Optimal size of sample pooling for RNA pool testing: an avant-garde for scaling up SARS CoV 2 testing

Abstract

Introduction-Timely diagnosis is essential for the containment of the disease and breaks in the chain of transmission of SARS-CoV-2. The present situation demands countries to scale up their testing and design innovative strategies to conserve diagnostic kits and reagents. The pooling of samples saves time, manpower, and most importantly diagnostic kits and reagents. In the present study, we tried to define the pool size that could be applied with acceptable confidence for testing. Material and methods- We used repeatedly tested positive clinical sample elutes having different levels of SARS CoV 2 RNA and negative sample elutes to prepare seven series of 11 pools each, having pool sizes ranging from 2 to 48 samples to estimate the optimal pool size. Each pool had one positive sample elute in different compositions. All the pools were tested by SARS CoV 2 RT-qPCR. Results- Out of the 77 pools, only 53 (68.8%) were found positive. The sensitivity of pools of 2 to 48 samples was decreased from 100% (95% CL; 98.4-100) to 41.41% (95% CL; 34.9-48.1). The maximum size of the pool with acceptable sensitivity (>95%) was found to be of 6 samples. For the pool size of 6 samples, the sensitivity was 97.8% and the efficiency of pooling was 0.38. Conclusion- The pooling of samples is a practical way for scaling up testing and ultimately containing the further spread of the COVID-19 pandemic.

Evaluation of RT-PCR pooling test for the detection of SARS-CoV-2 in low-resource setting

“…Further, the pooling process can be done by pooling the nasopharyngeal/ oropharyngeal swab containing VTM samples and perform RNA extraction for pooled VTM samples [16]. The major bottleneck of RNA extraction can be removed by this method [17]. Here, we also demonstrated that pooling also works well when applied before RNA extraction by serial dilution process.…”

“…The study showed that the extracted RNA elutes can be detected in a pool of up to 64 samples with high viral load (Ct value [16][17][18][19][20][21][22][23] in pooling prior to RT-PCR. The result correlated with the study done in Israel where a single positive RNA elute sample could be detected in pools of up to 32 samples, with an estimated false-negative rate of 10% and showed experimentally that 64 samples could be pooled together that contains enough RNA copies for detection [4].…”

“…Further, the pooling process can be done by pooling the nasopharyngeal/ oropharyngeal swab containing VTM samples and perform RNA extraction for pooled VTM samples [16]. The major bottleneck of RNA extraction can be removed by this method [17].…”

“…To increase testing throughput, consuming fewer reagents and faster results yield, multiple sample pooling can be a good alternative [4,[16][17][18]. The present gold standard for detection of SARS-CoV-2 is qPCR, which requires resources that are currently limited [5].…”

“…For this, 3 previously confirmed positive VTM samples were taken with cycle threshold (Ct) value low (16)(17)(18)(19)(20)(21)(22)(23) and high (29)(30)(31). Then, 17 negative VTM samples were used to prepare 5 pools of 1, 2, 4, 8 and 16 negative pools with an equal volume of each negative sample.…”