Can dropouts in Polymerase Chain Reaction (PCR) assays for COVID-19 occur with any new variant or only with specific mutations?

COVID-19 PCR Assay Dropouts Can Occur with Any Variant Due to Viral Mutations

Yes, dropouts for COVID-19 variant PCR assays can occur with any variant that has mutations in the target regions of the PCR test, not just specific variants. This phenomenon is a direct result of viral evolution and can affect the reliability of diagnostic testing for SARS-CoV-2 1.

Mechanism of PCR Dropouts

• Primer/Probe Binding Issues: PCR dropouts occur when mutations in the viral genome affect the regions where PCR primers and probes bind 2
• Single Nucleotide Changes: Even single base mutations in the nucleocapsid gene or other target regions can alter amplification efficiency or cause complete assay failure 2
• Target Regions: Most commonly affected genes include:
  • S gene (spike protein) - notable examples include the del69-70 mutation in Alpha and Omicron variants 3
  • N gene (nucleocapsid)
  • E gene (envelope)
  • RdRp gene (RNA-dependent RNA polymerase)

Evidence of Variant-Related PCR Issues

• The emergence of Alpha, Beta, Delta, and Omicron variants has demonstrated how viral evolution can affect PCR test performance 1, 3
• S-gene target failure (SGTF) has been observed with variants carrying specific deletions like del69-70, which has been used as a surrogate marker for certain variants 3
• Reduced S-gene target performance (rSGTP) can occur even when complete dropout doesn't happen 3

Mitigation Strategies

1. Multi-Target Assays: The IDSA recommends using SARS-CoV-2 nucleic acid amplification tests that target at least two distinct viral gene sequences 4

   • This approach helps maintain diagnostic sensitivity even as the virus evolves
   • If one target region is affected by mutations, other targets can still detect the virus

2. Continuous Surveillance:

   • Regular genomic surveillance is essential to monitor for mutations that might impact diagnostic sensitivity 1
   • Public health genomics played a crucial role in identifying new variants like Omicron in South Africa and Botswana 1

3. Visual Inspection of Amplification Curves:

   • Laboratory quality procedures should include visual observation of amplification curves, not just relying on automatic threshold settings 2
   • This can help identify subtle changes in amplification efficiency that might indicate variant-related issues

4. Multiplex PCR Approaches:

   • Novel multiplex real-time RT-PCR assays have been developed to simultaneously detect multiple SARS-CoV-2 variants 5
   • These can identify specific mutations associated with variants of concern

Clinical Implications

• False negative results may occur due to viral mutations affecting PCR target regions 6, 7
• The false negative rate of COVID-19 PCR testing has been estimated at approximately 9.3% in some studies 6
• For patients with high clinical suspicion but negative initial tests, repeat testing may be warranted 4
• Clinical indicators such as CT images should be considered alongside PCR results, especially when test results are inconsistent 7

Best Practices for Testing

• Use assays targeting conserved regions of the viral genome
• Employ multiple target assays to reduce the risk of false negatives due to mutations
• Consider the possibility of PCR dropouts when interpreting negative results in the context of strong clinical suspicion
• Update testing protocols as new variants emerge and are characterized

Conclusion

PCR dropouts can theoretically occur with any SARS-CoV-2 variant that develops mutations in regions targeted by diagnostic assays. This is not limited to specific variants but is an inherent risk with any evolving virus. Using multi-target PCR assays and maintaining vigilant genomic surveillance are essential strategies to minimize the impact of viral mutations on diagnostic accuracy.