Could automated, saliva-based PCR testing be the future of COVID-19 surveillance?

By Pooja Toshniwal PahariaAug 28 2023

In a recent study posted to the medRxiv preprint* server, researchers established a polymerase chain reaction (PCR)-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing program in an educational institution, analyzing saliva samples obtained from asymptomatic students and staff.

SARS-CoV-2 testing in the community was crucial during the coronavirus disease 2019 (COVID-19) pandemic, with extensive facilities and volunteers from universities setting up mass testing programs. However, contact tracing and management of these large facilities were complex, and testing was limited to symptomatic individuals. Universities offer an advantage by offering a flexible workforce, space availability, and experience in service provision and management.

Study: KCL TEST: an open-source inspired asymptomatic SARS-CoV-2 surveillance programme in an academic institution. Image Credit: Dizfoto / Shutterstock

*Important notice: medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

About the study

In the present prospective cohort study, researchers developed an automated, PCR-based service to detect SARS-CoV-2 from saliva samples and enable surveillance of asymptomatic community residents.

The study was conducted between December 2020 and July 2022. KCL test, funded by Kings College London, comprised several teams, including operations, laboratory, and management. In total, there were 18 staffed hubs located around London, United Kingdom (UK), where students and workers could pick up saliva sampling kits and submit their saliva samples. The authors created their laboratory information management system (LIMS) to log samples and save handling times by utilizing the barcodes present on the tubes. SARS-CoV-2 ribonucleic acid (RNA) was detected in the saliva samples of asymptomatic students, staff members, and their home contacts by real-time quantitative PCR (RT-qPCR).

The exam was created following the UK Department of Health and Social Care (DHSC) criteria. Asymptomatic individuals were motivated to test every day (and twice a week on campus). The primary outcome measures were the number of COVID-19 tests performed, the number of individuals tested, and the rate of SARS-CoV-2 positivity. The samples were processed using an automated, open source-low-cost framework using non-commercial-type reagents. Sample processing was anonymized since laboratory personnel solely used the barcodes on the tubes.

The results were delivered to the service users (who were matched using barcode data) as well as to the National Health Service's Test and Trace program, which contributed to national testing efforts. The management team was also provided information on SARS-CoV-2-positive people and the likelihood of possible outbreaks, such as whether positive samples were grouped in certain areas. Triplex assays were done to identify SARS-CoV-2 RNA utilizing the SARS-CoV-2 nucleocapsid (N) and envelope (E) genes and the human ribonuclease P (RNAseP) as a negative outcome control and primer sequences provided by the United States Centers for Disease Control and Prevention (US CDC).

Data acquired from hundreds of RNA samples were used to generate detection and cycle threshold (Ct) values to determine the best range of human ribonuclease P amplification. As part of the accreditation criteria, the team completed competency exams, including theoretical questions concerning the assays. The researchers created a "homebrew" technique for RNA extraction from nasal swabs which was improved to extract RNA from salivary samples, and its performance was evaluated using 980 samples. The researchers also assessed the assay's specificity and sensitivity, testing 250 negative and 150 positive samples using commercial kits, comparing the findings to those of a United Kingdom Accreditation Service (UKAS)-accredited testing laboratory, and running 67 of the samples using the in-house technique. The researchers compared saliva and swab samples of the same individual with swab specimens processed in a different UKAS-accredited facility.

Results

In total, 58,277 PCR tests were performed on saliva, with 2,989 positive (1.9%), collected from 20,186 individuals. During the study period, 160,000 samples were processed using the KCL methodology. Every day for 18.0 months, between 10.0 and 30.0% of campus footfall was evaluated. To detect SARS-CoV-2, the turnaround time was eight hours, and the detection limit was 100 copies per mL. Except for the period of SARS-CoV-2 Delta variant of concern (VOC) predominance, the SARS-CoV-2 positive rate was comparable to that reported by the Office for National Statistics in the United Kingdom; Delta infections were minimal in the study cohort.

The swiftly built automated SARS-CoV-2 surveillance service received the United Kingdom Accreditation Service (UKAS) accreditation under the International Organization for Standardization (ISO) 15189 standard. The system allowed for down- and up-scaling, making it adaptable and applicable in different contexts and for various objectives. The homebrew approach matched the commercial RNA extraction method for detecting SARS-CoV-2 in saliva, indicating that non-commercial reagents provided equivalent results at a fraction of the cost of commercial reagents and may be utilized and accredited for diagnostic purposes.

According to UK government criteria for healthcare and public health screening and testing, 85% sensitivity and 100% specificity were observed. Restricting the analysis to samples with more than 10⁶ copies/mL increased the sensitivity to 95%. SARS-CoV-2 identification differed between combined saliva samples and nasopharyngeal and oropharyngeal swabs, with salivary detection dropping quicker than the combined nasopharyngeal and oropharyngeal swabs. This is owing to a faster SARS-CoV-2 clearance from the oral mucosa, indicating that saliva may provide a more precise measure of infectiousness than swab samples.

Despite testing numbers being consistent and the return to university gatherings that occurred in September, the proportion of SARS-CoV-2-positive samples showed similar patterns compared to that documented by the United Kingdom's Office for National Statistics (ONS), with the exception of a practically missing delta wave. The findings also showed a significant elevation in SARS-CoV-2 positivity between May end and the beginning of June 2022.

The study findings showed that the SARS-CoV-2 RNA detection framework provided minimally invasive sampling, accurate reporting, and low-cost diagnostics. Asymptomatic COVID-19 testing in the community is valuable, and governments should engage with academic centers to develop pipelines and alleviate overstretched healthcare systems.

*Important notice: 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.