key: cord-0814648-ati4ym9b
authors: Becker, Michael G.; Taylor, Tracy; Kiazyk, Sandra; Cabiles, Dana R.; Meyers, Adrienne F.A.; Sandstrom, Paul A.
title: Recommendations for sample pooling on the Cepheid GeneXpert® system using the Cepheid Xpert® Xpress SARS-CoV-2 assay
date: 2020-07-15
journal: bioRxiv
DOI: 10.1101/2020.05.14.097287
sha: 26dc82b70f4c81b144bee1d191db41eb932333b3
doc_id: 814648
cord_uid: ati4ym9b

The coronavirus disease 2019 (Covid-19) pandemic, caused by SARS-CoV-2, has resulted in a global testing supply shortage. In response, pooled testing has emerged as a promising strategy that can immediately increase testing capacity. Here, we provide support for the adoption of sample pooling with the point-of-care Cepheid Xpert® Xpress SARS-CoV-2 molecular assay. Corroborating previous findings, the Xpert® Xpress SARS-CoV-2 assay limit of detection was comparable to central laboratory reverse-transcription quantitative PCR tests with observed SARS-CoV-2 detection below 100 copies/mL. The Xpert® Xpress assay detected SARS-CoV-2 after samples with minimum viral loads of 461 copies/mL were diluted into six sample pools. Based on these data, we recommend the adoption of pooled testing with the Xpert® Xpress SARS-CoV-2 assay where warranted by population public health needs. The suggested number of samples per pool, or pooling depth, is unique for each point-of-care test site and should be determined by assessing positive test rates. To statistically determine appropriate pooling depth, we have calculated the pooling efficiency for numerous combinations of pool sizes and test rates. This information is included as a supplemental dataset that we encourage public health authorities to use as a guide to make recommendations that will maximize testing capacity and resource conservation.

The coronavirus disease 2019 pandemic has caused an unprecedented demand for global 29 testing supplies. In response, public health officials are searching for innovative ways to increase testing capacity 30 in the face of limited resources. One approach that could be rapidly deployed to achieve increased SARS-CoV-2 31 testing capacity is pooled sample testing. The number of samples to be combined into each test pool, or pooling 32 depth, is determined by test sensitivity and community disease prevalence, with some laboratories pooling up to 33 10 (1), 30 (2), or 48 (3) samples using the Corman quantitative reverse transcription PCR (RT-qPCR) test (4).

Similar strategies should be explored for currently deployed SARS-CoV-2 point-of-care tests, such as the

Cepheid Xpert ® Xpress SARS-CoV-2 assay.

The Cepheid Xpert ® Xpress SARS-CoV-2 assay is a rapid, fully-automated, and self-contained multiplex 37 qualitative RT-qPCR test for SARS-CoV-2 detection. The Cepheid Xpert Xpress SARS-CoV-2 assay targets two 38 regions of the SARS-CoV-2 genome: the N (nucleocapsid) region and the E (envelope) region. The test is 39 interpreted as positive for SARS-CoV-2 if either of the two analytes produce a Ct below 45. The test is performed 40 on the Cepheid GeneXpert system in single-use cartridges, with an approximate run time of 50 minutes. This

Cepheid Xpert ® Xpress SARS-CoV-2 assay received approval from Health Canada on March 24, 2020 under 42 interim order authorization. Evaluation of the Cepheid SARS-CoV-2 assay is ongoing, with higher reported 43 sensitivity than the Abbott ID Now SARS-CoV-2 Assay (5, 6), and high agreement (>99%) with the Roche Cobas 44 6800 system (5, 7, 8) and the Centers for Disease Control and Prevention (CDC) . Using viral 45 recombinants to contrive samples, Cepheid reports 100% sensitivity (n=35) at 250 copies (cp)/mL. Using 46 synthetic RNA controls, Zhen et al. (6) reported 100% sensitivity at 100 cp/mL (n=10) and a 87.5% sensitivity at 47 50 cp/mL (n=8).

