Increasing SARS-CoV-2 RT-qPCR testing capacity by sample pooling

Alcoba-Flórez, Julia; Gil-Campesino, Helena; Artola, Diego García-Martínez de; Díez-Gil, Oscar; Valenzuela-Fernández, Agustı́n; González-Montelongo, Rafaela; Ciuffreda, Laura; Flores, Carlos

doi:10.1016/j.ijid.2020.11.155

Cited by 34 publications

(33 citation statements)

References 16 publications

(8 reference statements)

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“…Results from this retrospective study are consistent with recent reports confirming an insignificant effect on the false negative rate for pools of similar size; additionally, our average CT value shifts were precise [14,15]. Probit analysis is applicable to the bimodal output of RT-qPCR for LoD analysis.…”

Section: Discussionsupporting

confidence: 92%

Two Stage Hierarchical Group Testing Strategy to Increase SARS-CoV-2 Testing Capacity at an Institution of Higher Education: A Retrospective Analysis

Ganz

Waithe-Alleyne

Slate

et al. 2021

Preprint

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Population testing for severe acute respiratory syndrome 2 (SAR-CoV-2) is necessary owing to the possibility of viral transmission from asymptomatic cases, yet scarcity of reagents and equipment has added to the cost prohibitive implementation of screening campaigns at institutions of higher education. The high analytical sensitivities of leading nucleic acid amplification diagnostic methods allow for group testing to increase testing capacity. A feasibility study was performed using an optimized testing configuration model for pooling three, five, and ten samples. Following the standard RNA extraction and purification workflow for quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) method using Thermo Fisher TaqPath COVID-19 multiplex primers and probes for the ORF1ab, N, and S genes, matrix and dilution effects were assessed using pooled negative samples as the diluent. Probit analysis produced a limit of detection of 16075 (ORF1ab), 1308 (N), and 1180182 (S) genomic copy equivalents per milliliter. Trials comparing neat to 1:5 dilution for 34 weak-to-strongly positive samples demonstrated average threshold cycle (CT) shifts of 2.31+/-1.16 (ORF1ab), 2.23+/-1.12 (N), and 2.79+/-1.40 (S). Notwithstanding observed S gene dropouts, the false negative rate was unaffected. As the ratio of asymptomatic positive to symptomatic positive SARS-CoV-2 infected individuals was approximately 4:1 and the average prevalence was 0.16% since we started testing in August 2020, pooled testing was identified as a viable, cost-effective option for monitoring the Northeastern University community.

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Section: Discussionsupporting

confidence: 92%

Two Stage Hierarchical Group Testing Strategy to Increase SARS-CoV-2 Testing Capacity at an Institution of Higher Education: A Retrospective Analysis

Ganz

Waithe-Alleyne

Slate

et al. 2021

Preprint

View full text Add to dashboard Cite

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“…There can be no doubt that, in principle, RT-PCR in general and RT-qPCR in particular, are technologies eminently suitable for fast, accurate, reliable and high throughput molecular diagnostic testing, especially when employed creatively, for example by pooling samples [ 98 ]. RT-qPCR has proven its proficiency by having been the benchmark diagnostic technology for the identification of numerous pathogens, including viruses, for many years.…”

Section: Discussionmentioning

confidence: 99%

COVID-19 and Diagnostic Testing for SARS-CoV-2 by RT-qPCR—Facts and Fallacies

Bustin

Mueller²,

Shipley³

et al. 2021

IJMS

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Although molecular testing, and RT-qPCR in particular, has been an indispensable component in the scientific armoury targeting SARS-CoV-2, there are numerous falsehoods, misconceptions, assumptions and exaggerated expectations with regards to capability, performance and usefulness of the technology. It is essential that the true strengths and limitations, although publicised for at least twenty years, are restated in the context of the current COVID-19 epidemic. The main objective of this commentary is to address and help stop the unfounded and debilitating speculation surrounding its use.

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“…al. (2020) where they concluded that pooling of 5 samples is sufficient to detect the presence of SARS-CoV-2, especially when pooled in pre-RNA extraction phase (Alcoba-Florez et al, 2020; Volpato et al, 2020). However, this is in contrary to Yelin et.…”

Section: Discussionmentioning

confidence: 99%

Diagnostic Performance of Pooled RT-PCR Testing for SARS-CoV-2 Detection

Ricarte¹,

Gador²,

Mendiola³

et al. 2021

Preprint

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Background: With the high number of COVID-19 cases, a need to optimize testing strategy must be regarded to obtain a timely diagnosis for early containment measures. With this, several studies have employed pooled RT-PCR testing for SARS-CoV-2 as this could potentially conserve laboratory resources while has the capacity to test several individuals. However, this was recommended to firstly validate the method as different laboratory reagents and equipment vary with its diagnostic performance. Objective: The aim of this study was to determine the diagnostic performance of pooled SARS-CoV-2 nasopharyngeal/oropharyngeal swabbed samples using the RT-PCR technique. Methods: A records review of two-staged pooled RT-PCR testing data from August 10, 26, 30, and September 5, 2020, was utilized from CHDNM TB Regional Center. For the first stage, using known samples, a total of 30 pools were made for each of the pooling sizes, 5- and 10-pooled, on both pooling phase, pre-and post-RNA extraction. One positive individual was used to represent each of the Cycle threshold values given (<24, 25-28, 29-32, 33-36, and 37-40) while the rest of the samples were negative. For the second stage, 54 pools of five from 270 random unknown samples were used to validate the results. Target gene performance of N gene and RdRp was also determined. Key Results: Results show that 5-pooled sample has higher sensitivity (SN), specificity (SP), positive predictive value (PPV), and negative predictive value (NPV) of 100% (95% confidence interval (CI) 88.97-100), 66.95% (95% CI, 60.75-72.6), 28.18% (95% CI, 20.62-37.22), and 100% (95% CI, 97.66-100) compared to 10-pooled sample that has 87.1% (95% CI, 71.15-94.87), 56.9% (50.57-63.02), 20.77% (95% CI, 14.68-28.53) and 97.14% (95% CI, 92.88-98.88). Further, these Ct values were only from the N gene, emphasizing its higher diagnostic performance as well to detect SARS-CoV-2 compared to RdRp as only a few samples were detected, thus, no analysis was made. Conclusion: This study found out that a 5-pooled sample has better diagnostic performance compared to 10-pooled samples. Specifically, all positive individual samples were detected in 5-pooled samples in the pre-RNA extraction phase which these results are evident and consistent on both known and unknown samples. N gene was found out to detect more SARS-CoV-2 samples compared to RdRp.

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Increasing SARS-CoV-2 RT-qPCR testing capacity by sample pooling

Cited by 34 publications

References 16 publications

Two Stage Hierarchical Group Testing Strategy to Increase SARS-CoV-2 Testing Capacity at an Institution of Higher Education: A Retrospective Analysis

Two Stage Hierarchical Group Testing Strategy to Increase SARS-CoV-2 Testing Capacity at an Institution of Higher Education: A Retrospective Analysis

COVID-19 and Diagnostic Testing for SARS-CoV-2 by RT-qPCR—Facts and Fallacies

Diagnostic Performance of Pooled RT-PCR Testing for SARS-CoV-2 Detection

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