key: cord-342765-rw8valjp authors: Wacharapluesadee, Supaporn; Kaewpom, Thongchai; Ampoot, Weenassarin; Ghai, Siriporn; Khamhang, Worrawat; Worachotsueptrakun, Kanthita; Wanthong, Phanni; Nopvichai, Chatchai; Supharatpariyakorn, Thirawat; Putcharoen, Opass; Paitoonpong, Leilani; Suwanpimolkul, Gompol; Jantarabenjakul, Watsamon; Hemachudha, Pasin; Krichphiphat, Artit; Buathong, Rome; Plipat, Tanarak; Hemachudha, Thiravat title: Evaluating the efficiency of specimen pooling for PCR‐based detection of COVID‐19 date: 2020-05-13 journal: J Med Virol DOI: 10.1002/jmv.26005 sha: doc_id: 342765 cord_uid: rw8valjp In the age of a pandemic, such as the ongoing one caused by SARS‐CoV‐2, the world faces a limited supply of tests, personal protective equipment, and factories and supply chains are struggling to meet the growing demands. This study aimed to evaluate the efficacy of specimen pooling for testing of SARS‐CoV‐2 virus, to determine whether costs and resource savings could be achieved without impacting the sensitivity of the testing. Ten previously tested nasopharyngeal and throat swab specimens by real‐time PCR, were pooled for testing, containing either one or two known positive specimens of varying viral concentrations. Specimen pooling did not affect the sensitivity of detecting SARS‐CoV‐2 when the PCR cycle threshold (Ct) of original specimen was lower than 35. In specimens with low viral load (Ct>35), 2 out of 15 pools (13.3%) were false negative. Pooling specimens to test for COVID‐19 infection in low prevalence (≤1%) areas or in low risk populations can dramatically decrease the resource burden on laboratory operations by up to 80%. This paves the way for large‐scale population screening, allowing for assured policy decisions by governmental bodies to ease lockdown restrictions in areas with a low incidence of infection, or with lower risk populations. This article is protected by copyright. All rights reserved. The ongoing Coronavirus Disease 2019 (COVID-19) pandemic has highlighted the need for early diagnosis of emerging infectious diseases to better contain an outbreak. Testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, has been limited due to the considerable strain on Accepted Article global supply chains for reagents, personal protective equipment (PPE) and other consumables 1, 2 . To date, countries that are able to screen patients swiftly have fared better in containing the COVID-19 outbreak and suppressing the mortality rate associated with the disease 3 . The rapid diagnosis of COVID-19 in both symptomatic and asymptomatic patients can shed light on transmission patterns and facilitate contact tracing 2, 3 . Large scale population screening for COVID-19 infection is generally considered a necessary part of an exit strategy from the coronavirus lockdown. Specimen pooling is a method of screening large number of patients for an infection, and typically involves combining multiple patient specimens into a single test sample, then testing multiple such samples. This approach has the advantage of cost-effectiveness and speed, and was used to retrospectively screen for COVID-19 in specimens that were negative for common respiratory viruses earlier in the course of the pandemic in the United States 4 . Specimen pooling has also been used in screening efforts for several other infectious diseases, including donated blood samples for HIV [6] [7] [8] [9] . Pooling nasopharyngeal and throat swab (NT) specimens would be more economical than individually testing all specimens from low-risk populations, particularly in limited-resource settings 10 . It is unclear how pooling biological samples would affect the sensitivity and the false-negative rate of PCR assays. The current study compares laboratory results from pooled testing (10 samples) with individually tested samples using the standard real-time polymerase chain reaction (qPCR) to ensure that detection accuracy is not compromised. Additionally, NT specimens with PCR cycle threshold (Ct) greater than 35 were pooled to determine the limit of detection and sensitivity of pooling samples to test for SARS-CoV-2. This study is an evaluation of laboratory techniques using archived clinical specimens and was exempted from Chulalongkorn University Institutional Review Board (IRB) review. NT specimens used in this study had been collected from patients under investigation (PUI) for COVID-19 infection at King Chulalongkorn Memorial Hospital, placed in 2.0mL viral transport media (VTM) and sent to the Thai