key: cord-275360-uphdzj5l
authors: Sahajpal, Nikhil Shri; Mondal, Ashis K.; Njau, Allan; Ananth, Sudha; Jones, Kimya; Ahluwalia, Pankaj K.; Ahluwalia, Meenakshi; Jilani, Yasmeen; Chaubey, Alka; Hegde, Madhuri; Kota, Vamsi; Rojiani, Amyn; Kolhe, Ravindra
title: Proposal of Reverse Transcription-PCR–Based Mass Population Screening for SARS-CoV-2 (COVID-19)
date: 2020-07-30
journal: J Mol Diagn
DOI: 10.1016/j.jmoldx.2020.07.001
sha: 
doc_id: 275360
cord_uid: uphdzj5l

Testing for SARS-CoV-2 has lagged behind in many countries due to lack of adequate test kits and bottlenecks in the analytical process. The aim of this study was to investigate the feasibility and accuracy of a sample pooling approach for wide-scale population screening for COVID-19. A total of 940 nasopharyngeal-swab samples (934 negative and 6 positive) previously tested for SARS-CoV-2 were de-identified and assigned random numbers for analysis. From this, 94 pools of 10 samples each were created. Automated RNA extraction followed by RT-PCR was carried out in a 96 well plate. Positive pools were identified and the individual samples were re-analyzed.

The outbreak of COVID-19 (caused by SARS-CoV-2) is now a pandemic that has caused mass disruption of the world order, impacting public health care systems, social lifestyle, governance and economics. Since its identification in the region of Wuhan, China, over 6.24 M confirmed cases with over 374,452 COVID-19 related deaths have been reported globally (https://coronavirus.jhu.edu/map.html, last accessed June 1, 2020). The incidence of disease is highly varied across the globe, with the incidence rates ranging from 5618 per 1M in US, 617 per 1M in Spain and ~50 per 1M in Africa, compared to the global incidence rate of 816 per 1M (https://www.worldometers.info/coronavirus/#countries, last accessed June 1, 2020).

In an attempt to contain the spread of disease, multidisciplinary strategies have been launched in different regions of the world, including implementing social distancing, maintaining personal hygiene, contact tracing, quarantine, travel restrictions and lockdowns. 1 A widely accepted method, though not effectively implemented as a measure to control its spread is testing for SARS-CoV-2, typically utilizing nasopharyngeal swab specimens. Patients tested positive require appropriate clinical management by either effective isolation or quarantine at home for mild symptoms or within healthcare facilities for moderate to severe symptoms. Wide-scale testing approaches such as those implemented in South Korea have resulted in great success at reducing community spread and lowering mortality rates. 2 In addition, wide scale testing provides more informative epidemiological data for drafting policies on disease monitoring and control. Currently, at least 85 manufacturers of diagnostic assays have received Emergency Use Authorization (EUA) from the Federal Drug and Food Administration (FDA) for COVID-19 testing (https://www.fda.gov/medical-devices/emergency-situations-medical-devices/emergencyuse-authorizations#COVID19ivd, last accessed June 1, 2020). 4 However, testing has lagged behind in many countries due to various factors, most significant of which being supply chain issues with lack of reagents and adequate test kits. Therefore, many patients (both symptomatic and asymptomatic) remain untested and hence are potentially contributing to community spread of the virus. Furthermore, many countries including high income countries, have logically resorted to prioritize testing for the hospitalized, symptomatic and high risk population. With this approach, absence of testing or long turnaround times among exposed but asymptomatic individuals and patients exhibiting mild symptoms have been observed; a factor that likely contributes to exponential community spread.

Herein, we propose a mass population screening approach, based on sample pooling strategy for rapid and wide-scale population screening that may be adopted by laboratories currently using RT-PCR based methods to test for SARS-CoV-2. The strategy we propose leverages on existing high throughput systems that employ high analytically sensitive [limit of detection (LOD) 5-20 copies/ml] real-time PCR chemistries, coupled with pooling of samples based on current COVID-19 incidence rates. Pooling of samples compared to individual testing has been investigated previously such as in screening blood donations, infectious and genetic diseases. 3, 4, 5 Pooling when carefully executed, has been found to be useful and more cost effective for estimating incidence rates in specific cases. 6 The advantages of this approach include the potential to catch up with huge testing deficits, reducing turnaround times and most importantly ensuring enormous savings through the most efficient use of RNA extraction and/or testing kits, which even today are in significant short supply.

The RT-PCR based methods have two primary components, first: RNA extraction from clinical specimens and second: RT-PCR based detection of SARS-CoV-2 nucleic acid region(s 

The assay is based on RNA extraction followed by TaqMan Table S1 ). Table S2 ).

