Intricacies in characterizing positivity in pooled sample testing for SARS-CoV-2.

Area of Science:

• Virology
• Molecular Diagnostics
• Public Health

Background:

• The COVID-19 pandemic led to reagent shortages, prompting sample pooling strategies.
• Sample pooling in RT-PCR can introduce dilution effects, potentially affecting sensitivity.
• Low prevalence areas benefit from pooling, but detection of low viral loads is challenging.

Purpose of the Study:

• To investigate the detection of true positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples within pooled RT-PCR tests, particularly those showing late amplification.
• To assess the impact of including "probable positive" pool criteria on identifying missed true positive cases.

Main Methods:

• Real-time polymerase chain reaction (RT-PCR) was used to test pooled nasal/nasopharyngeal/throat swabs (4 samples/pool).
• Pools identified as "probable positive" (Ct > 35, non-sigmoid curve, or late amplification) were de-convoluted for individual sample testing.
• Comparison of results using classical positive criteria (Ct ≤ 35, sigmoid curve) versus expanded criteria.

Main Results:

• An additional 39 true positive samples (15.5%) were identified by including "probable positive" pools with late amplification signals.
• These positive cases would have been missed using only the classical positive criteria for pooled RT-PCR.
• The study analyzed samples from various collection centers and hospital settings in Odisha.

Conclusions:

• Considering "probable positive" pools with late amplification in RT-PCR is crucial for accurate COVID-19 detection.
• This approach enhances the sensitivity of pooled testing, preventing the omission of true positive cases.
• Optimizing pooled testing strategies is vital for managing reagent shortages during pandemics.