Dataset Information

Pooling of SARS-CoV-2 samples to increase molecular testing throughput.

ABSTRACT: BACKGROUND:SARS-CoV-2 testing demand has outpaced its supply. Pooling samples for lower risk populations has the potential to accommodate increased demand for SARS-CoV-2 molecular testing. OBJECTIVE:To evaluate the sensitivity, specificity, and reproducibility of 4-way pooling of SARS-CoV-2 specimens for high-throughput RT-PCR. STUDY DESIGN:Individual samples were pooled 1:4 through automated liquid handling, extracted, and assayed by our emergency use authorized CDC-based RT-PCR laboratory developed test. Positive samples were serially diluted and theoretical and empirical PCR cycle thresholds were evaluated. Thirty-two distinct positive samples were pooled into negative specimens and individual CTs were compared to pooled CTs. Low positive samples were repeated for reproducibility and 32 four-way pools of negative specimens were assayed to determine specificity. RESULTS:Four-way pooling was associated with a loss of sensitivity of 1.7 and 2.0 CTs for our N1 and N2 targets, respectively. Pooling correctly identified SARS-CoV-2 in 94 % (n = 30/32) of samples tested. The two low positive specimens (neat CT > 35) not detected by pooling were individually repeated and detected 75 % (n=6/8) and 37.5 % (n = 3/8) of the time, respectively. All specimens individually determined negative were also negative by pooling. CONCLUSION:We report that 1:4 pooling of samples is specific and associated with an expected 2 CT loss in analytical sensitivity. Instead of running each sample individually, pooling of four samples will allow for a greater throughput and conserve scarce reagents.

SUBMITTER: Perchetti GA

PROVIDER: S-EPMC7396208 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Pooling of SARS-CoV-2 samples to increase molecular testing throughput.

Perchetti Garrett A GA Sullivan Ka-Wing KW Pepper Greg G Huang Meei-Li ML Breit Nathan N Mathias Patrick P Jerome Keith R KR Greninger Alexander L AL

Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 20200802

<h4>Background</h4>SARS-CoV-2 testing demand has outpaced its supply. Pooling samples for lower risk populations has the potential to accommodate increased demand for SARS-CoV-2 molecular testing.<h4>Objective</h4>To evaluate the sensitivity, specificity, and reproducibility of 4-way pooling of SARS-CoV-2 specimens for high-throughput RT-PCR.<h4>Study design</h4>Individual samples were pooled 1:4 through automated liquid handling, extracted, and assayed by our emergency use authorized CDC-based ...[more]

PMID: 32805524

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Project description:Tests to diagnose acute SARS-CoV-2 infection are at the center of controlling the COVID-19 pandemic. Rapid tests benefit from providing quick results but suffer from lower sensitivity, while PCR tests usually take longer to provide more reliable results and can be difficult to scale to meet population needs. We evaluated the diagnostic efficacy of a Molecular Mirror assay (MMA) using nucleic acid extraction and a nucleic acid extraction-free method to determine its ability to identify SARS-CoV-2 in nasal specimens from individuals suspected of having SARS-CoV-2. We compared the MMA using nucleic acid extraction to the emergency use authorization (EUA)-approved TaqPath reverse transcriptase PCR (RT-PCR) assay to determine its performance characteristics. From 412 total specimens (including 115 previous positives and 297 previous negatives), we found that the positive percent agreement (PPA) was 99.1% (confidence interval [CI], 97.4% to 100.0%) and the negative percent agreement (NPA) was 99.3% (95% CI, 98.4% to 100.0%) for SARS-CoV-2 detection. Using the extraction-free method, we analyzed 109 specimens (51 previous positives and 58 previous negatives) and found that the PPA for the more rapid version of the assay was 87.8% (95% CI, 78.5% to 96.9%) and the NPA was 100.0% (95% CI, 100.0%) for virus detection. The extraction method has performance comparable to what is observed in many PCR-based assays. The extraction-free method has lower PPA but has the advantage of being more rapid and having a higher throughput. Our data offer a proof of concept that nuclear magnetic resonance (NMR) detection can be used in SARS-CoV-2 diagnostic testing and may allow for alternative supply chains to increase testing options. IMPORTANCE Accurate diagnostics for SARS-CoV-2 infections have been critical for responding to the COVID-19 pandemic. Both high-sensitivity/specificity PCR-based tests and lower-sensitivity/specificity rapid antigen assays have been the subject of worldwide supply chain limitations as individual facilities and countries have struggled to meet their population testing needs. We evaluated the diagnostic efficacy of a Molecular Mirror assay (MMA), which uses nuclear magnetic resonance to detect the presence of SARS-CoV-2 nucleic acids both with and without full nucleic acid extractions. We found that compared to a U.S. emergency use authorization (EUA) approved assay (TaqPath) that uses reverse transcriptase PCR (RT-PCR), the MMA had high PPA and NPA with full nucleic acid extractions, and acceptable positive percent agreement (PPA) and negative percent agreement (NPA) with an extraction-free protocol. In a landscape marred by supply chain shortages across the world, altered SARS-CoV-2 detection methods such as the MMA can add to testing supplies while providing quality SARS-CoV-2 testing results.

Dataset Information

Pooling of SARS-CoV-2 samples to increase molecular testing throughput.

Publications

Pooling of SARS-CoV-2 samples to increase molecular testing throughput.

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