key: cord-0849386-3zu782x0
authors: Ganz, T.; Sanderson, S.; Baush, C.; Mejia, M.; Gandhi, M.; Auclair, J.
title: Performance of the TaqMan COVID-19 Pooling Kit for detection of SARS-CoV-2 in Asymptomatic and Symptomatic populations at an Institution of Higher Education
date: 2021-05-21
journal: nan
DOI: 10.1101/2021.05.20.21257523
sha: 453dc3cc5e7377cfccf25309fa37babdda848e05
doc_id: 849386
cord_uid: 3zu782x0

Clinical evidence for asymptomatic cases of coronavirus disease (COVID-19) has reinforced the significance of effective surveillance testing programs. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays are considered the gold standard for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. However, the labor and resource requirements can be prohibitive with respect to large testing volumes associated with the pandemic. Pooled testing algorithms may serve to increase testing capacity with more efficient resource utilization. Due to the lack of carefully curated cohorts, there is limited evidence for the applicability of RT-PCR pooling in asymptomatic COVID-19 cases. In this study, we compared the analytical sensitivity of the TaqMan SARS-CoV-2 Pooling Assay to detect one positive sample in a pool of five anterior nare swabs in symptomatic and asymptomatic cohorts at an institute of higher education. Positive pools were deconvoluted and each individual sample was retested using the TaqPath COVID-19 Combo Kit. Both assays target the open reading frame (ORF) 1ab, nucleocapsid (N), and spike (S) gene of the strain that originated in Wuhan, Hubei, China. Qualitative results demonstrated absolute agreement between pooled and deconvoluted samples in both cohorts. Independent t-test performed on Ct shifts confirmed an insignificant difference between cohorts with p-values of 0.306 (Orf1ab), 0.147 (N), and 0.052 (S). All negative pools were correctly reported as negative. Thus, pooled PCR testing up to five samples is a valid method for surveillance testing of students and staff in a university setting, especially when the prevalence is expected to be low.

negative pools were correctly reported as negative. Thus, pooled PCR testing up to five samples is a valid 28 method for surveillance testing of students and staff in a university setting, especially when the 29 prevalence is expected to be low. 30

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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 21, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 3 Introduction: 31

Efforts to mitigate the reproductive number (R0) for the coronavirus disease 2019 (COVID-19) pandemic,  32   an emergent respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-33 CoV-2), has been undermined by the potential for sustained transmission through pre-symptomatic, 34 paucisymptomatic (subclinical), and asymptomatic carriers [1] . Growing evidence from immunological 35 case studies and bioinformatic investigations have substantiated clinical diagnostic testing that have 36 reported insignificant variance in results among patients that differ in symptomology. Asymptomatic 37 patients have reportedly developed ground-glass opacities in lung tissue and significant adaptive 38 immune (IgG and IgM) responses, however cell-mediated immune response from asymptomatic cases 39 have shown to be less significant compared to symptomatic cases [2, 3] . A recent investigation noted a 40 correlation between globally reported asymptomatic cases and a single nucleotide polymorphism (SNP) 41 in SARS-CoV-2 RNA at position 11083G>T translating to a phenylalanine mutation (L37F) on non-42 structural protein 6 (NSP6) [4] . While a less severe immune response may be elicited in asymptomatic 43 cases, the replication mechanisms in these individuals remain intact, and this necessitates effective risk 44 management measures [5] . 45

The analytical sensitivity of RT-qPCR has propelled its utility as the 'gold standard' to accurately detect 46 SARS-CoV-2 RNA from upper respiratory specimens (e.g., nasopharyngeal, anterior nare, and saliva); 47 however, the high-complexity format has merged with demands of a pandemic to generate severe 48 shortages in materials and technical personnel. A resurgence of interest in group testing, or sample 49 pooling, whereby the ratio of specimens to result is greater than one, has led investigators to optimize 50 protocols that would allow an increase in testing capacity without requiring additional resources [6] . 51

Caveats include a linear loss of sensitivity with sample dilution, and, in hierarchical systems, disease 52 prevalence may determine efficiency as positive pools are deconvoluted to retest each patient 53 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. March 24, 2020, Thermo Fisher Scientific, Inc. has developed a specialized RT-qPCR testing kit with 57 enhanced sensitivity for the purposes of pooling up to five samples known as the TaqMan™ SARS-CoV-2 58

Pooling Assay. In the following study, we compared the sensitivity of the TaqMan™ assay to detect one 59 positive sample in a pool of five from 30 symptomatic and 30 asymptomatic individuals that were 60 previously characterized as positive using the TaqPath™ COVID-19 Combo Kit; 10 negative pools of five 61 were included as a control group. 62

