key: cord-1029454-ptkzvkxd
authors: Chung, Hsing-Yi; Jian, Ming-Jr; Chang, Chih-Kai; Lin, Jung-Chung; Yeh, Kuo-Ming; Chen, Chien-Wen; Yang, Ya-Sung; Hsieh, Shan-Shan; Chen, En-Sung; Yang, Mei-Hsiu; Tang, Sheng-Hui; Perng, Cherng-Lih; Yang, Ji-Rong; Liu, Ming-Tsan; Chang, Feng-Yee; Shang, Hung-Sheng
title: Multicenter study evaluating novel multi-specimen pooling assay for the detection of SARS-CoV-2: high sensitivity and high throughput testing
date: 2021-09-02
journal: J Microbiol Immunol Infect
DOI: 10.1016/j.jmii.2021.08.003
sha: 9a9302a7247202a17cd678565e8cafcf23e2977d
doc_id: 1029454
cord_uid: ptkzvkxd

Background/Purpose Mass screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important to prevent the spread of coronavirus disease 2019 (COVID-19). Pooling samples can increase the number of tests processed. LabTurbo AIO 48 is an automated platform that allows ribonucleic acid extraction and sample analysis on the same instrument. We created a novel pooling assay on this platform for SARS-CoV-2 detection and demonstrated that the pooling strategy increases testing capacity without affecting accuracy and sensitivity. Methods Comparative limit of detection (LoD) assessment was performed on the LabTurbo AIO 48 platform and the current standard detection system based on real-time reverse transcription polymerase chain reaction (rRT-PCR) using 55 clinically positive samples. An additional 330 primary clinical samples were assessed. Results Six samples pooled into one reaction tube were detected in approximately 2.5 h using the World Health Organization rRT-PCR protocol. LabTurbo AIO 48 also demonstrated a higher throughput than our reference rRT-PCR assay, with an LoD of 1000 copies/mL. The overall percentage agreement between the methods for the 330 samples was 100%. Conclusion We created a novel multi-specimen pooling assay using LabTurbo AIO 48 for the robust detection of SARS-CoV-2, allowing high-throughput results; this assay will aid in better control and prevention of COVID-19. The diagnostic assay was cost-effective and time-efficient; thus, the pooling strategy is a practical and effective method for diagnosing large quantities of specimens without compromising precision.

Six samples pooled into one reaction tube were detected in approximately 2.5 h using the World 19 Health Organization rRT-PCR protocol. LabTurbo AIO 48 also demonstrated a higher throughput 20 than our reference rRT-PCR assay, with an LoD of 1000 copies/mL. The overall percentage 21 agreement between the methods for the 330 samples was 100%.

We created a novel multi-specimen pooling assay using LabTurbo AIO 48 for the robust detection 24 of SARS-CoV-2, allowing high-throughput results; this assay will aid in better control and 25 prevention of COVID-19. The diagnostic assay was cost-effective and time-efficient; thus, the 26 pooling strategy is a practical and effective method for diagnosing large quantities of specimens 27 without compromising precision. problem as it causes social disruption due to high morbidity and mortality. Moreover, its 36 rapid spread significantly increases the demand on healthcare systems. 2 Therefore, the 37 identification of SARS-CoV-2 in the initial stage is crucial for COVID-19 diagnosis. 3,4 38 Several diagnostic methods for SARS-CoV-2 have been developed; however, they 39 have different infrastructure requirements, which affect the capacity of batch production. 40

In addition, these methods have variable throughput, turnaround times (ranging from a few 41 minutes to several hours), and analytical performances. 5-7 Real-time reverse-transcription 42 PCR (rRT-PCR) for the detection of SARS-CoV-2 is one of the confirmatory diagnostic 43 methods for COVID-19. However, implementation of SARS-CoV-2 testing has been 44 difficult in some areas due to the shortage of reagents. 8 Therefore, it is important to consider 45 how rRT-PCR testing can be efficiently conducted with high accuracy, when the 46 availability of reagents and well-trained staff is limited during this global disaster. 6,7,9 47

Pooling samples as a cost-and time-saving approach may help overcome these 48 limitations. 10-12 Studies have suggested two protocols for pooling: (1) extracting RNA from 49 a mixture of the original samples and (2) mixing the extracted RNA from individual 50 samples. 13-14 However, this pooling approach directly affects the analytical sensitivity of 51 the rRT-PCR assay, potentially leading to reduced diagnostic sensitivity. 13 52 J o u r n a l P r e -p r o o f

