Researchers have developed an automated method of RNA extraction from saliva samples to allow faster processing times and high test sensitivity. Saliva testing could be used as an alternative to swab testing for coronavirus disease 2019 (COVID-19) surveillance.
Overview of nucleic acid extraction from saliva specimens — a , Overview of Innovative Genomics Institute’s (IGI’s) specimen processing pipeline for both swab and saliva samples. OP = oropharyngeal. OP-MT = oropharyngeal-mid turbinate. b , Cultured SARS-CoV-2 (1.58x10 6 TCID50/ml) was mixed 1:1 with OMNIgene solution present in OM-505 collection tubes to test incubation conditions that inactivate viral replication. Samples were either held at room temperature (RT) or incubated at 65°C for the indicated length of time before being applied to Vero-E6 cells. Cytopathic effect (CPE) was quantified at 3 and 7 days post treatment (dpt). c , 3:2 dilution of saliva samples with DNA/RNA Shield improves detection of spiked-in SARS-CoV-2 RNA or MS2 in four saliva donors.
Many cases of COVID-19, the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are asymptomatic. Detecting such asymptomatic cases is important for curbing the spread of the disease.
SARS-CoV-2 testing is usually performed by health practitioners through the collection of nasal or throat swabs or saliva samples. However, since this can be time-consuming and expensive, several labs have recently studied the use of self-collected samples. Saliva is an attractive option as it is easier to collect and has been proved to detect SARS-CoV-2 ribonucleic acid (RNA), which is its genetic material. Self-collection of saliva samples will increase the samples collected in a population but will also require faster testing methods.
Several saliva tests use direct RT-qPCR, eliminating nucleic acid extraction, but this reduces sensitivity. To enable faster testing of SARS-CoV-2 in saliva while improving sensitivity, US-based researchers from the University of California, Berkeley, and Washington Hospital Healthcare System Clinical Laboratory in California report an automated extraction method for SARS-CoV-2 RNA in saliva. They reported their results on the medRxiv* preprint server.
Robotic RNA extraction from saliva
Participants in the IGI-FAST study at the University of California, Berkeley, self-collected about 1 mL of saliva sample in DNA Genotek OMNIgene collection tubes at kiosks set up on campus. The team used a two-step pre-extraction procedure, by incubating the samples at 50°C for two hours and at 65°C for 30 minutes, to inactivate the virus.
For robotic sample plating and RNA extraction, the authors diluted the saliva sample in a solution that disrupts the DNA structure, or a chaotropic solution. This allowed improved RNA detection.
Using saliva samples of COVID-19 negative participants, the researchers determined the limit of detection of the assay by adding a positive control RNA or inactivated SARS-CoV-2 virus to the samples. They found the assay could detect down to 3000 RNA copies/mL and 50% tissue culture infective dose (TCID50)/mL. The team tested 20 more samples with added virus RNA to confirm the robustness of the robotic test method.
The team then tested their laboratory procedure in a real-world setting (on campus), using the IGI-FAST study. The study ran from June to October 2020, involving 3,653 participants and about 12,000 saliva samples. During the first three weeks, about 6% of the samples gave errors during testing. So, the team modified their laboratory procedure to make it more robust.
Although the team tested pooled saliva samples, they found many specimen-insufficient errors, negating any advantages of pooling the samples. In addition, it increased the test turnaround times and was logistically difficult to manage. Thus, the team went back to testing individual saliva samples.
The authors compared their saliva test with nasopharyngeal samples of the same individuals; the latter tested using both standard methods and the robotic method. There was about 73% positive agreement between the swab and saliva samples.
The good and bad of testing saliva
The results allowed the team to identify several advantages and disadvantages of testing saliva for SARS-CoV-2. Automated nucleic acid extraction and processing from saliva was very sensitive, but the saliva quality was highly variable, which affects extracting and amplification processes. Furthermore, specimen amounts were insufficient from saliva samples compared to nose and throat swabs, which prevented running pooled samples.
The authors and others have found that saliva samples tested after two or more days after collection are easier to extract and process. Thus, “A key takeaway from this work is the importance of performing validation experiments with freshly-collected saliva samples from the target population to ensure assay performance is assessed according to real-world applications of the assay,” write the authors.
The saliva samples were also less clinically sensitive than the swab samples. This could be because due to variance in viral loads at different sites in the body or the stage of infection during the sample collection period. The authors also had difficulty obtaining consent from inpatients, as many were too weak to provide a sample. Thus, saliva sample collection requires active patient participation. Several other labs have also had difficulty testing saliva samples and continued with testing swabs.
Despite the challenges, the authors write that saliva testing is an important and complementary alternative to swab tests. The team continues to offer their saliva test as a surveillance method at the UC Berkeley campus, usually provided as an in-home test to allow testing smaller target groups or people who are not able to tolerate swab tests.
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.