Dataset Information

Sample pooling for SARS-CoV-2 RT-PCR screening.

ABSTRACT:

Objective

To evaluate the efficacy of sample pooling compared to the individual analysis for the diagnosis of coronavirus disease 2019 (COVID-19) by using different commercial platforms for nucleic acid extraction and amplification.

Methods

A total of 3519 nasopharyngeal samples received at nine Spanish clinical microbiology laboratories were processed individually and in pools (342 pools of ten samples and 11 pools of nine samples) according to the existing methodology in place at each centre.

Results

We found that 253 pools (2519 samples) were negative and 99 pools (990 samples) were positive; with 241 positive samples (6.85%), our pooling strategy would have saved 2167 PCR tests. For 29 pools (made out of 290 samples), we found discordant results when compared to their correspondent individual samples, as follows: in 22 of 29 pools (28 samples), minor discordances were found; for seven pools (7 samples), we found major discordances. Sensitivity, specificity and positive and negative predictive values for pooling were 97.10% (95% confidence interval (CI), 94.11-98.82), 100%, 100% and 99.79% (95% CI, 99.56-99.90) respectively; accuracy was 99.80% (95% CI, 99.59-99.92), and the kappa concordant coefficient was 0.984. The dilution of samples in our pooling strategy resulted in a median loss of 2.87 (95% CI, 2.46-3.28) cycle threshold (C_t) for E gene, 3.36 (95% CI, 2.89-3.85) C_t for the RdRP gene and 2.99 (95% CI, 2.56-3.43) C_t for the N gene.

Conclusions

We found a high efficiency of pooling strategies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA testing across different RNA extraction and amplification platforms, with excellent performance in terms of sensitivity, specificity and positive and negative predictive values.

SUBMITTER: de Salazar A

PROVIDER: S-EPMC7481316 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Publications

Sample pooling for SARS-CoV-2 RT-PCR screening.

de Salazar Adolfo A Aguilera Antonio A Trastoy Rocio R Fuentes Ana A Alados Juan Carlos JC Causse Manuel M Galán Juan Carlos JC Moreno Antonio A Trigo Matilde M Pérez-Ruiz Mercedes M Roldán Carolina C Pena Maria José MJ Bernal Samuel S Serrano-Conde Esther E Barbeito Gema G Torres Eva E Riazzo Cristina C Cortes-Cuevas Jose Luis JL Chueca Natalia N Coira Amparo A Sanchez-Calvo Juan Manuel JM Marfil Eduardo E Becerra Federico F Gude María José MJ Pallarés Ángeles Á Pérez Del Molino María Luisa ML García Federico F

Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 20200910 12

<h4>Objective</h4>To evaluate the efficacy of sample pooling compared to the individual analysis for the diagnosis of coronavirus disease 2019 (COVID-19) by using different commercial platforms for nucleic acid extraction and amplification.<h4>Methods</h4>A total of 3519 nasopharyngeal samples received at nine Spanish clinical microbiology laboratories were processed individually and in pools (342 pools of ten samples and 11 pools of nine samples) according to the existing methodology in place a ...[more]

PMID: 32919074

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Project description:During the COVID-19 pandemic, veterinary diagnostic laboratories tested both human and animal samples and needed to ensure that they could accurately perform large numbers of diagnostic tests in a timely manner. Sample pooling, a methodology used effectively for over 80 years as a surveillance tool for screening large numbers of potentially infected individuals, was employed. Given its sensitivity, real-time polymerase chain reaction (PCR) is more suitable for employing this strategy, as compared to other less sensitive testing methods. In this study, we evaluated the capability of detecting SARS-CoV-2 in both 5-sample and 10-sample pools of feces using real-time reverse transcriptase polymerase chain reaction (rRT-PCR) as well as determined the level of sensitivity. A blinded method test (BMT) by an independent laboratory was conducted to assess the five-sample fecal pool. To complement detection capability, the stability of the genome within a PBS fecal suspension was measured under various time and temperature conditions across a 28-day period. Our results showed that the limit of detection for 5-sample and 10-sample fecal pools is 12.8 and 6.4 genome copies in a 25 µL PCR, respectively. The 5-sample and 10-sample pooling resulted in a cycle threshold (Ct) value loss of 2.35 and 3.45, as compared to Ct values of known positive individual samples, but consistent detection was still achieved in pools containing positive samples with an original Ct below 36 and 34, respectively. The simulation of clinical five-sample pooling showed that all positive samples could be detected regardless of the number (1-3) of positive samples in each pool. The BMT results demonstrated excellent sensitivity (100 copies/reaction) in five-sample pools for the detection of SARS-CoV-2 RNA even though a fecal matrix effect was observed. Finally, our results show that the SARS-CoV-2 genome remains stable over a wide range of time and temperature variations. Overall, our findings provide solid data to scale up SARS-CoV-2 testing capacity in veterinary diagnostic laboratories.

Project description:BackgroundThe coronavirus disease 2019 (COVID-19) pandemic has revealed the global public health importance of robust diagnostic testing. To overcome the challenge of nucleic acid (NA) extraction and testing kit availability, an efficient method is urgently needed.ObjectivesTo establish an efficient, time and resource-saving and cost-effective methods, and to propose an ad hoc pooling approach for mass screening of SARS-CoV-2.MethodsWe evaluated pooling approach on both direct clinical and NA samples. The standard reverse transcriptase polymerase chain reaction (RT-PCR) test of the SARS CoV-2 was employed targeting the nucleocapsid (N) and open reading frame (ORF1ab) genomic region of the virus. The experimental pools were created using SARS CoV-2 positive clinical samples and extracted RNA spiked with up to 9 negative samples. For the direct clinical samples viral NA was extracted from each pool to a final extraction volume of 200μL, and subsequently both samples tested using the SARS CoV-2 RT-PCR assay.ResultsWe found that a single positive sample can be amplified and detected in pools of up to 7 samples depending on the cycle threshold (Ct) value of the original sample, corresponding to high, and low SARS CoV-2 viral copies per reaction. However, to minimize false negativity of the assay with pooling strategies and with unknown false negativity rate of the assay under validation, we recommend pooling of 4/5 in 1 using the standard protocols of the assay, reagents and equipment. The predictive algorithm indicated a pooling ratio of 5 in 1 was expected to retain accuracy of the test irrespective of the Ct value samples spiked, and result in a 137% increase in testing efficiency.ConclusionsThe approaches showed its concept in easily customized and resource-saving manner and would allow expanding of current screening capacities and enable the expansion of detection in the community. We recommend clinical sample pooling of 4 or 5 in 1. However, we don't advise pooling of clinical samples when disease prevalence is greater than 7%; particularly when sample size is large.

Dataset Information

Sample pooling for SARS-CoV-2 RT-PCR screening.

Objective

Methods

Results

Conclusions

Publications

Sample pooling for SARS-CoV-2 RT-PCR screening.

Similar Datasets

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