AUTHOR=Millward Georgia G. , Popelka Shane M. , Gutierrez Anthony G. , Kowallis William J. , von Tersch Robert L. , Yerramilli Subrahmanyam V. 

TITLE=A novel strategy to avoid sensitivity loss in pooled testing for SARS-CoV-2 surveillance: validation using nasopharyngeal swab and saliva samples

JOURNAL=Frontiers in Public Health

VOLUME=Volume 11 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/public-health/articles/10.3389/fpubh.2023.1190308

DOI=10.3389/fpubh.2023.1190308

ISSN=2296-2565

ABSTRACT=In the peak of the COVID-19 pandemic, pooled surveillance strategies were employed to alleviate overwhelming demand on clinical testing facilities. A major drawback in most pooled-testing methods is the dilution of positive samples leading to loss of detection sensitivity and potential for false negatives. We developed a novel pooling strategy that compensates the initial dilution by appropriate concentration during nucleic acid extraction and real-time PCR. We demonstrated the proof of principle using laboratory created 10-sample pools with one positive and corresponding individual positive samples by spiking a known amount of heat-inactivated SARS-CoV-2 into viral transport medium (VTM) or pooled negative saliva. No Ct difference was observed between a 10-sample pool with one positive vs. the corresponding individually analyzed positive sample by this method, suggesting that there is no detectable loss of sensitivity. We further validated this approach by using nasopharyngeal swab (NPS) specimens and showed that there is no loss of sensitivity. Serial dilutions of the virus were spiked into VTM and pooled negative saliva in simulated 10-samples pools containing one positive to determine the LOD and process efficiency of this pooling methodology. LOD of this approach was 10 copies/PCR and the process efficiencies are ~95-103% for N1 and ~87-98% for N2 with samples in different matrices and with two different master mixes tested.Relative to TaqPath 1-step master mix, TaqMan Fast Virus 1-Step master mix showed better sensitivity for N2 assay while N1 assay showed no Ct difference. Our pooled testing strategy can facilitate large scale, cost-effective SARS-CoV-2 surveillance screening and maintains same level of sensitivity when analyzed individually or in a pool. This approach is highly relevant for public health surveillance efforts in mitigating SARS-CoV-2 spread.