Given the high sensitivity of the Xpert ® Xpress SARS-CoV-2 assay, it is reasonable to propose that it could be 49 suitable for pooled testing. Here, the potential for pooled SARS-CoV-2 testing was assessed on the GeneXpert 50 system using a small panel of clinical specimens with low-to mid-range viral loads that were diluted with 51 . CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted May 15, 2020 . . https://doi.org/10.1101 /2020 Page 3 of 13 known clinical negative samples. The results here corroborate previous findings that the LOD for the Cepheid 52 test is likely <100 cp/mL. Additionally, data generated by this study suggest that the GeneXpert device can be 53 effectively applied for SARS-CoV-2 pooled sample testing in pools containing up to at least six individual 54 samples. Finally, a reference dataset is provided that can be used by public health authorities to advise point-of-55 care test sites on the optimal number of samples to combine per pool given their current positive test rates. 

. CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a 

CoV-2 were prepared in viral transport media to yield a series from 6 x 10 8 cp/mL to 6 x 10 0 cp/mL. For each step 77 of the dilution series, 300 µL was pipetted into an Xpert ® Xpress SARS-CoV-2 cartridge. The linear equation The Xpert ® Xpress SARS-CoV-2 assay can be used to provide quantitative results

Although the Xpert ® Xpress SARS-CoV-2 assay is considered a qualitative test, it does provide output Ct 109 values that can be used to approximate viral loads using a standard curve. To produce a standard curve, 10-fold 110 serial dilutions of inactivated high-titre SARS-CoV-2 were prepared in viral transport media from 6 x 10 8 cp/mL 111 to 6 x 10 0 cp/mL. All dilutions above 6 x 10 1 were recorded as SARS-CoV-2 positive by the assay (Supplemental 112 

Though not clinically relevant, this would likely affect accurate quantitation of viral load at higher Ct values.

To better observe the effects of sample pooling near the Xpert ® Xpress SARS-CoV-2 assay's LOD, an 

CoV-2 assay detected SARS-CoV-2 after six-fold pooling with negative specimens, while our weak positive (64 132 cp/mL) returned a negative result (Table 2) . Additionally, the E target was not detected in one of the pools;

133 however, only one detected analyte is needed to return an actionable positive test result.

An objective of this study is to provide guidance for when sample pooling is a viable option for SARS-

CoV-2 testing with the Xpert ® Xpress SARS-CoV-2 assay, or any sensitive SARS-CoV-2 test in general. At high 137 positive testing rates, pooling may actually increase the number of tests required to screen samples and increase The copyright holder for this preprint (which this version posted May 15, 2020. . https://doi.org/10.1101/2020.05.14.097287 doi: bioRxiv preprint Page 7 of 13 between pooling depths and positive test rates. We determined testing capacity using various combinations of 140 pool sizes (1-10) and test rates (0-100% in increments of 0.1%). A complete summary of all combinations can be 141 found in Supplemental Table 2 , and a graphical representation of a subset of these data is shown in Figure 2 .

This information can help medical authorities provide informed recommendations pertaining to sample 143 pooling. For example, no pooling strategy is effective when positive test rates exceed ~30%. Additionally, at no 144 combination of pool size and positive test rates is two sample pooling more efficient than three sample pooling 145 ( Figure 2 ). Although at low positive test rates aggressive pooling is favored, this quickly changes when test rates 146 increase above 1%. For example, if the positive test rate at a site is ~3% the ideal pool size would be six samples.

The results of this study strongly suggest that sample pooling is a viable option for SARS-CoV-2 testing 149 using the Xpert ® Xpress SARS-CoV-2 assay. All samples tested positive after pooling, except for a high-Ct discordant 

One challenge that may prevent some point-of-care testing sites from adopting a pooled testing strategy 157 is the lack of mechanical pipettes. The Xpert ® Xpress SARS-CoV-2 assay is provided with single-use transfer pipettes 158 that dispense 300 µL of sample. With small pool sizes, multiple samples can be combined into a 5 mL specimen 159 tube or 15 mL canonical tube, and inverted to mix. Subsequently 300 µL of this pool can be transferred into a test 160 cartridge. With this approach, pooled testing with the Xpert ® Xpress SARS-CoV-2 assay could be readily achieved 161 in a resource-limited setting with the provision of additional 300 µL transfer pipettes.

. CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted May 15, 2020. . https://doi.org/10.1101/2020.05.14.097287 doi: bioRxiv preprint

Page 8 of 13

Several other strategies for SARS-CoV-2 pooled testing are being investigated such as combinatorial 163 testing, or matrix testing (3, 15) . In this approach, samples are combined into multiple pools, such that each sample 164 is tested multiple times across multiple pools. The combination of SARS-CoV-2 positive pools can identify 165 individual positives with limited retesting required. Although this strategy is promising, it works best for high-166 throughput laboratories processing batches of hundreds of samples using 96-or 384-well plates and real-time PCR 167 machines. Because of the need for larger batch sizes and its more complicated testing design, a combinatorial 168 approach is unlikely to be feasible with point-of-care tests that perform only a few tests in a single run, such as the 169 Xpert ® Xpress SARS-CoV-2 assay.

Another pooling strategy proposed by the German Red Cross Blood Donor Service and Geothe University 171 is swab pooling, or the mini-pool method (16). Multiple swabs can be combined into a single tube at the point of 172 collection, rather than the traditional method of pooling transport media or extracted RNA. As a result, there is 173 minimal loss of sensitivity as no dilution is occurring. The main disadvantage of this approach is that the pooled 174 samples need to be collected simultaneously and at the same location; however, the swab pool approach could be 175 applied in certain scenarios. For example, this strategy may be appropriate for door-to-door household testing, 176 workplace screening, or its intended of purpose of blood donor screening. This approach could easily be combined 177 with traditional pooling to substantially increase testing capacity with the Xpert ® Xpress SARS-CoV-2 assay or other 178 validated molecular test method.

This study provides a resource that can be used to determine the appropriate pool size to use at each 181 testing site. Public health authorities can approximate positive tests rates, and use this information with the 182 reference table (Supplemental Table 2 ) to make appropriate recommendations on pooling strategies. The The copyright holder for this preprint (which this version posted May 15, 2020 . . https://doi.org/10.1101 /2020 . CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted May 15, 2020 . . https://doi.org/10.1101 /2020 .05.14.097287 doi: bioRxiv preprint medRxiv 2020 CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted May 15, 2020. . https://doi.org/10.1101/2020.05.14.097287 doi: bioRxiv preprint 

Sample Pooling as a Strategy to Detect Community 206 Transmission of SARS-CoV-2

Pooling of samples for testing for SARS-CoV-2 in asymptomatic people

Efficient high 211 throughput SARS-CoV-2 testing to detect asymptomatic carriers

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Comparison of Cepheid Xpert Xpress

Abbott ID Now to Roche cobas for the Rapid Detection of SARS-CoV-2

Clinical Evaluation of Three Sample-To-Answer 219 Platforms for the Detection of SARS-CoV-2

The Detection of SARS-221

CoV-2 using the Cepheid Xpert Xpress SARS-CoV-2 and Roche cobas SARS-CoV-2 Assays

Commercially Available and Laboratory Developed Assays for in vitro Detection of SARS-CoV-2 in 225 Clinical Laboratories

Boosting test-efficiency by pooled testing strategies for SARS-CoV-2

Viral load of SARS-CoV-2 in clinical samples

Viral dynamics of SARS-CoV-2 across a 231 spectrum of disease severity in COVID-19

Asymptomatic and presymptomatic SARS-COV-2 infections in residents of a long-term 241 care skilled nursing facility

SARS-CoV-2 asymptomatic and symptomatic patients and risk for transfusion 245 transmission

SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected 248 Patients

Evaluation of Group Testing for SARS-CoV-2 RNA

Page 9 of 13 pooling is viable on the GeneXpert ® system, similar to the aggressive pooling strategies being explored for the 186 laboratory-based RT-qPCR tests.

The authors would like to acknowledge partners who provided the clinical specimens used in this study.