Evaluation of pooling of samples for testing SARS-CoV- 2 for mass screening of COVID-19

Abstract

Background The current pandemic of the SARS-CoV-2 virus, widely known as COVID-19, has affected millions of people around the world. The World Health Organization (WHO) has recommended vigorous testing to differentiate SARS-CoV-2 from other respiratory infections to aid in guiding appropriate care and management. Situations like this have demanded robust testing strategies and pooled testing of samples for SARS-CoV-2 virus has provided the solution to mass screening of people for COVID-19. A pooled testing strategy can be very effective in testing when resources are limited, yet it comes with its own limitations. These benefits and limitations need critical consideration when it comes to testing highly infectious diseases like COVID-19. Methods This study evaluated the pooled testing of nasopharyngeal swabs for SARS-COV-2 by comparing the sensitivity of individual sample testing with 4-and 8-pool sample testing. Median cycle threshold (Ct) values were compared, and the precision of pooled testing was assessed through an inter- and intra-assay of pooled samples. Coefficient of variance was calculated for inter- and intra-assay variability. Results The sensitivity becomes considerably lower when the samples are pooled. There is a high percentage of false negative reports with larger sample pool size and when the patient viral load is low or weak positive samples. High variability was seen in the intra- and inter-assay, especially among weak positive samples and when more number of samples are pooled together. Conclusion As COVID − 19 infection numbers and need for testing remain high, we must meticulously evaluate the testing strategy for each country depending on its testing capacity, infrastructure, economic strength, and need to determine the optimal balance on the cost-effective strategy of resource saving and risk/ cost of missing positive patients.

Implementation of Sample Pooling Procedure Using a Rapid SARS-CoV-2 Diagnostic Real-Time PCR Test Performed Prior to Hospital Admission of People with Intellectual Disabilities

“…To our knowledge, this is the first study reporting the pooling test applied in point-of-care testing thanks to a semiquantitative instrument. Similar studies have already detailed the qPCR sample pooling method [8,9]. In these studies, unlike ours, the pooled samples are diluted, resulting in less viral genetic material available to detect and thus in a greater likelihood of false-negative results.…”

“…When the sample tested negative, we excluded the entire population subjected to the test thanks to that single analysis; when the sample tested positive, the search for the positive subject was carried out by dividing the population and reanalyzing them individually. This strategy has already been demonstrated and widely successfully adopted in a number of studies performed with another method [5][6][7][8][9].…”

Evaluation of the influenza and respiratory syncytial virus (RSV) targets in the AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well assay: sample pooling increases testing throughput

“…This is because pooling may increase the Ct, exceeding the LOD threshold and may result in a false negative error. 31 , 32 Another limitation to consider is that only one influenza A (containing the H1 surface haemagglutinin) and influenza B strain was used. Therefore, it is considered in future studies to test the assay's sensitivity and specificity to identify or detect different influenza A strains (e.g., H1N1, H5N1o or H3N2) and influenza B variants (Yamagata or Victoria lineages).…”

Pooling of Samples for SARS-CoV-2 Detection Using a Rapid Antigen Test

“…Based on a number of published studies for sample pooling using RT-PCR, we opted for nasal swab eluate pools consisting of a total of 20 combined samples (14)(15)(16). 50 ml from a single RT-PCR confirmed positive nasal swab specimen dilution was mixed with 50 ml from nineteen RT-PCR confirmed negative nasal swab specimens.…”