In a recent study published in Med, researchers monitored the remarkably rapid Omicron BA.1 to BA.2 sublineage dominance transition in the Swedish population in early 2022 on a day-to-day basis using a customized lineage-typing reverse transcriptase-polymerase chain reaction (RT-PCR) assay.
In January 2022, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) new variant of concern (VOC) Omicron caused around 50 million infections worldwide daily, far exceeding the peak of 14 million Delta VOC cases during April 2021. The figures signify an unprecedented level of Omicron transmission worldwide.
In late December 2021, amid the Omicron BA.1 wave in Sweden, the Omicron sublineage BA.2 (B.1.1.529.2) rapidly spread through the Swedish population. Swedish health authorities used an RT-PCR assay capable of genotyping Omicron BA.1 cases directly in primary SARS-CoV-2 RT-PCR tests, enabling day-to-day tracking of the BA.1 cases at the population scale.
About the study
In the present study, researchers analyzed 174,933 clinical upper airway samples, including throat swabs, nose swabs, and saliva collected in Sweden from January to March 2022 using an RT-PCR assay directly classifying SARS-CoV-2 sublineage status. They demonstrated how the Omicron BA.1 sub-variant was consistently outcompeted by BA.2.
The team developed a modified version of the Centers for Disease Control and Prevention (CDC) SARS-CoV-2 RT-PCR assay. They designed combinatorial SBA1-primer-probe sets, matching CDC N1 properties but with minimal amplicon length to attain a multiplex assay of high sensitivity and specificity. This modified assay was apt for RNA-extraction-free RT-PCR on heat-inactivated samples used in mass testing. Furthermore, it simultaneously detected general SARS-CoV-2 infection status, human RNaseP sample integrity (RP-HEX), and Omicron BA.1-lineage status (SBA1-Cy5) leveraging BA.1-specific mutations in the spike (S) gene.
Furthermore, the researchers obtained the first in vitro expanded Omicron BA.1 inoculate in Sweden from the global initiative on sharing avian flu data (GISAID) database. They used 185 clinical upper airway samples of known SARS-CoV-2-infection status to validate that the addition of SBA1-Cy5 probes did not affect nucleocapsid (N)1 cycle thresholds (CT) values and the sensitivity to detect general SARS-CoV-2 infection in primary RT-PCR. The C28311T substitution at the third base position of the N1 probe had a negligible effect on CT values by tests using CDC N1 and custom Omicron N1 probes.
The authors observed that the detection sensitivity and log-linear CT range were similar for both the N1 and SBA1 sets in the assay conditions. Parallel genotyping of SARS-CoV-2-positive samples using SARS-CoV-2 mutation panel assay and whole-genome sequencing (WGS) provided Omicron-BA.1-case classification that was 100% consistent with the study used direct RT-PCR assay.
The authors also observed that the predominant Omicron BA.1 lineage was gradually and consistently outcompeted by Omicron BA.2 across nine Swedish healthcare districts. The study data also showed that BA.2 cases presented nearly double the levels of viral ribonucleic acid (RNA) in the upper airways compared to BA.1 cases, suggesting that a higher viral load most likely contributes to the BA.2 lineage’s increased transmissibility. Given SARS-CoV-2 droplet and aerosol transmission, these results validated the notion that an elevated viral load contributes to increased transmissibility of BA.2, as immune evasion of both the sub-variants was similar.
The study demonstrated the viability and usefulness of lineage typing of select SARS-CoV-2 lineages or variants for population monitoring directly in the primary SARS-CoV-2 RT-PCR, without burdening the clinical laboratory with separate genotyping RT-PCR assays. Backtracking viral RNA levels after obtaining WGS results biases sample inclusion towards strongly positive samples. The current study showed a way to avoid the time-lag and logistic bottlenecks of genotyping by WGS sequencing.
The direct Omicron BA.1-typing assay used in the current study appeared like a compatible addition to the well-established extraction-free SARS-CoV-2 RT-qPCR. Therefore, it could easily be deployed for instant monitoring of the Omicron BA.1/BA.2 transition and lineage assignment. Also, it could quantify viral RNA levels in all the samples of a large population. Importantly, it used a novel SBA1-Cy5 primer-probe set that could readily be replaced to target and monitor other SARS-CoV-2 variants and adapted to other viruses in the future.