Here, we evaluated a novel sample-pooling strategy using an rRT-PCR platform 53 without any volume loss. We investigated the extent to which this was possible, while 54 detecting weakly positive samples in the largest pool of samples thus far. Taipei, Taiwan) composed of reverse transcriptase, the primer/probe mixture, and 2× PCR 84 master mix solutions was utilized according to the manufacturer's instructions. 13 We used 85 three different reagents to detect the target genes (E, RdRp, and N1) in the specimens from 86

Tri-Service General Hospital (TSGH) and Cathay General Hospital (CGH). 87

We purchased purified RNA controls (Vircell, Granada, Spain) of the above viral 89 genes for absolute quantification. These controls were used to prepare a serial dilution panel 90 with approximately 5-20 replicates. We serially diluted the RNA controls (2000, 1000, and 91 500 copies/mL) using nuclease-free water to assess the limit of detection (LoD). We mixed 92 300 μL of each serial dilution of the SARS-CoV-2 RNA controls with different specimens 93 using nuclease-free water. Samples were prepared as 1-, 3-, 5-, 6-, and 7-sample pools, of 94 total volumes 300, 900, 1500, 1800, and 2100 μL, respectively (Table 1) . For example, the 95 3p-sample pool of 1000 copies/mL was prepared by mixing 300 μL of 1000 copies/mL 96 SARS-CoV-2 RNA control with 600 μL of nuclease-free water. These samples were then 97 designated as 1P, 3P, 5P, 6P, and 7P and used in the RNA extraction and rRT-PCR steps. 98

LoD was defined as a 95% probability of 20 replicates testing positive. The LoD was determined for solutions with 2000, 1000, and 500 copies/mL SARS-116

CoV-2 RNA-positive controls by testing 5-20 replicates. The LoD of 20 replicate tests at 117 1000 copies/mL was 100% and 95% for the N1 and E genes, respectively, on the LabTurbo 118 AIO 48 platform (Table1). The LOD did not appear to change in different pooled strategies. 119

In our method, we pooled 300 µL of each specimen, and subjected the pooled mixture to 120 the extraction procedure. The last method is RNA pooling. RNA is extracted from each sample, and the 171 extracted RNA from different samples are mixed and used for rRT-PCR. After obtaining a 172 positive response in the pool, it is easy to identify the sample responsible for the positive 173 signal. Usually, resampling is not required for confirmation. However, this method is time-174 consuming, particularly, the loading process during extraction. It also exhibits decreased 175 sensitivity when too many specimens are pooled. These three methods demonstrate that 176 multi-sample pools are a good method to increase testing throughput, while using fewer 177 reagents and offering faster results. 22-23 However, the implementation of these methods 178 might be negatively affected by the resulting low sensitivity. The above methods were 179 summarized in Supplementary Table 1 11,16,19-23 .  180 Here, we propose a solution for sample pooling by expanding the volume limit. 181

Though this could cause double dilution, we propose an increase in the tolerance of sample 182 volume without directly reducing the number of samples and subsequently mixing them for 183 simultaneous analyses. During the analysis of high-throughput samples, we can observe 184 whether there is a decline in sensitivity, because the pooled specimens may be used as 185 screening tools, and the clinical sensitivities differ by pool size. In Table 1 , for the N1 and 186 E genes, the number of samples that can be mixed is 1, 3, 5, and 6, and the obtained 187 sensitivity is 100%. The performance of N1 (100%) better than E (95%) in the 6P strategy. 188

In our study, we selected the 6P strategy as the clinical pooling strategy. When the 6p-189 sample is screened positive, even if just one gene is positive, each specimen should be re-190 tested following the WHO protocol using the RdRp and E genes. 191

Our protocol will be helpful for quickly screening individuals in groups at a high 192 risk for COVID-19, enabling quarantining of confirmed positive people, even in situations 193 with limited time and resources. 194

In conclusion, we propose a pooling strategy to detect SARS-CoV-2. The LoD was 195 estimated according to the US Food and Drug Association (N1 gene) and WHO (E gene) 196 guidelines, respectively. We propose that a six-in-one mixture might be a feasible strategy 197 (Table 1) . For clinical efficiency analysis, we spiked one positive SARS-CoV-2 sample 198 with five negative SARS-CoV-2 samples in the pool. We poured the mixture into the 199

LabTurbo AIO system without volume loss for SARS-CoV-2 detection by rRT-PCR. 2000 copies/mL 1000 copies/mL 500 copies/mL 

Weekly epidemiological update on COVID-19 -31

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