key: cord-0969068-lwh3rww4
authors: Anderson, Cole; Castillo, Fritz; Koenig, Michael; Managbanag, Jim
title: Pooling nasopharyngeal swab specimens to increase testing capacity for SARS-CoV-2
date: 2020-05-22
journal: bioRxiv
DOI: 10.1101/2020.05.22.110932
sha: 0794a0afcec438b3b5bc5f6f7ac9a8d9079fc967
doc_id: 969068
cord_uid: lwh3rww4

The recent emergence of SARS-CoV-2 has lead to a global pandemic of unprecedented proportions. Current diagnosis of COVID-19 relies on the detection of SARS-CoV-2 RNA by RT-PCR in upper and lower respiratory specimens. While sensitive and specific, these RT-PCR assays require considerable supplies and reagents, which are often limited during global pandemics and surge testing. Here, we show that a nasopharyngeal swab pooling strategy can detect a single positive sample in pools of up to 10 samples without sacrificing RT-PCR sensitivity and specificity. We also report that this pooling strategy can be applied to rapid, moderate complexity assays, such as the BioFire COVID-19 test. Implementing a pooling strategy can significantly increase laboratory testing capacity while simultaneously reducing turnaround times for rapid identification and isolation of positive COVID-19 cases in high risk populations.

patients with viral pneumonia 2 . Pneumonia associated with SARS-CoV-2 was later designated 26 as coronavirus disease 2019 (COVID-19) by the World Health Organization in February 2020 3 . 27

It was determined that after a zoonotic transmission event in Wuhan city 4 , widespread person-28 to-person transmission quickly occurred that led to the infection and death of over 80,000 and 29 3,000 people in China, respectively. To date, according to the WHO, there have been 4,258,666 30 reported cases of COVID-19, including 294,190 deaths worldwide 5 . 31

Since the initial outbreak in China, COVID-19 has been declared a global pandemic affecting at 32 least 216 other countries, territories or areas. To monitor and diagnose COVID-19, the US Food 33 and Drug Administration (FDA) approved an emergency use authorization (EUA) for the CDC 34 2019-nCoV Real-Time RT-PCR Diagnostic Panel on February 4, 2020 6 . This protocol allows for 35 the rapid detection of SARS-CoV-2 RNA from clinical specimens such as, nasopharyngeal and 36 oropharyngeal swabs, sputum, bronchoalveolar lavage, and tracheal aspirates. As evidenced by 37 the ongoing SARS-CoV-2 pandemic, increased demand for testing can overwhelm diagnostic 38 laboratories and lead to drastic shortages in supplies and reagents. A strategy to overcome 39 high testing demand is to pool specimens before RNA extraction, test pools, and then retest 40 individual specimens from positive pools. Similar strategies have shown to increase testing 41 capacity for the detection of common infectious diseases such as influenza, HIV, Hepatitis, and 42 Chlamydia trachomatis 7-11 . 43 In this study, we examined the feasibility of pooling nasopharyngeal swab specimens submitted 44 for COVID-19 testing using the CDC 2019-nCoV RT-PCR diagnostic panel without compromising 45

clinical sensitivity. Our data shows that pooling respiratory samples during times of increased 46 volume and low disease prevalence can save time and reagents without significant 47 modifications to laboratory infrastructure or workflow. 48 49

This study was determined to meet the exempt criteria listed in 32CFR219.104(d) from the 51 Landstuhl Regional Medical Center Exempt Determination Official. 52

During an outbreak cluster of SARS-CoV-2 in Stuttgart, Germany, 494 nasopharyngeal (NP) 53 swabs were collected and placed into 1.0 ml of normal saline. Specimens were submitted to 54 the Virology laboratory at Landstuhl Regional Medical Center for routine SARS-CoV-2 testing 55 using the CDC 2019-nCoV RT-PCR assay. Post clinical testing, specimens were de-identified and 56 randomly assigned into pools of 10 to create 50 distinct pools (the 50 th pool contained 4 57 specimens diluted in 0.6 ml of transport media). Pools were created by combining 100 ul of 58 each specimen to create 1.0 ml pools. Viral transport media was added to each pool at a 1:1 59 ratio for nucleic acid extraction performed on the Roche MagNA Pure 24 platform using the 60 MagNA Pure 24 Total NA Isolation kit (Roche). Elution volume was set to 50 ul to concentrate 61 viral RNA. Each round of extraction contained a human specimen control to monitor for PCR 62 inhibition and specimen quality. Table 2) . The mean C T value and standard deviation for N1 and N2 of the pools were 84 29.2 (4.4) and 29.4 (4.3), respectively. Similarly, the mean C T values of individual positive 85 specimens were 28.0 (4.5) and 29.9 (4.8) for N1 and N2, respectively. Despite dilution, there 86 was no significant difference in mean C T value between the pooled and individually tested 87 specimens (Figure 1) . 88

To determine if a pooling approach is feasible with rapid, moderate complexity tests, we tested (Table 3) . 

A new coronavirus associated with human respiratory disease in China

Clinical features of patients infected with 2019 novel coronavirus in 160

WHO Director-General's remarks at the media briefing on 2019-nCoV on 11 February

A pneumonia outbreak associated with a new coronavirus of probable bat 166 origin

Coronavirus disease (COVID-19) Situation Report-116. 168 Available at

Update: FDA Issues first Emergency Use Authorization for Point 172 of Care Diagnostic | FDA

Pooling nasopharyngeal/throat swab specimens to increase testing 176 capacity for influenza viruses by PCR

Pooling of sera for human 178 immunodeficiency virus (HIV) testing: An economical method for use in developing 179 countries

Pooling of clinical 181 specimens prior to testing for Chlamydia trachomatis by PCR is accurate and cost saving

High throughput screening of 16 million serologically negative blood 184 donors for hepatitis B virus, hepatitis C virus and human immunodeficiency virus type-1 185 by nucleic acid amplification testing with specific and sensitive multiplex reagent in 186

Utility of pooled urine specimens for detection of Chlamydia trachomatis 188 and Neisseria gonorrhoeae in men attending public sexually transmitted infection clinics 189 in Mumbai, India, by PCR

Evaluation of COVID-19 RT-qPCR test in multi-sample pools

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

Increasing testing throughput and case detection with a 195 pooled-sample Bayesian approach in the context of COVID-19

BioFire COVID-19 Test Emergency Use Authorization