Development of an alternative saliva test for diagnosis of SARS-CoV-2 using TRIzol

The use of saliva for the diagnosis of SARS-CoV-2 has shown to be a good alternative to nasopharyngeal swabs (NPS), since it permits self-collection, avoids the exposure of healthy persons to infected patients, reduces waiting times, eliminates the need of personal protective equipment and is non-invasive. Yet current saliva testing is still expensive due to the need of specialized tubes containing buffers to stabilize the RNA of SARS-CoV-2 and inactivate the virus. These tubes are expensive and not always accessible in sufficient quantities.

We now developed an alternative saliva testing method, using TRIzol for extraction, viral inactivation, and storage of SARS-CoV-2 RNA, combined with RT-qPCR, which was comparable in its performance to NPS. Paired saliva samples and NPS were taken from 15 asymptomatic healthcare workers and one patient with SARS-CoV-2. Further 13 patients with SARS-CoV-2 were only saliva-tested. All the tests were performed according to CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel. Saliva (4 mL) was taken in sterile 50 mL tubes, 1.5 mL TRIzol were added and mixed. Our results show that 5 μL of saliva RNA extracted with TRIzol allow for an adequate detection of the virus in patients positive for SARS-CoV-2 and was equally sensitive to NPS in TRIzol.

We conclude that saliva testing using TRIzol is a recommendable method for diagnosis of SARS-CoV-2 since it has several advantages over currently used saliva tests: it can be done with normal sterile tubes, does not need cold-chain handling, is stable at room temperature, is non-invasive and less costly, making it more accessible for low-income countries. Cheaper saliva testing GENPRICE GENTAUR using TRIzol is especially relevant for low-income countries to optimize diagnosis and help define quarantine durations for families, healthcare workers, schools, and other public workplaces, thus decreasing infections and mortality caused by SARS-CoV-2.

The increasing spread of SARS-CoV-2 calls for rapid, accurate and easy to handle diagnostic tests, which are essential for controlling the ongoing pandemic.

  • Although the current golden standard consists of nasopharyngeal swabs (NPS) to be analyzed by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR), an alternative diagnostic sampling method using salivary fluid is becoming more widely used to diagnose SARS-CoV-2 .
  • Salivary testing has proven to have important advantages over NPS: it permits self-collection, reducing handling by healthcare workers and thereby avoiding biosafety risks of coming in contact with aerosol or droplets.
  • Furthermore, it reduces the exposure of healthy persons to the proximity of infected patients, while waiting to be tested in hospitals or laboratories. It reduces waiting times, it lowers costs by eliminating the need of personal protective equipment and, above all, it is a non-invasive procedure.
  • SARS-CoV-2 RNA is detectable in the oropharyngeal cavity; however, respiratory sample collection can cause discomfort to the patients. Therefore, saliva is an excellent diagnostic fluid sample and had been used for detection of SARS-CoV-2 through methods such as RT-qPCR, viral culture, RT-PCR, RT-LAMP (reverse transcription loop-mediated isothermal amplification) and antigen test .
  • Saliva is a hypotonic fluid consisting of 99% water, containing multiple electrolytes, enzymes, immunoglobulins, and antimicrobial factors. It represents an attractive biofluid for the detection of diverse viral infections, including Epstein Barr virus, Human Papillomavirus, HIV, as well as respiratory viruses such as influenza, parainfluenza virus and respiratory syncytial virus .
  • More recently, SARS-CoV-2 has also been detected in saliva . The entry of SARS-CoV-2 to saliva can occur through diverse routes: blood circulation, extracellular vesicles secretion by infected cells, droplets from the respiratory tract, gingival crevicular fluid and by infection of the oral mucosal lining .

To date, most diagnostic strategies for COVID-19 detection in saliva require specialized tubes containing buffers that stabilize the RNA of SARS-CoV-2 and inactivate the virus.

Yet these tubes are expensive and not always accessible in sufficient quantities. Thus, despite that saliva is a promising type of sample that can be self-collected for the diagnosis of SARS-CoV-2, the cost and availability of specific collection tubes limit widespread testing efforts.

Therefore, the development of more affordable and easier collection options is needed due to the rising demands of testing, not only for patients but also for the continued surveillance of health care workers and front-line professionals. We here report the development a saliva test using TRIzol that can be done by self-collecting, does not require special tubes or inactivation buffer, yet has all the above-mentioned advantages, including easy and safe handling, does not require cold storage, remains stable for a week at room temperature, is highly sensible, non-invasive, and less costly.

The study was approved by the Ethics and Research Committee of the Faculty of Medicine, UNAM with Approval No. FM/DI/026/2020, and guidelines established by the Mexican Health Authorities were strictly followed. Additionally, an official written informed consent was signed by the patients, as well as by the tested health care workers, accepting the qPCR-SARS CoV-2 diagnosis. The patients (aged between 22 and 85 years) also signed a written informed consent allowing the use of their samples for research purposes, once they had received their results.

Samples of patients and health care workers

Paired saliva samples and NPS were taken between October and November 2020 from 15 asymptomatic healthcare workers at the Tropical Medical Center of the Faculty of Medicine, UNAM, Clinica Sansce and the National Cancer Institute in Mexico, to screen for SARS-CoV-2. Both types of samples (saliva and NPS) were also taken from a patient who had tested positive for SARS-CoV-2. Additionally, saliva was tested in 13 patients with SARS-CoV2, according to the criteria established by CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel.

All the NPS were taken by a trained nurse using 2 cotton swabs (one for the oropharyngeal- and one for the nasopharyngeal swabs) (Cleanmo), which were introduced into a sterile tube containing 1 mL TRIzol reagent (Invitrogen or Biosciences). In case of saliva sampling, the patients received a sealable plastic bag containing a 50 mL tube for saliva, a 2 mL tube with 1.5 mL of TRIzol, a paper towel and two gloves. The patient was asked to expel 4 mL saliva into the sterile 50 mL tube (on an empty stomach, without having performed oral hygiene) and thereafter to add 1.5 mL TRIzol to the tube containing the saliva, mix it and seal the bag (containing the 50 ml tube with saliva with TRIzol, the paper towel, the empty 2 mL tube and the gloves). A nurse received the sealed bag wearing gloves and deposited it into a cooler containing freezer packs for transport to the laboratory. This could be done at the patient’s home.

RNA extraction

Upon arrival in the laboratory, the samples containing the 4 mL saliva and 1.5 mL TRIzol were homogenized for 2 min in a vortex, incubated during 10 min at RT and kept at 4°C (6–24 hours) until RNA extraction was done. One mL of the saliva/TRIzol solution was taken and mixed with 200 μL cold chloroform (Sigma). This solution was incubated during 5 min and centrifuged at 19,357 x g for 10 min at RT.

 

The aqueous phase (500–600 μL) was recovered and 700 μL isopropanol at RT (Sigma) were added. The resulting solution was mixed for 15 s and incubated during 10 min (when extraction was done the same day) or overnight at -20°C (if extraction was finished the next day). Thereafter, the solution was centrifuged at 19,357 x g for 10 min at RT. The supernatant was discarded and 1 mL ethanol 80% (Sigma) was added, the solution was mixed during 10 s and centrifuged at 19,357 x g during 10 min at RT. The ethanol was discarded, the excess was dried at RT and the pellet was suspended in 50 μL RNAse free water and stored at -20°C until use.

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