When False-Positives Arise: Troubleshooting a SARS-Coronavirus-2 (SARS-CoV-2) Detection Assay on a Semi-Automated...

Area of Science:

• Molecular Diagnostics
• Infectious Disease Testing
• Laboratory Automation

Background:

• High-throughput SARS-CoV-2 testing became common during the COVID-19 pandemic, utilizing automated workflows.
• Vaccination campaigns led to increased testing of vaccinated individuals, raising questions about positive results.
• A quality improvement initiative was launched to investigate and address false-positive results in semi-automated testing.

Purpose of the Study:

• To investigate the causes of false-positive results in a semi-automated SARS-CoV-2 molecular diagnostic workflow.
• To implement procedural improvements to mitigate contamination and improve accuracy.
• To establish a reliable testing protocol for molecular diagnostic laboratories.

Main Methods:

• Utilized the MagMax-96 Viral RNA kit and CDC 2019-nCoV RT-qPCR Panel on the Agilent Bravo Liquid-Handler.
• Conducted environmental, personnel, and instrumentation screening using checkerboard challenges to detect cross-contamination.
• Evaluated assay design and reporting algorithms, adjusting them based on identified contamination indicators.

Main Results:

• Specimen contamination was identified during the viral extraction process on the Bravo system.
• Program modifications reduced plate contamination by 50% and revealed consistent hallmarks of contaminated samples.
• Adjusted reporting algorithm identified false positives, which constituted 0.11% of over 45,000 tests.

Conclusions:

• Implemented adjustments ensured confident, quality results while maintaining timely patient reporting.
• The corrected false-positive rate aligns with published data from automated systems, potentially setting a laboratory performance standard.
• The improved workflow serves as a resource for other laboratories facing similar testing anomalies.