Making molecular assays accessible

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With the ongoing SARS-CoV-2 pandemic, mass testing of patient samples using RT-PCR has been essential to control the spread of the virus. Testing is key to determining if individuals are positive for SARS-COV-2 and isolating those patients.  However, the high costs of molecular testing relating to the need to transport samples, skilled technicians, cost of reagents for sample extraction, and long turnaround times are barriers that limit the use of RT-PCR assays. Whilst the cost of RT-PCR testing may be bearable for developed nations, this is not the same for those in resource-limited environments.

The alternative to RT-PCR tests is lateral flow tests/rapid antigen tests.  These tests have been widely used in the fight against COVID-19, owing to their ease of use, rapid turnaround times, and low cost. However, lateral flow tests demonstrate a lower sensitivity relative to RT-PCR assays, increasing the risk of false negatives, particularly with asymptomatic cases.  Sensitivity is important to limit the number of infected individuals circulating in the population and support efforts to control the spread of the virus.  Thus, despite the challenges, molecular testing via RT-PCR provides the benefit of improved sensitivity, minimizing the risk of false negatives. To promote widespread use of such sensitive molecular tests, we need to find ways to make them more accessible to all.

To deal with these needs, Erba Molecular developed the ErbaMDx SARS-CoV-2 RT-PCR Kit (EM kit) that addressed the challenges associated with cost, limited adaptability, ease of use, and shelf life whilst also providing high sensitivity. One such method adopted to reduce costs included the use of a sample pooling strategy, as reported in Jayakody et al, 2022.

Pooling refers to the process of combining multiple patient samples and analysing these combined samples using a single RT-PCR test. This process offers several benefits including high throughput testing, reduced costs, lower usage of resources, and reduced time for sample testing. The benefits of pooling can be reaped in environments where the prevalence of SARS-CoV-2 is low. This is because pooling relies on the majority of individual samples testing negative. In the event of a positive pool, retesting is required to analyse each sample and determine which of those are positive.

Pooling can be performed at different stages within the sample testing process. The two main approaches used are swab and sample/media pooling. Swab pooling combines swabs from multiple patients within a single vial of transport medium whereas sample pooling mixes transport medium (each containing material from a single patient) from multiple patients. Swab pooling does not demonstrate viral dilution however, this requires multiple samples to be collected from a patient if re-testing is required, which may reduce compliance. On the other hand, sample pooling uses a small volume of each sample and therefore does not require multiple samples from a single patient. However, this approach results in sample dilution and reduced sensitivity. The effect of reduced sensitivity can be addressed by using highly sensitive molecular technologies, such as RT-PCR. Sensitive tests such as the EM kit, with a 200cp/ml limit of detection, will be less impacted in these circumstances, as the majority of positive samples demonstrate a viral load of >1000cp/ml thus, pooling 1 in 5 still achieved accurate detection of positive samples in a pool.

The recent publication by Jayakody et al., 2022 demonstrates the process of pooling and the methods used to validate the EM kit for pooling against an FDA emergency use approved (EUA) comparator assay. The EM kit was assessed to determine its ability to accurately detect positive samples when using a 1 in 5 sample pooling strategy. This pooling strategy was selected as the appropriate method to strike a balance between loss in sensitivity whilst maximising resource efficiency. The FDA requires new pooling assays to demonstrate 85% positive percent agreement (PPA) or greater when validated against a comparator test in both pooled and non-pooled samples, to receive FDA EUA approval for pooling. The EM kit demonstrated 100% PPA in comparison to an FDA EUA kit, indicating the kit’s high sensitivity to detect low positive samples within a pool of five samples.

Other key challenges observed with many commercial diagnostic tests are the need for cold chain transport and storage along with limited adaptability, preventing their use on external sample extraction and RT-PCR platforms.  The EM kit addresses these challenges by using a lyophilized master mix. This assay format eliminates the need for cold storage, extends shelf life, and removes the need for mixing test reagents, thereby limiting both reagent cost and sources of human error. The EM kit has been validated for use with widely used extraction workflows and RT-PCR platforms – characteristics that together make the test accessible for use in multiple settings. This enables laboratories to mix and match their sample extraction workflow with their RT-PCR instruments, improving accessibility.

As the pandemic progresses, with infection rates steadily waning and a greater number of individuals being vaccinated, testing could shift to a situation where samples are mostly negative. In such circumstances, pooling could prove to be an attractive option for use in laboratories across the world. This will enable cost-effective monitoring of SARS-CoV-2 which will be of particular benefit to those in both high-resource and resource-limited environments.



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Harindi Jayakody

Scientist II, Erba Molecular