key: cord-1019902-vun23m1c
authors: Lee, Hao-Ru; Wang, Fang-Yu; Li, Jia-Ling; Chou, Teh-Ying; Ho, Hsiang-Ling
title: Real-world Evaluation of a Sample Pooling Strategy for Large-Scale Rapid COVID-19 Testing
date: 2022-03-15
journal: J Clin Virol
DOI: 10.1016/j.jcv.2022.105133
sha: d356c7e86304ea758fbcb5e5f1ea7fc33f56d8bc
doc_id: 1019902
cord_uid: vun23m1c

BACKGROUND: The worldwide outbreak of COVID-19 has become a public health crisis of unprecedented proportions. The fast spread of emerging variants increases the needs of rapid diagnostic and screening testing. Sample pooling efficiently expands the testing capacity under limited resources. OBJECTIVES: We evaluated the performance of sample pooling on the Point-of-Care (POC) Liat® and cobas® 6800 systems and provided real-world experiences for implementing these systems in large-scale screenings. METHODS: Positive nasopharyngeal (NP) specimens with Ct values < 25, 25∼30 or > 30 were tested individually and in pools to optimize the POC Liat® and cobas® 6800 systems, which were then implemented in community screenings. RESULTS: The 5-sample pooling strategy did not affect the positive detection rates on Liat® or cobas® 6800 in samples with Ct values <25 or 25∼30. However, in samples with low viral loads (Ct values >30), five-sample pooling has a higher positive detection rate on POC Liat® (20/20; 100%), compared to cobas® 6800 (9/20; 45%). Five-sample pooled on POC Liat® and two-sample pooled on cobas® 6800 appear to be appropriate for SARS-CoV-2 detection. By implementing the pooling strategies in two large-scale community screenings, 7,606 NP specimens was tested within 36 hours; the average turn-around time was 4.8 hours for cobas® 6800 and 1.3 hours for POC Liat®. Eight positive specimens (0.11%; 8/7,606) were identified, with Ct values ranging from 18.85 to 37.68. CONCLUSION: The performance of sample pooling on POC Liat® was demonstrated to be an effective, accurate, and economical approach for large-scale community screenings for COVID-19.

The worldwide outbreak of COVID-19 has become a public health crisis of unprecedented 23

proportions. The fast spread of emerging variants increases the needs of rapid diagnostic and 24 screening testing. Sample pooling efficiently expands the testing capacity under limited 25

Objectives 27

We evaluated the performance of sample pooling on the Point-of-Care (POC) Liat® and cobas® 28 6800 systems and provided real-world experiences for implementing these systems in large-scale 29 screenings. 30

Positive nasopharyngeal (NP) specimens with Ct values < 25, 25~30 or > 30 were tested 32 individually and in pools to optimize the POC Liat® and cobas® 6800 systems, which were then 33 implemented in community screenings. 34

Results 35 9 (Thermo fisher scientific inc.) targeting the ORF1ab, N and S genes was used as a confirmatory 97 assay. 98

The limit of detection (LoD) of cobas® 6800 and Liat® systems was assessed by using the 99 inactivated and quantitated SARS-CoV-2 standard obtained from Taiwan Food and Drug 100

Administration (Lot number 109-06, 1.26E+06 CCID 50 /mL). The quantitated SARS-CoV-2 101

standard was serially diluted and tested with 5 replicates. The experimental LoD was defined as 102 the lowest concentration with a detection rate of 100%, and the Probit LoD was estimated at 95% 103 detection rate using probit analysis by SPSS v22.0. 104

For sample pooling, two to five individual samples were pooled into a total volume of 400 105 μl, and after mixing with 400 μl lysis buffer, the pooled samples were subjected to the cobas® 106 6800 system for SARS-CoV-2 testing. For sample pooling in the Liat® system, an equal amount 107 of five individual samples was pooled to a total volume of 200 μl and then transferred to Liat® 108 cartridges for analysis. 109

To increase the working efficiency and reduce manual errors, we implemented a pooling 110 program in the laboratory informatics system (LIS) by which the individual patient samples 111 within a pool are automatically associated with a unique pooling barcode during sample 112 10 reception. The pooling barcode was recognized by the cobas® 6800 and Liat® systems. After 113 analysis, if the result of the pool is negative, the "negative" result corresponding to the individual 114 patient samples is automatically reported; if the result of the pool is positive, there will be a flag 115 on the LIS to suspend the reporting and further deconvolution will be requested to identify 116 positive samples. 117

The study was approved by the Institutional Review Board of Taipei Veterans General 118

