Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has caused the deaths of over 6.2 million people to date, with numbers rising every day¹. The gold standard of testing for SARS-CoV-2 is quantitative real-time (RT-q) PCR, with primers designed to target the viral genome, such as nucleocapsid, envelope, spike, and RNA-dependent RNA polymerase genes². At the beginning of the pandemic, sufficient capacity for SARS-CoV-2 testing was severely lacking. It arose from a lack of validated assays, testing components, clinical personnel, and an infrastructure unprepared to rapidly expand to accommodate pandemic-level, mass testing. Due to shortages, testing centers often required a physician's referral to be test eligible. These shortages resulted in delays for testing approval, long lines for uncomfortable nasopharyngeal sample collection, and lengthy wait times for results. Additionally, because of these constraints, testing efforts could not accommodate pre-symptomatic, mild, or asymptomatic carriers unknowingly spreading the SARS-CoV-2. The lack of easily accessible, widespread testing likely contributed to the uncontrolled spread of COVID-19.

Large-scale interval testing can be performed either as surveillance or screening. Both can be used to monitor local positivity rates in high-density or high-risk transmission areas and can be utilized to make public health decisions. Surveillance testing is intended to monitor the incidence and prevalence of disease in a population and is not used for individual diagnostics³. Surveillance results typically are de-identified and not returned to participants; laboratories conducting surveillance testing need not be clinically certified, nor are they required to use an FDA-authorized assay. Screening allows for results to be returned to individual participants, but screening laboratories in the United States must have a Clinical Laboratory Improvement Amendments (CLIA) certificate and meet all applicable CLIA requirements.

The Michigan State University (MSU) early detection program began in September 2020 and has processed over 350,000 samples. The program arose out of a research group's efforts to design a highly sensitive SARS-CoV-2 assay that did not require high demand testing supplies^4,5,6,7,8. The goals were to aid clinical labs to increase capacity and develop flexible processes to accommodate supply shortages while also developing a screening strategy to establish a return-to-work plan for the MSU College of Human Medicine. The initial efforts focused on alternative collection, extraction, and quantitation methods for SARS-CoV-2. High demand and subsequent shortages of nasopharyngeal swabs led to the evaluation of anterior nares samples collected with buccal swabs, and reagent shortages resulted in development of a sample extraction method adapted from early reports out of Wuhan, China⁹. To increase sensitivity for detecting SARS-CoV-2 in anterior nares samples, droplet digital PCR was substituted for RT-qPCR^6,7. Though droplet digital PCR is highly sensitive and can provide absolute values with an endpoint readout, it was determined that its use was not feasible for large-scale testing due to the lack of reliable high-throughput instrumentation for the technology. Additionally, self-collection of anterior nares samples based on levels of human RNase P was extremely variable, suggesting that it was not sufficiently reliable for mass testing.

An alternative to nasopharyngeal and anterior nares swabs is the collection of saliva. Respiratory viruses such as SARS-CoV, H1N1, and MERS were all historically detected in saliva^10,11,12,13. This was subsequently proven true for SARS-CoV-2^14,15,16,17. Direct comparison between saliva and nasopharyngeal samples showed saliva yields higher viral titers than nasopharyngeal swabs in matched samples, and that saliva is less variable with repeated sample collection¹⁴. Saliva has also been reported to be more sensitive in certain variants, such as Omicron, compared to Delta¹⁶. Added benefits to saliva collection are the relative ease of off-site self-collection without high-demand supplies, the ability to repeatedly retest the sample if needed, the elimination of on-site staffing requirements for sample collection, and the avoidance of participant queues which could increase the potential for viral transmission. The lab-assembled saliva kit was developed as a collaboration among lab assay developers, experts in the school of packaging, university branding experts, safety officers, and external manufacturing partners that produced the box and labeling system.

While saliva samples offer ample genetic starting material and RT-qPCR provides sensitive, reliable outcomes, the cost of reagents (primer/probes and master mix) made large-scale testing of individual samples a costly endeavor on an individual, per sample basis. Since the primer/probes and master mix are the most expensive components of the process, the goal was to seek solutions that would stretch their use and therefore decrease the per sample cost. Systemically optimizing sample pool size based upon incidence in the community and assay sensitivity has been proposed for SARS-CoV-2 testing¹⁸. However, when pools of any size indicate presence of SARS-CoV-2, all participants in the pool must be retested, resulting in lost time and increased opportunities for spread. To address these limitations, a two-dimensional pooling method was employed, like the process proposed by Zilinskas and others¹⁹ to conduct a first pass under the strictures of surveillance testing. In this process, 96 individual samples are placed in a 96-well plate consisting of 12 columns and eight rows. Each sample is included in a pool of eight and a pool of 12 on two different reaction plates. This results in every sample being uniquely represented with the two pools. Deconvolution of the pools based on the coordinates identifies potentially positive samples. Samples in pools where SARS-CoV-2 was not detected, do not move from surveillance testing to screening. Meanwhile, samples from individuals testing positive in the surveillance process are re-extracted through a CLIA-approved screening process. If confirmed positive, individuals are given their result, referred to the university physician's office, contact tracing is initiated, and the health department is notified. In total, an individual's sample is tested in three separate reactions before being declared positive, twice in surveillance pools and once as a single confirmatory screen, reducing the chances of a false positive. Sample pooling uses ~80% less reagents than running samples individually, resulting in a cost of ~$12 per sample.

Beyond the saliva kit, pooling strategy, and assay development process, the team also developed a logistics plan for distribution of kits, collection of samples, and reporting of results. Participants in the program pick up their kit, register the unique alphanumeric code on their tube, produce their sample and deposit it in one of the many drop-off bins where they are picked up daily and transported to the lab. The lab processes the samples, the technical supervisor reviews and uploads results, and participants are notified to check the results portal. This process has a turnaround time of 24-48 h from the time a sample is deposited. The collaboration from all parts of the institution were key for a successful large-scale implementation of this hybrid surveillance and screening process. The following procedures and descriptions of the testing program and infrastructure needed are intended as blueprints on how to scale-up testing for future surveillance and/or screening purposes.