Sample Collection, Storage, and Preanalytical Processing: 64

The study was conducted at the Northeastern University COVID-19 response laboratory, the Life Science 65 Testing Center (LSTC; Burlington, MA, USA), which performs population testing for university affiliates. 66

Students on campus are required to test every three days while staff and faculty on campus test twice a 67

week. Affiliates attending campus are required to complete a daily wellness tracker, or a two-question 68 survey, that identifies whether they have experienced COVID-19 related symptoms or have been in close 69 contact with a positive COVID-19 case in the past 24 hours. Depending on the survey results, they are 70 directed to get tested at either the non-symptomatic site (Cabot Center), where observed self-collection 71 is performed, or the symptomatic site (Marino Center), where collection is performed by a healthcare 72 professional. Temperature checks and general symptom screening were performed by trained personnel 73 on the subjects at both sites to determine presence or absence of symptoms. Anterior nasal swabs 74 (polyester) are collected in 3 mL BD Vacutainer® (without additive) tubes and transported dry to the 75 LSTC facility. Upon logging into the laboratory information system (LIMS), 3 mL of viral transport media 76 (VTM; Redoxica, Little Rock, AR, USA) is added and the sample is shaken at 1000 rpm for 5 minutes. 77 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. binding beads, and 5 µL MS2 phage extraction control. Wash procedure was performed using an Agilent 99

Bravo liquid handler (Agilent Technologies, Santa Clara, CA, USA) whereby, after initial shaking 2 minutes 100 at 1050 rpm and 5 minutes incubation at 65°C, the beads underwent three cycles of 101 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 21, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 6 resuspension/aspiration in 165 µL wash buffer, 165 µL 80% ethanol, and 50 µL elution buffer, 102 respectively. RT-qPCR was performed using 10 µL of eluate added to 15 µL of reaction mix that 103 contained 6.25 µL TaqPath™ 

Containment of COVID-19: Simulating the impact of different policies and testing 167 capacities for contact tracing, testing, and isolation

Immune memory in 171 convalescent patients with asymptomatic or mild COVID-19

Clinical and immunological assessment of 174 asymptomatic SARS-CoV-2 infections

Decoding Asymptomatic COVID-19 Infection 176 and Transmission

Duration of infectiousness and correlation with RT-PCR cycle threshold values in 179 cases of COVID-19

Pooled testing for COVID-19 diagnosis by 184 real-time RT-PCR: A multi-site comparative evaluation of 5-& 10-sample pooling

Cost Effectiveness of Sample Pooling to Test 187 for SARS-CoV-2

Two-step strategy for the identification of SARS-CoV-2 191 variant of concern 202012/01 and other variants with spike deletion H69-V70

Increasing SARS-CoV-2 RT-qPCR testing 195 capacity by sample pooling

All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted May 21, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 7 ) and the deconvoluted asymptomatic samples were 20.64 test did not show a statistically significant Ct shift between the asymptomatic and symptomatic cohorts 136 with p-values of 0.306, 0.147, and 0.052 for ORF1ab, N, and S genes, respectively ( Figure 1g ). Each 137 negative pool reported negative and were not deconvoluted. 138

This study sought to confirm SARS-CoV-2 infections that cause mild immune responses proliferate to 140 similar extents as those that elicit severe cases of COVID-19, which enables equal probability of 141 detection in RT-qPCR assay. Furthermore, the TaqMan™ multiplex assay identified each positive sample 142 in pools of five with minimal loss of sensitivity. Prevalence of SARS-CoV-2 variants, apparent by S gene 143 target loss, has increased by an order of magnitude since November 2020 at the LSTC and cases 144 represented 7% of asymptomatic and 20% of symptomatic cases in the sample size for this study [8] . It is 145 important to note, significant dilution effects observed in all genes was a result of interpreting one 146 positive per pool of five and may indicate a limitation of the experimental design due to probability of 147 more than one positive per pool in practice [9] . Thus, the practical significance observed by the average 148 All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This strategy of increased extraction and elution volumes for group tests reported absolute qualitative 151 agreement to the deconvoluted counterpart, including a 1/3 gene positive case, and produced a more 152 conserved Ct shift [7, 9] . Since symptomatic subjects tend to have a significantly higher prevalence of 153 COVID-19 in a pandemic setting, from a resource efficiency standpoint, it is advisable to consider pooling 154 samples when testing subjects who are non-symptomatic such as in vaccinated populations, where the 155 prevalence rates are expected to be significantly lower. 

The authors declare no conflict of interest. 161

The authors would like to acknowledge the efforts of the LSTC technical staff, including M.K., R.M. who 163 made contributions to this work. 164 All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted May 21, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 9 References: 165 All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted May 21, 2021. b. c.d. e. f.

All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted May 21, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021