Red Cross Emerging Infectious Diseases Health Science Centre Laboratory for testing between February 1, and March 31, 2020. All specimens had been stored at -80 o C. A total of 50 leftover specimens that had tested negative for SARS-CoV-2 by qPCR amplifying the ORF1ab gene (BGI, Shenzhen, China) were combined into a single sample, which was then used as the negative portion of all pooling tests. The purpose of homogenized negative pooled specimen was to standardize and eliminate the possibility of variations between pools, which could have potentially affected our results. This pooled negative NT-VTM was re-tested for SARS-CoV-2 using qPCR to confirm the negative result prior to pooling with selected positive specimens. This study used Boom method's magnetic extraction-based assay (NucliSens®, easyMag®, bioMérieux, Marcy-l'Étoile, France) to extract DNA and RNA, which allows a maximum specimen volume of 1.0mL 11 . By using magnetic beads to capture DNA and RNA during the extraction step, pooling 10 specimens of 0.1mL each (total of This article is protected by copyright. All rights reserved. 1.0mL extraction sample) can result in the same extraction capability as 0.1mL if the elution volume at the end is equal and there is no PCR interference from the specimen such as lipid, protein or cell debris. Two pooling ratios were evaluated in this study, termed 1X and 2X. In the 1X ratio, 0.1mL of NT-VTM from one SARS-CoV-2 positive specimen was combined with 0.9mL pooled negative NT-VTM, thus modeling a 10% infection rate. Correspondingly, in the 2X ratio, 0.1mL of NT-VTM each from two SARS-CoV-2 positive specimens were pooled with 0.8mL pooled negative NT-VTM, thus modeling a 20% infection rate (see Figure 1 ). All pooled samples (1.0mL each) were added to 2.0mL of lysis buffer (total 3.0mL) and processed for nucleic acid extraction using NucliSens® easyMAG® instrument (bioMérieux). In addition, 0.1mL of the same positive specimens that were used in the pooled samples were re-tested individually for sensitivity comparison using a separate extraction system (EZ1, Qiagen, Hilden, Germany). Realtime PCR (qPCR) for detection of SARS-CoV-2 was performed using a commercial kit which targets the ORF1ab gene as per the manufacturer's protocol (BGI, Shenzhen, China). The protocol's stated limit of detection of ORF1ab real-time PCR was 100 copies/mL and the cutoff PCR cycle threshold (Ct) was 38. Previously positive specimens with high and low-concentrations of RNA, as determined by PCR Ct values at the time of detection, were selected to determine the effect of viral load on pooling to ensure that the sensitivity and accuracy of the assay was maintained (Table 1) The fifteen 1X (L>35) pools were tested by performing duplicate (replicates I and II) qPCR assays to determine the limit of detection of specimen pooling when compared to individual testing. The 2X ratio pools had two positive specimens each (Positive NT 1 and 2 in Table 1 The sensitivity of viral RNA detection for each pool was compared with the sensitivity of qPCR results for the individually tested positive specimen in that pool. For 2X ratio This article is protected by copyright. All rights reserved. pools, the positive specimen with the lower Ct value (Positive NT 1), when individually tested, was used for comparison. All 1X ratio pools (Ct<35) were positive, with Ct value difference within a range of -1.36 to +1.66 when compared to individual (non-pooled) testing. All 2X ratio pools were positive, with Ct value difference within a range of -1.72 to +1.81 when compared to individual testing (Table 1 ). Statistical paired t-test was calculated to compare the Ct value differences between pooled (including all patterns in Figure 1 Table 1 ). During the individual testing, 3 samples (out of 15) had 1 undetected result. Cost effectiveness of the pooling strategy was calculated, based on varying disease prevalence rates (0.1-10%) ( Table 2 ). Pooling appears most cost-effective when This article is protected by copyright. All rights reserved. testing among populations with lower COVID-19 prevalence. Estimated laboratory costs were reduced from $35 per patient to $3.85, $6.85, $17.54, $26.30 at prevalences of COVID-19 in the tested population of 0.1%, 1, 5%, and 10% respectively. By this estimation, pooled-specimen testing of 1,000,000 subjects in a population with 1% COVID-19 prevalence would save approximately $28.15 million, assuming evenly distributed positive specimens in each pool ( Table 2) . This study