Pooled RNA sample reverse transcriptase real time PCR assay for SARS CoV-2 infection: A reliable, faster and economical method

Gupta, Ekta; Padhi, Abhishek; Khodare, Arvind; Agarwal, Reshu; Ramachandran, Krithiga; Mehta, Vibha; Kilikdar, Mousumi; Dubey, Shantanu; Kumar, Guresh; Sarin, Shiv Kumar

doi:10.1371/journal.pone.0236859

Cited by 54 publications

(68 citation statements)

References 8 publications

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“…Further unpublished data from our center suggest that less than 37 and by following the above‐mentioned precautions we picked maximum possible positive cases. Similar cases have been reported by another recent study 10 …”

Section: Discussionsupporting

confidence: 93%

Evaluation of sample pooling for diagnosis of COVID‐19 by real time‐PCR: A resource‐saving combat strategy

Garg

Singh

Pandey

et al. 2020

Journal of Medical Virology

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Background and Objectives Although about 80% of coronavirus disease‐2019 (COVID‐19) cases are reported to be mild, the remaining 20% of cases often result in severe disease with the potential of crushing already overstrained health care services. There has been sustainable growth of COVID‐19 cases worldwide since mid‐May 2020. To keep tabs on community transmission of COVID‐19 infection screening of the samples from a large population is needed which includes asymptomatic/symptomatic individuals along with the migrant population. This requires extra resources, man power, and time for detection of severe acute respiratory syndrome coronavirus 2 by real‐time polymerase chain reaction (RT‐PCR). In the current scenario, the pooled sample testing strategy advocated by the Indian Council of Medical Research, New Delhi is a new approach that is very promising in resource‐limited settings. In this study, we have evaluated the pooled strategy in terms of accurate testing results, utilization of consumables, and identification of borderline positive cases. Materials and Methods Between April and June 2020, we performed COVID‐19 testing by RT‐PCR from areas with varying prevalence of population referred to COVID laboratory, Dr Ram Manohar Lohia Institute of Medical Sciences, Lucknow. In the first step, the samples are collated into pools of 5 or 10. These pools are tested by RT‐PCR. Negative pools were reported as negative whereas positive pools of 5 and 10 are then deconvoluted and each sample is tested individually. Results In the present study, we tested 4620 samples in 462 pools of 10 and 14 940 samples in 2990 pools of 5. Among 10 samples pool, 61 (13%) pools flagged positive in the first step. In the second step, among 61 pools (610 samples) deconvoluted strategy was followed in which 72 individual samples came positive. The pooled‐sample testing strategy helps saves substantial resources and time during surge testing and enhanced pandemic surveillance. This approach requires around 76% to 93% fewer tests done in low to moderate prevalence settings and group sizes up to 5–10 in a population, compared to individual testing. Conclusions Pooled‐sample PCR analysis strategies can save substantial resources and time for COVID‐19 mass testing in comparison with individual testing without compromising the resulting outcome of the test. In particular, the pooled‐sample approach can facilitate mass screening in the early coming stages of COVID‐19 outbreaks, especially in low‐ and middle‐income settings, and control the spread by meticulous testing of all risk groups.

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Section: Discussionsupporting

confidence: 93%

Evaluation of sample pooling for diagnosis of COVID‐19 by real time‐PCR: A resource‐saving combat strategy

Garg

Singh

Pandey

et al. 2020

Journal of Medical Virology

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“…In our study, after samples were concentrated by centrifugation, the range of Ct differences was 0.5 to 3.01. The third pooling option was described by Gupta et al [17] who recommended pooling of RNA samples before PCR as a faster and more economical method since otherwise when a pool turns out to be positive, repeat sample collection or RNA extraction has to be done [17]. Our results proved that the size of pooled specimens effectively diagnosed was equal to those pooled after RNA extraction the same being more cost-effective.…”

