Dec 14, 2021
The winter holiday season is in full swing. For many, this means getting together with friends and family to celebrate indoors as temperatures drop. Following interruptions caused by COVID-19 in 2020, people are eager to get back to their usual family traditions, but in addition to COVID-19, conditions are ripe for influenza and other respiratory viruses.
With various levels of mask mandates, there is an even higher risk of exposure to seasonal flu. Even with people receiving both flu and COVID-19 vaccines, people will still become ill this season, often with indistinguishable symptoms that resemble the flu, COVID-19, and respiratory syncytial virus (RSV).
To manage this situation effectively, it is crucial to use combination tests that detect and differentiate SARS-CoV-2, influenza A, influenza B, and RSV, as individuals can be infected with none or multiple viruses at once. In a July 2021 communication regarding SARS-CoV-2 testing, the CDC encourages laboratories to consider adoption of a multiplexed method that can facilitate detection and differentiation of SARS-CoV-2 and influenza viruses.
Specifically, a multianalyte RT-PCR test allows labs to conserve precious resources by avoiding multiple tests on samples collected from individuals suspected of respiratory viral infection consistent with COVID-19. These tests will help alleviate confusion arising from similar symptoms caused by these infections and reduce further strain on the health care system.
Impact of SARS-CoV-2 variants
As variants of concern (VOC) like Omicron (B.1.1.529) emerge rapidly, ensuring the accuracy of multianalyte diagnostic assays is an important aspect of test development. With dozens of variants spreading at different speeds, developers must update the arsenal of available tests to outpace the mutations.
Second tier RT-PCR tests designed to identify mutations associated with specific variants have provided labs another method of tracking the VOCs in SARS-CoV-2 positive samples. As the COVID-19 pandemic eventually scales down to an endemic, such tools can complement next-generation sequencing (NGS) and facilitate future surveillance of mutations.
Designing multiplex assays
Target identification is the first step in designing a multiplex assay. This is done with information gathered from scientific publications, databases like NCBI and GISAID, and agencies such as the World Health Organization. Care is taken when choosing the targets for pathogen identification. For example, the SARS-CoV-2 S gene (coding for the spike protein) is prone to mutation and should be prudently avoided during assay design. After the targets are identified, assay developers typically take the following steps into consideration:
- Primer design using bioinformatics algorithms
- Wet lab testing to ensure the primer/probe designs work well
- Optimization of cycling and other conditions to increase specificity and sensitivity
- Design iterations as needed
- Assay validation with commercially available reference materials and/or patient samples
Another aspect that clinical lab managers should take into consideration is the possibility of incorporating RT-PCR tests into an automated workflow that streamlines sample analysis. An ideal workflow is characterized by modular design, flexible extraction, and liquid handling options to increase throughput, while enhancing safety and saving time with automation.
The commendable work done by clinical laboratories and health care agencies across the globe has enabled combination tests to evolve as a reliable method to detect and differentiate SARS-CoV-2 from influenza and other viruses that cause similar symptoms. These diagnostic tools boost our confidence in navigating the flu season successfully and delivering optimal outcomes for all.