
, which uses three different specific sequences of 
SARS-CoV-2 in the ORF1ab, N and S genes of the virus, for a multiplex detection focus.   
The cobas® SARS-CoV-2 (PCR) test, by
, is a PCR detection system which is 
fully automated for the cobas® 6800/8800 laboratory systems. This PCR detects in parallel a 
fragment of the E gene of the pan-Sarbecovirus and a specific ORF1 sequence of SARS-CoV-2, 
and is validated for nasopharyngeal and oropharyngeal samples taken with swabs and collected in 
UTM. 
The three PCR kits include an internal positive control (IPC) which targets the RNA of human β-
actin as an endogenous or externally added control, or externally added control targets in each 
reaction to validate the polymerase reaction.  
1.3 
JUSTIFICATION  
In order to effectively test and monitor, reliable diagnostic tools must be selected, implemented and 
regularly performed. The results must be evaluated in relation to the medical history and clinical 
evidence. RT-qPCR is the standard for identifying SARS-CoV-2 infection and for detecting viral RNA, 
generally when the medical staff take nasopharyngeal swab samples. This is considered to be an 
established procedure and is expected to provide reliable results; however, it has been found that the 
procedure depends on participant cooperation and staff experience. Furthermore, self-sampling is 
not considered optimal and may produce false-negative results due to the difficulty of the technique 

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and the high level of variability due to human factors. In some cases it is difficult or impossible to 
obtain suitable samples. Furthermore, as there is no need to go to special centres, there is a lower 
risk of spreading the virus. It also significantly reduces biosafety and risk of infections for medical staff 
when taking swab samples. 
The Salivette® Cortisol device will be used to collect saliva from the patient due to its high capacity 
to stimulate salivation and absorb saliva. In this saliva sample, it is expected to find SARS-CoV-2 viral 
RNA which will be identified through processing with the RT-qPCR technique, as described for the 
nasopharyngeal mucosa swab samples. No cortisol measurements will be performed in the collected 
sample. 
1.4 
RISK-BENEFIT ASSESSMENT 
The Salivette® Cortisol device, which is placed in the mouth and gently chewed for approximately 60 
seconds, is considered to be very low risk. Its taste may be considered unpleasant. It is handled by 
the participant and, as such, there is no additional risk expected for the medical staff. During the tests, 
everyone wears masks in the rooms while the swab samples are taken, and the medical staff are 
protected with FFP2 masks, protective clothing and double gloves. Sampling with Salivette® Cortisol 
can be performed on-site. The layout of the room will follow the general distancing and hygiene 
measures in place, and surfaces will be cleaned and disinfected in line with procedure after each 
participant leaves. The risk of harm, such as sequelae due to the medical procedure, and the risk of 
infection of all participants, including the occupational health care team, was assessed before the 
tests and was considered highly unlikely.  
There is recent evidence (Wyllie, AL et al. 2020. https://doi.org/10.1101/2020.04.16.20067835doi) 
that saliva sampling potentially produces more reliable results when compared to swab sampling. 
Conclusions: It is considered necessary to establish sampling methods other than swab samples ion 
order to reduce the biosafety risk and increase the flexibility of tests and their acceptance among 
potentially infected individuals, as taking swab samples is more invasive than collecting samples with 
Salivette® Cortisol. The sampling method established should allow for regular tests and be easy and 
well accepted by patients, while providing comparable results. 
Risks and methods related to the medical procedure 
There are some risks involved in the nasopharyngeal mucosa sampling procedure using swabs, such 
as mild bleeding of the nasal mucosa, pharyngeal pain after the sample is taken, sneezing, cough, 
and gag reflex when taking the pharyngeal mucosa sample.  There are no known risks for patients 
related to the Salivette® Cortisol device, and even accidental ingestion of the device has not led to 
any adverse reactions being reported, as it is made of inert material and does not contain any active 
or reactive substances. 
Sample taking with swab/Salivette® Cortisol 
SARS-CoV-2 sampling: 
 
The nasopharyngeal mucosa swab samples and those taken with the Salivette® Cortisol device will 
be collected in line with the SARS-CoV-2 biological sample collection standards in place at the 
Research Site. 
 
 
Sampling 

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1. 
Only the health care staff and the individual undergoing the test should be in the room 
when the test is performed. 
2. 
All of the non-aerosol generating procedures (documentation, interviews, blood 
sampling) must be performed first. The aerosol generating procedures (collection of 
oropharyngeal swab samples and saliva samples) must be the final activity performed. 
3. 
It is likely that the collection of oropharyngeal swab samples will cause a cough 
4. 
All samples must be identified with a unique code, using a permanent marker or a label 
5. 
The identities of all samples must be listed separately, without including additional 
personal data of the patients alongside the samples 
6. 
Collection of oropharyngeal swab samples 
1. 
Use a tongue depressor to prevent the tongue from interfering with the sample 
collection 
2. 
Insert the swab into the mouth 
3. 
Rub the swab on the tonsillar pillars and the posterior oropharynx, avoiding 
contact with the tongue, teeth and gums. 
 