Section: Resultsmentioning

confidence: 53%

Sample pooling as a strategy for community monitoring for SARS-CoV-2

Sawicki

Korona-Głowniak

Boguszewska

et al. 2020

Preprint

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Sample pooling strategy was intended to determine the optimal parameters for group testing of pooled specimens for the detection of SARS-CoV-2 and process them without significant loss of test usability. Standard molecular diagnostic laboratory equipment and commercially available centrifugal filters, RNA isolation kits and SARS Cov2 PCR tests were used. The basic idea was to combine and concentrate several samples to the maximal volume, which can be extracted with the single extraction column. Out of 16 tested pools 12 were positive with cycle threshold (Ct) values within 0.5 and 3.01 Ct of the original individual specimens. The analysis of 112 specimens determined that 12 pools were positive followed by identification of 6 positive individual specimens among the 112 tested. This testing was accomplished with the use of 16 extractions/PCR tests, resulting in saving of 96 reactions but adding the 40 centrifugal filters. The present study demonstrated that pool testing can detect a single positive sample with Ct value as high as 34. According to the standard protocols, reagents and equipment, this pooling method can be applied easily in current clinical testing laboratories.

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“…Pool testing has gained importance to fight the COVID-19 pandemic, as challenges involving cost and logistics are at the core of shared struggles to promote large-scale screenings worldwide 10 . Traditional pooling methods proposed 9-15 rely on the combination of multiple individual samples prior to RNA extraction or RT-qPCR, leading to a dilution factor directly related to the number of samples in the pool 9,[11][12][13][14][15] . This dilution effect has been of major concern over the diagnostic performance of pool testing procedures 26 .…”

Section: Discussionmentioning

confidence: 99%

Swab pooling for large-scale RT-qPCR screening of SARS-CoV-2

Christoff¹,

Cruz²,

Sereia³

et al. 2020

Preprint

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Pool testing has been proposed as an alternative for large-scale SARS-CoV-2 screening. However, dilution factors proportional to the number of pooled samples have been a source of major concern regarding its diagnostic performance. Further, sample pooling can lead to increased laboratory workload and operational complexity. Therefore, pooling strategies that minimize sample dilution, loss of sensitivity, and laboratory overload are needed to allow reliable and large-scale screenings of SARS-CoV-2. Here, we describe a pooling procedure in which nasopharyngeal swabs are pooled together at the time of sample collection (swab pooling), decreasing laboratory manipulation and minimizing dilution of the viral RNA present in the samples. Paired analysis of pooled and individual samples from 613 patients revealed 94 positive individual tests. Having individual testing as a reference, no false-positives or false-negatives were observed for swab pooling. A Bayesian model estimated a sensitivity of 99% (Cr.I. 96.9% to 100%) and a specificity of 99.8% (Cr.I. 99.4% to 100%) for the swab pooling procedure. Data from additional 18,922 patients screened with swab pooling were included for further quantitative analysis. Mean Cq differences between individual and corresponding pool samples ranged from 0.1 Cq (Cr.I. -0.98 to 1.17) to 2.09 Cq (Cr.I. 1.24 to 2.94). Overall, 19,535 asymptomatic and presymptomatic patients were screened using 4,400 RT-qPCR assays, resulting in 246 positive patients (positivity rate 1.26%). This corresponds to an increase of 4.4 times in laboratory capacity and a reduction of 77% in required tests. Finally, these data demonstrate that swab pooling can significantly minimize sample dilution and sensitivity issues commonly seen in its traditional counterpart. Therefore, swab pooling represents a major alternative for reliable and large-scale screening of SARS-CoV-2 in low prevalence populations.

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Pooled RNA sample reverse transcriptase real time PCR assay for SARS CoV-2 infection: A reliable, faster and economical method

Cited by 54 publications

References 8 publications

Evaluation of sample pooling for diagnosis of COVID‐19 by real time‐PCR: A resource‐saving combat strategy

Evaluation of sample pooling for diagnosis of COVID‐19 by real time‐PCR: A resource‐saving combat strategy

Sample pooling as a strategy for community monitoring for SARS-CoV-2

Swab pooling for large-scale RT-qPCR screening of SARS-CoV-2

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