Pooling of Upper Respiratory Specimens Using a SARS-CoV-2 Real-time RT-PCR Assay Authorized for Emergency Use in...

Area of Science:

• Infectious Diseases
• Molecular Diagnostics
• Public Health

Background:

• Nucleic acid amplification testing is crucial for managing the SARS-CoV-2 pandemic.
• Specimen pooling can enhance testing throughput and resource conservation.
• Validation is essential to ensure pooled testing doesn't compromise sensitivity and increase false-negative rates.

Purpose of the Study:

• Evaluate the performance of an FDA-authorized RT-PCR test for pooled upper respiratory specimens.
• Determine the accuracy and reliability of pooled testing for SARS-CoV-2 detection.
• Assess the impact of pooling on test sensitivity and false-negative rates.

Main Methods:

• Real-time reverse transcription polymerase chain reaction (RT-PCR) was used.
• Single-positive specimens were pooled with three negative specimens across different prevalence groups.
• Passing-Bablok regression and cycle threshold (Ct) value analysis were employed to assess performance.

Main Results:

• Pooled testing demonstrated 100% positive percent agreement (PPA) with individual testing.
• A strong linear relationship was observed between Ct values of pooled and single specimens (r=0.96-0.99).
• Minimal Ct shifts were noted, with a low percentage of positive specimens falling near the assay's limit of detection.

Conclusions:

• Pooled and individual SARS-CoV-2 testing showed 100% agreement, validating pooled testing.
• Dual-target RT-PCR systems are suitable for pooled specimen testing.
• Pooled testing offers a viable strategy to expand SARS-CoV-2 testing capacity with low false-negative risk.