Hospital. 119 120

A total of 46 nasopharyngeal specimens were used for comparing the performance between 122 the Liat® and cobas® 6800 systems, of which 26 were detected as positive and 20 as negative 123 using the Liat® system, while 16 as positive, 10 as equivocal (Ct values of the E gene over 35 on 124 cobas® 6800 but less than 35 on Liat®) and 20 as negative using the cobas® 6800 system 125 (Table 1 ). The 10 equivocal cases were further confirmed to be positive by the TaqPath assay 126 (Table 2) . 127

To evaluate the detection sensitivity of Liat® and cobas® 6800 systems, the SARS-CoV-2 128 standard with known viral concentration was serially diluted and subjected to LoD analysis 129 (Supplemental Table S1 ). The Ct values of tests with varied viral dilutions on the Liat® and 130 cobas® 6800 systems were shown in Supplemental Figure S1 . These results showed that the 131 LoD toward SARS-CoV-2 detection of Liat® is lower than that of cobas® 6800. (Table 3 and Supplemental Table S2 ). These findings demonstrated that the 5-sample pooling 149 strategy did not affect the positive detection rate on Liat® but had a compromise on cobas® 150

To further validate the performance of the 5-sample pooling strategy on the Liat® system, 152 363 nasopharyngeal specimens, including 12 positive and 351 negative cases, were analyzed. As 153 13 shown in Table 4 showed only E gene positivity with Ct values over 37 using the cobas® 6800 system, and they 188 were further confirmed as positive using the Liat® system. The TAT from sample reception to 189 reporting was 1.3 hours for the Liat® system and 4.8 hours for the cobas® 6800 system (Figure  190 2). In this study, we demonstrated that the sample pooling strategy significantly increases the 194 testing capacity of SARS-CoV-2, in which a total of 7,606 tests with an average TAT of 3 hours 195 (4.8 hours for cobas® 6800 and 1.3 hours for Liat®) from sample reception to reporting were 196 completed within 36 hours. This is also the first study to evaluate the sample pooling on the 197

Liat® system, demonstrating that it could serve as an effective, accurate, and economical 198 approach for large-community screenings. 199

The LoD assessment on the Liat® and cobas® 6800 systems showed that the LoD of Liat® 200 is lower than that of cobas® 6800 (Supplemental Table S1 ). By comparing these two systems, 201 as shown in Table 2 and Table 4 , our data suggest that the detection sensitivity of Liat® toward 202 SARS-CoV-2 may be higher than that of cobas® 6800. However, without head-to-head clinical 203 trials, it is immature to make assumptions about relative sensitivity between these two systems. 204

In Table 3 , we found the false negative rates among samples with Ct > 30 was 40% (8/20) on 205 cobas® 6800, but no false negatives were identified on Liat®, indicating with 5-sample pooling, 206

Liat®, compared to cobas® 6800, has a higher positive detection rate for samples with low viral 207 loads. 208 17 The advantages of using Liat® for pooling include its lower LoD than cobas® 6800 and it 209 is more economical. For individual testing, the reagent cost is around US$ 60/sample for Liat®, 210 US$ 24/sample for cobas® 6800, US$ 24/sample for TaqPath assay, and US$ 12/sample for the 211 antigen test. The cost of reagents and instruments (quoted by the local distributors) regarding 212 sample pooling used in this study were provided in Supplemental Table S3 . The application of 213 sample pooling will reduce the PCR reagent cost to be comparable to that of an antigen test. In 214 addition, the testing capacity for Liat® (5-sample pooling; 10 Liat® machines) was 150 tests/hr 215 with 20 min/run, but for cobas® 6800 (2-sample pooling; 2 cobas® 6800 machines) was 376 216

Although the US FDA has granted EUA to the cobas® SARS-CoV-2 test on cobas® 218 6800/8800 systems with pooled specimens of up to six in a single pool, in our study, the 219 appropriate number of pools on cobas® 6800 is two. This discrepancy is most likely due to that 220 in Taiwan, we usually add an external lysis buffer in a 1:1 ratio for virus inactivation, which may 221 result in a twofold dilution. In this study, by assessing the LoD, we found the use of external 222 lysis in cobas® 6800 may slightly impair the assay LoD (Supplemental Table S1 ). 223

The use of pooling in large-community screenings could effectively increase testing 224 capacity and preserve testing reagents/resources. However, in high prevalence situations, e.g., 225 18 when 5-sample pooling is used in the areas with a prevalence > 25%, it may become less 226 efficient and lead to delayed test results since positive pools will need to be deconvoluted. The 

The authors declare that they have no conflicts of interest related to the subject matter or 255 materials discussed in this article. 256 257 

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