 
4. 
Place the swab in the sterile viral transport tube and remove the applicator. Ensure that 
the cap is tightly sealed.  
 
 
 
5. 
Transfer the closed tube into a biohazard transportation bag and store the 
sample in a refrigerator (2-8°C) or freeze the tubes (< 20°C). 
 
 
Use of throat swab 
Swab 
Rub the throat in 
the tonsil area 
Tonsil 
Image courtesy of the US National Library of Medicine 

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7. 
Saliva (Salivette® Cortisol) 
1. 
Instructions for use 
 
…and replace the cap (see Fig. 5). 
Telephone 
Fax 

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Sample transportation 
The biological samples obtained from the participant will be processed at a central laboratory in 
Germany, as described in section 2.1.4 TEST METHODS, and transported from Mexico in cold chain 
to ensure their stability. Sample transportation will be carried out in line with IATA specifications as 
category B biological samples. 
 
1. Classification: UN3373 Biological Substance, Category B 
2. Correct name for shipping: UN3373 Biological Substance, Category B 
3. Packaging for international transportation: Samples must be packaged, shipped and 
transported in line with the current edition of the International Air Transport Association 
(IATA) Dangerous Goods Regulation: packing instruction 6504.  
4. Swab sample tubes to be sent between 4 and 8 degrees Celsius or in dry ice, as indicated 
by the carrier depending on the transportation time. (see section 2.1.6 Storage and 
transportation conditions – Mexico to Germany). 
5. Each shipment must contain a full list of every sample's identity.  
6. To ensure the traceability of the shipments, all of these will be organised by the receiving 
 in 
 using the World Courier service.  
  

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2. 
OBJECTIVES AND GOALS 
2.1 
MAIN OBJECTIVES AND PRIMARY GOALS 
2.1.1 
Main objectives 
The main objective of the tests is to establish saliva collection with Salivette® Cortisol.   
For PCR: 
Real-time RT-PCR (
) 
+/- 
Real-time RT-PCR 
 
+/- 
 
2.1.2 
Main diagnosis for admission 
Suspected or confirmed cases of SARS-CoV-2 infections (including COVID-19), confirmed with PCR 
within the past five days. 
 
2.1.3 
Inclusion criteria 
Only participants who meet the following criteria will be included in the programme: 
1. Informed consent form signed and dated before any samples have been taken.  
2. Suspected or confirmed SARS-CoV-2 infection by PCR within the past five days. 
3. Male and/or female patients aged 18 years and over.  
 
2.1.4 
Withdrawal of consent or decision by BI to discontinue the test programme  
Participants may withdraw their consent to participate at any time, without having to justify their 
decision. 
 
2.2 
TEST METHODS 
The in vitro diagnostics have CE marking, are commercially available and are established for 
diagnostic purposes at the laboratories indicated. They were selected based on their reported 
sensitivity and specificity criteria, as well as their availability on the market. The laboratory 
 
was chosen due to being a professional, certified laboratory which is highly experienced in serology 
methods and with experience in COVID-19 diagnostics since the start of the SARS-CoV-2 pandemic. 
 

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2.2.1 
Identity of the in vitro diagnosis at BI 
The test product characteristics are shown below: 
Name: 
Kylt® for detection/confirmation of SARS-CoV-2 
Method: 
Real-time RT-PCR 
Source: 
Diagnosis site: 
Sample: 
Swab (throat) and Salivette® Cortisol 
Duration: 
4-6 hours 
Name: 
cobas® SARS-CoV-2 (PCR) test 
Method: 
Automated real-time RT-PCR for cobas® systems 
Source: 
Diagnosis site: 
Sample: 
Swab (throat) 
Duration: 
3 hours 
Name: 
TaqPath™ COVID-19 CE-IVD RT-PCR kit 
Method: 
Multiplex RT-PCR 
Source: 
 
Diagnosis site: 
Sample: 
Salivette® Cortisol 
Duration: 
4-6 hours 
 
 

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Real-time PCR detection of SARS-CoV-2 
For the qualitative detection of SARS-CoV-2, a single-step RT-PCR method based on specific kits for 
SARS-CoV-2 or kits which include a primer and a probe (RT-PCR assay). 
RT-PCR assays are suitable for implementing a multiplex RT-PCR focus in real time: in the case of 
a reaction, the RNA target sequences of the gene(s) of interest (e.g., SARS-CoV-2), and an 
endogenous control (e.g., β-actin) or an IPC (e.g., RNA phage control), are reversely transcribed and 
amplified in parallel with the corresponding pairs of primers in the polymerase chain reaction. The 
amplified target gene fragments are detected using probes marked with fluorescence during the real-
time PCR reaction. In order to monitor each analysis run, general controls will be provided for 
performing the run (e.g. positive control and negative control of synthesis). 
The specific probes for the gene(s) of interest and the internal control(s) (endogenous or loaded) are 
each marked with different fluorescent dyes (e.g. FAM and HEX), and their fluorescence emitted is 
optically measured separately with the real-time PCR thermocycler. Through the individual analyses 
in one reaction vessel per sample and the positive and negative controls per run, the status of a 
sample can be evaluated in terms of the gene(s) of interest [Corman et al. 2020]. 
 
