Self-Collected Saliva Samples Without Viral Transport Media for COVID-19 Testing Via RT-PCR

This is a descriptive study that will attempt to validate the procedure for detecting
SARS-CoV-2 on the Eppendorf Thermal Cycler PCR system using self-collected saliva as the
specimen. Investigators will calculate the percent agreement and the 95% confidence intervals
(ie, overall agreement, percent positive agreement, and percent negative agreement) between
self-collected saliva samples (evaluated on the Eppendorf Thermal Cycler PCR system) and
healthcare-worker collected nasopharyngeal samples (evaluated on either the BioFire®
FilmArray® Torch® system or the Cepheid® GeneXpert® as the gold standard). Participants
without usable results for both the saliva and nasopharyngeal methods will not be included in
the analyses. Currently, DGMC uses both FDA Emergency Use Authorization systems
interchangeably to test for SARS-CoV-2.

Using human volunteers to obtain positive and negative nasopharyngeal and saliva specimens of
SARS-COV-2, this is a test validation study. It uses the Eppendorf Thermal Cycler PCR system
to compare the test performance of saliva as a specimen to the standard nasopharyngeal
specimen.

This protocol utilizes two different convenience samples of DGMC patients. Patients (i.e.
inpatient, outpatients) with a positive COVID-19 test in the past 72 hours, and those who
present for COVID-19 testing at DGMC and have negative results. Both groups of patients are
willing to provide a saliva sample for COVID-19 testing within 72 hours. The populations will
be comprised of active duty, DoD dependents, and retirees who are DGMC patients. This study
will enroll at least 60 individuals. Only saliva samples from 30 participants with negative
and 30 with positive NP results will be processed. The study will stop recruiting positive
patients after obtaining the paired saliva and NP results from 30 participants who are
COVID-19 positive by NP test. The study will stop actively recruiting negative patients after
obtaining the paired saliva and NP results from 30 participants who are COVID-19 negative by
NP test. More than 30 negative patients may be recruited in an attempt to capture 30 positive
patients.

Recruitment efforts for positive patients will be conducted utilizing a HIPAA waiver to
identify potential study participants, as well as, targeting patients exposed to and/or with
symptoms related to COVID-19 who present for COVID-19 testing. The HIPAA Waiver will be used
to identify patients (inpatient, outpatient) who have had a positive COVID-19 NP test within
the past 72 hours. These patients, as well as others who will have COVID-19 testing at DGMC,
will be informed of the study.

Patients interested in participation will make contact with study staff. Those eligible to
participate will be given study documents (consent and HIPAA Authorization Form), that will
later be reviewed and signed by the patient and study staff. Thereafter, additional
information (contact, demographic), and a self-collected saliva specimen will be obtained.

To minimize contact with COVID-19 positive or potentially infected patients when signing
documents and obtaining the self-collected saliva sample, staff will wear scrubs (or a lab
coat), gloves, eye protection and N95 mask. Documents requiring signature will be signed by
the patient while wearing a mask and maintaining a distance of at least 6 feet from study
staff or from their vehicle. Consent and HIPAA Authorization Forms will be signed. The
original, signed documents will be maintained by study staff and a copy will be provided to
the participant. After both documents have been signed, study staff will obtain demographic
(age, ethnicity, race and sex) and contact information (name, email address if future contact
is desired), date of birth, and the self-collected saliva sample.

Prior to collecting the saliva sample, study staff will ensure the participant has not
smoked, eaten or drank anything for the previous 10 minutes. Participants will be given a
biohazard bag, label (with a 6-digit numerical code), 25 ml plastic specimen tube without
transport medium inside. Then asked to fill it with approximately 2 mL of saliva. There will
be a marking on the tube to ensure a sufficient sample is collected. The participant will be
instructed to close the lid, place the label on the outside of the tube, then put the labeled
tube into the biohazard bag. Participants will then place the bagged specimen inside a second
biohazard bag held by either DGMC or a member of the study team. The double-bagged
self-collected saliva sample will then be placed into the study cooler containing an ice
pack. Saliva specimens will then be transported to the CIF in batch.

After obtaining the paired saliva and NP results from 30 participants who are COVID-19
negative by NP test, saliva testing will only be performed on samples collected from NP
positive participants. Patients with unknown COVID-19 status, enrolled in an attempt to
capture a COVID-19 positive patient, will have the medical record accessed to retrieve the NP
result prior to performing saliva testing. If the NP result is positive, saliva testing will
be performed.

Testing will be performed on saliva samples using reverse-transcription polymerase chain
reaction (RTPCR). Natural and/or synthetic RNA will be acquired for use as a positive
control. RT-PCR will target the E-gene and N-gene of SARS-CoV-2.

1. Within 5 minutes of sample collection the sample will be kept cold for stabilization. A
cooler with an ice pack inside will be taken to the location where the participant
provides the sample. The sample will be put in a biohazard bag, then immediately be
placed into the cooler for transport to the CIF Laboratory in batch.

2. Ribonucleic Acid (RNA) Extraction - Within 12 hours, RNA will be extracted from the
specimens using Invitrogen PureLink ® Viral RNA/DNA Mini Kit (Life Technologies). Sample
RNA will be stored at -80oC until used.

3. SuperScriptTM III One-Step RT-PCR with PlatinumTM Taq (Life Technologies) will be used
for detection and amplification of viral RNA present in specimens using Eppendorf
Mastercyler Pro S (Eppendorf). Up to seven primer pairs that are based from CDC and
other publications will be used.18-19 Three from the envelope protein gene and four from
the nucleocapsid protein gene.

4. Frozen samples will be tested and run in batches.

5. PCR products will be evaluated with gel electrophoresis using the Mini-Protein® Tetra
Cell (Bio-Rad).

6. Sequencing - All PCR reactions that produce acceptable amplicons will be stored at -80oC
for possible sequencing confirmation using the ABI 3130 (Life Technologies).

Once testing is completed, specimens will be aliquoted and stored at -80oC.

Saliva samples will be interpreted as negative, positive or inconclusive. Negative will be
reported when there is no detection in any target; inconclusive when 1 target is detected;
and positive when 2 or more targets are detected.

Study staff will access the lab accession number associated with each SARS-CoV-2 positive NP
result to request a print out, from the DGMC clinical lab, of the raw data from the
instrument used to perform testing. This information will be used to evaluate any
discrepancies between the two testing methods.