Identifying the positive sample:

Step 2

Pool(s) that resulted positive were identified and the 10 samples comprising each positive pool were retrieved and processed for downstream extraction and RT-PCR analysis for the identification of the positive sample(s). The positive sample(s) were identified based on the Ct value specified by the manufacturer. In addition, one negative pool was selected randomly and each sample was re-analyzed individually as a QC measure.

In step 1, the QC of negative and positive sample was observed to be within the range Table S5 ). Therefore, 1000 samples resulted in an overall 91.6% positive (PPA) and 100% negative percent agreement (NPA) compared to individual testing approach.

The COVID-19 pandemic has resulted in an overwhelming number of infected patients, leading to a tremendous burden on health care resources to the extent of outstripping the current production capabilities for supplies. Innovative ideas in specimen collection, isolation, respiratory support and other patient care plans have been implemented. Scaling up testing has been identified as a key component to manage the pandemic. 9 Laboratories and manufactures of test kits across the world have also responded by ramping up testing. However, more innovative approaches are needed for wide-scale population testing and to side step the foreseeable shortages in test kits. In this study we demonstrate that pooling patient samples and testing them on high sensitivity, high throughput systems is both practical and accurate.

In our proof-of-concept study with 40 samples, 4 pools were formed with 10 samples Table S5 ). Another challenge to this approach is inaccurate designation of individual and pooled samples within the 96 well plate, resulting in sample mix-up. To minimize this, use of a minimum of two identifiers and well documented workflows to ensure traceability of all pooled samples is essential. In addition, use of bar code readers and automated sample processing would minimize the chances of such a mix-up. Further, selected samples from a randomly selected negative pool(s) should also be analyzed individually as a quality control monitor. In our study, all samples in one negative result pool/well were retested and were found to be in agreement with individually tested samples.

In terms of cost analysis, 1 million individuals can be tested for $9.1M with the proposed mass population screening approach compared to $58M with routine screening. More important however, is the potential to massively increase the number of people tested using the same quantity of reagents/test kits. This is a critical advantage given the short supply of test kits, a fact with the disparity that ensues especially in low and middle income countries (LMIC). The direct saving on reagent and test kits are complemented by indirect savings on laboratory supplies including personal protective equipment that are needed to perform testing on these infectious clinical samples. These savings will enhance sustainable laboratory operations throughout the pandemic or can be deployed to laboratories that are facing dire constraints in supplies.

In conclusion, we surmise that this unprecedented crisis requires innovative solutions at all levels. The strategy we propose leverages existing high throughput systems which employ analytically high sensitive RT-PCR chemistries, coupled with pooling of samples based on current COVID-19 incidence rates. In this study, we analyzed 1000 samples in a pooled approach using only two-extraction and -PCR runs and achieved 91.6% PPA and 100% NPA. In order to optimize the number of pooled samples, real time region specific data in websites such that hosted by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University, Baltimore, MD, is helpful. In addition, robust validation and knowledge of the analytical performance of the assay to be adopted as well as regional/laboratory positivity rates are critical in this approach. The number of samples pooled are inversely proportional the analytical sensitivity of the assay and the local positivity rate. 10, 11 The advantages of this innovative approach include potential of catching up with testing, clearing backlogged samples, reducing turnaround times and ensuring enormous savings on RNA extraction and/or testing kits and laboratory supplies that are in short supply. This would relieve the pressure mounting on laboratories for increased testing, hopefully making a significant contribution to control of this pandemic. In addition, this strategy may come in handy for effective and consistent disease surveillance as many states and countries begin to reopen businesses, airports, public gatherings and work environments. Monitoring spikes in the number of cases in groups of individuals in the same environment will facilitate rapid and early containment. 

COVID-19: towards controlling of a pandemic

Estimating the reproductive number and the outbreak size of Novel Coronavirus disease (COVID-19) using mathematical model in Republic of Korea

Impact of pooling on accuracy of hepatitis B virus surface antigen screening of blood donations

Sensitivity evaluation of the Gen-Probe AMP-CT assay by pooling urine samples for the screening of Chlamydia trachomatis urogenital infection

A pooling strategy for heterozygote screening of the delta F508 cystic fibrosis mutation

Feasibility of pooling sera for HIV-1 viral RNA to diagnose acute primary HIV-1 infection and estimate HIV incidence

Sample pooling as a strategy to detect community transmission of SARS-CoV-2

Assessment of specimen pooling to conserve SARS CoV-2 testing resources

The 3 Steps Needed to End the COVID-19 Pandemic: Bold Public Health Leadership, Rapid Innovations, and Courageous Political Will. JMIR public health and surveillance

Optimization of Group Size in Pool Testing Strategy for SARS-CoV-2: A Simple Mathematical Model

Test in Multi-Sample Pools

The authors thank Lisa Middleton for help with manuscript review.