2.2.2 
Storage and transportation conditions – Mexico to Germany 
The test supplies will be kept in their original packaging and in a secure storage area with restricted 
access, in line with the recommended storage conditions (labelled). It is recommended to transport 
and store probes such as swabs and Salivette® Cortisol at 4-8°C. 
For storage times of longer than 48 hours prior to shipment of swab and Salivette® Cortisol samples 
for storage in Germany, transportation at temperatures < 20°C in dry ice is recommended. 
Packaging of all shipments must comply with the international regulations for transporting dangerous 
goods (UN3373 for dry ice).  
 
2.2.3 
Responsibility for the in vitro diagnosis 
Only the documented authorised staff may receive and dispense in vitro diagnostic tests. These tests 
must be performed as stated in the product instructions or in the test protocol.  
The occupational health care team must keep records of the diagnostic tests entering the test site, 
the site’s inventory, use with each participant and the disposal of compromised devices. 
All compromised devices will be disposed of locally by the test site. The receipt, use and disposal of 
the devices must be duly documented. Any discrepancies must be reported. 
 
 

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2.3 
REFERENCE MEASURES 
 
2.3.1 
Restrictions 
There are no dietary or lifestyle restrictions in place. However, a condition for taking biological 
samples is that no food or medicinal products may been consumed for a period of at least 30 minutes 
before the procedure to prevent saliva contamination. 
 
2.3.2 
Handling of swabs and saliva samples 
The throat swab sample collected arrives in a transport medium. The swabs are washed within the 
transport media, by shaking the tubes for 10 minutes at approximately 500 rpm, in an orbital shaker 
at room temperature. The sample can be stored for up to 7 days in the transport medium, at +4°C. 
The saliva samples collected with Salivette® Cortisol are packaged in the corresponding primary 
container. 

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3. 
SAMPLE SIZE 
There is no calculated sample size but rather a general attempt to directly compare swab samples 
versus saliva samples collected with Salivette® Cortisol in a participant, with a minimum of 20 to 80 
participants. 
 
4. 
ADVERSE EVENT REPORTING 
 
Although this study is based on recently collected data, adverse event (AE) collection is not required. 
There is no Boehringer Ingelheim medicinal product intended or marketed for this disease. The 
investigator is encouraged to report all adverse events related to any product marketed by Boehringer 
Ingelheim, in accordance with the local regulatory requirements for spontaneous AE reporting at the 
investigator’s discretion using the locally established means and AE report forms. The term AE 
includes the exposure to medicinal products during pregnancy and, regardless of whether or not an 
AE occurs, any abuse, off-label use, misuse, medication error, occupational exposure, lack of efficacy 
and unexpected benefit.  
 
Other reports. As any reportable adverse events involving medical devices, technical complaints 
regarding the product and device deficiencies could cause harm to patients during clinical studies 
related to Salivette® Cortisol/swabs, the investigator will remain responsible for managing all 
activities, including reporting the medical device in line with the local regulatory requirements on 
reporting medical devices. 
According to its definition, a reportable adverse event involving a medical device means any 
unfavourable medical event, disease or injury, or any unfavourable clinical sign, including abnormal 
laboratory findings, in subjects, users or other individuals, in the context of a clinical study, regardless 
of whether or not it is related to the investigational device, and which meets the criteria for a reportable 
event as set forth in the applicable laws and regulations. 
 

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5. 
REFERENCES 
1. 
Robert Koch-Institute (RKI). SARS-CoV-2 Steckbrief zur Coronavirus-Krankheit-2019 
(COVID-19). 
RKI; 
29 
May 
2020, 
https://www.rki.de/DE/Content/InfAZ/N/Neuartiges_Coronavirus/Steckbrief.html 
[consulted 
in June 2020] 
 
2. 
Liu, J., Yang, D. et al. Overlapping and discrete aspects of the pathology and pathogenesis 
of the emerging human pathogenic coronaviruses SARS‐CoV, MERS‐CoV, and 2019‐nCoV. 
Journal of Medical Virology. 2020; 92: 491–494. 
 
3. 
Huang, C., Wang, Y., Li, X., Ren, L., Zhao, J., Hu, Y., Bin Cao et al. Clinical features of 
patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395: 497-506 
 
4. 
He, X., Lau, E., Cowling, B., Li, F., Leung, G., Zhao, L., Zhang, F., Cowling, B., Li F., Leung, 
G. et al. Temporal dynamics in viral shedding and transmissibility of COVID-19. Nature 
Medicine. 2020; 26: 672-675  
 
5. 
Corman, V., Drosten, C. et al. Diagnostic detection of 2019-nCoV by real-time RT-PCR. 
Charité Virology. 2020 
 
6. 
Udugama B., Kadhiresan, P. Kozlowski, H., Malekjahani, A.,Osborne, M., Li, V., Chen, H., 
Mubareka, S., Gubbay, J., and Chan, W. Diagnosing COVID-19: The Disease and Tools for 
Detection. ACS Nano. 2020; 14: 3822-3835