Sample Pooling as a Strategy of SARS-COV-2 Nucleic Acid Screening Increases the False-negative Rate

BackgroundIdentification of less costly and accurate methods for monitoring novel coronavirus disease 2019 (CoViD-19) transmission has attracted much interest in recent times. Here, we evaluated a pooling method to determine if this could improve screening efficiency and reduce costs while maintaining accuracy in Guangzhou, China. MethodsWe evaluated 8097 throat swap samples collected from individuals who came for a health check-up or fever clinic in The Third Affiliated Hospital, Southern Medical University between March 4, 2020 and April 26, 2020. Samples were screened for CoViD-19 infection using the WHO-approved quantitative reverse transcription PCR (RT-qPCR) primers. The positive samples were classified into two groups (high or low) based on viral load in accordance with the CT value of COVID-19 RT-qPCR results. Each positive RNA samples were mixed with COVID-19 negative RNA or ddH2O to form RNA pools. FindingsSamples with high viral load could be detected in pool negative samples (up to 1/1000 dilution fold). In contrast, the detection of RNA sample from positive patients with low viral load in a pool was difficult and not repeatable. InterpretationOur results show that the COVID-19 viral load significantly influences in pooling efficacy. COVID-19 has distinct viral load profile which depends on the timeline of infection. Thus, application of pooling for infection surveillance may lead to false negatives and hamper infection control efforts. FundingNational Natural Science Foundation of China; Hong Kong Scholars Program, Natural Science Foundation of Guangdong Province; Science and Technology Program of Guangzhou, China. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSSince it emergence in late 2019, CoViD-19 has dramatically increased the burden healthcare system worldwide. A research letter titled "Sample Pooling as a Strategy to Detect Community Transmission of SARS-CoV-2" which was recently published in JAMA journal proposed that sample pooling could be used for SARS-COV-2 community surveillance. Currently, the need for large-scale testing increases the number of 2019-nCOV nucleic acid analysis required for proper policy-making especially as work and normal school resumes. As far as we know, there are many countries and regions in the world, who are beginning to try this strategy for nucleic acid screening of SARS-CoV-2. Added value of this studyWe carried out a study using pooled samples formed from SARS-COV-2 negative samples and positive samples with high or low viral and assessed detection rate for the positive samples. We found that positive sample with high viral load could be detected in pools in a wide range of dilution folds (ranging from1/2 to 1/50). On the contrary, the sample with low viral load could only be detected in RNA "pools" at very low dilution ratio, and the repeatability was unsatisfactory. Our results show the application of the "pooling" strategy for large-scale community surveillance requires careful consideration and depends on the viral load of the positive samples. Implications of all the available evidenceAlthough the number of newly diagnosed cases has been reducing in some parts of the world, the possibility of a second wave of infection has made quick and efficient data gathering essential for policy-making, isolation and treatment of patients. Fast and efficient nucleic acid detection methods are encouraged, but sample pooling as a strategy of SARS-COV-2 nucleic acid screening increased the false-negative rate, especially those with asymptomatic infections have lower viral load. Therefore, the application of the "pooling" strategy for large-scale community surveillance requires careful consideration by policy makers.