demonstrates that specimen pooling (either 1X or 2X pooling ratios) does not compromise the sensitivity of detecting SARS-CoV-2 provided the Ct value of the individually tested sample is lower than 35. In 2X ratio pooling, qPCR testing It was previously demonstrated that when the prevalence of COVID-19 is 1%, the optimal specimen pool size is 11 with an overall increase in testing efficiency calculated at 400% 10 . In this study, a 10-specimen pool size (0.1mL each specimen) was chosen based on the capacity of the RNA extraction system in the laboratory where this study was performed, and the result was similar to 5 samples pooling 10, 12 . The capability of the extraction protocol can affect the sensitivity of pooled testing. In this study, the maximum volume of specimen for extraction was 1.0mL (0.1mL x 10 samples). The sensitivity of the assay can be improved if ratio of 1 to 5 (0.2mL of each specimen) is used. It can also be improved by collecting specimens directly in 1.0mL of lysis buffer (extraction buffer), where maximum of 0.3mL of 10 samples can be pooled (NucliSens® easyMAG® or miniMAG®, bioMérieux), instead of 2.0mL of VTM. The nucleic acid can therefore be directly extracted without diluting with VTM. Additionally, the lysis buffer inactivates the virus, making it safer to handle. Further, similar PCR Ct values (within ±2; statistically not significant) between pooled and individually tested specimens indicated there was no interference of PCR inhibitor from 1.0mL pooled specimens in one extraction tube. Beyond maintaining accuracy, specimen pooling will almost certainly reduce cost. For example, if 1% of the population is infected, pooling 10 specimens can reduce the cost of laboratory operation by about 80% (Table 2 ). However, in the case of 10% prevalence, specimen pooling will only save 24.87%, as positive pooled samples will This article is protected by copyright. All rights reserved. need to be individually tested. Therefore, pooling samples is especially useful in areas with low prevalence rates, or when conducting proactive surveillance in areas of low infection rate. Proactive surveillance, particularly in asymptomatic cases, remains a challenge to surmount in order to exit lockdown, as screening on a large scale is required. A limitation of this study is the maximum number of two positive specimens in the 10-specimen pool. In theory, more positive specimens in a pool could decrease the sensitivity of qPCR as it would result in too many viral copies, causing an insufficiency of PCR enzyme and other reagents in the mix to amplify all the viral copies. Practically, however, this does not affect the overall testing results, since positive pools would require individual testing in any case. pandemic. The present gold standard for testing SARS-CoV-2 is qPCR, which requires resources that are currently limited, along with specialized equipment and technically skilled labor. Shortage of testing reagents and equipment may result in delays in testing and result in reduced effectiveness in containing the outbreak. Pooled specimen testing would enable substantial savings in reagent costs, technical burden and time to generate laboratory results. This article is protected by copyright. All rights reserved. *% of pool with no infection = (% of non-infected samples in one pool)^number of samples per pools Figure Figure 1 Illustrates the experimental design of the pooling strategies tested in this study Covid-19: how doctors and healthcare systems are tackling coronavirus worldwide Fair allocation of scarce medical resources in the time of Covid-19 Countries test tactics in 'war'against COVID-19 Sample Pooling as a Strategy to Detect Community Transmission of SARS-CoV-2 Testing the repatriated for SARS-Cov2: Should laboratory-based quarantine replace traditional quarantine? Detection of HIV-1 and HCV infections among antibody-negative blood donors by nucleic acid-amplification testing Detection of acute infections during HIV testing in North Carolina Detection of acute HIV infections Acute HIV revisited: new opportunities for treatment and prevention Assessment of Specimen Pooling to Conserve SARS CoV-2 Testing Resources Rapid and simple method for purification of nucleic acids Pooling of Nasopharyngeal Swab Specimens for SARS-CoV-2 detection by RT-PCR Hospital's Excellent Center Program, National Research Council of Thailand (NRCT), This article is protected by copyright. All rights reserved. Reduction Agency (DTRA). We would also like to acknowledge Dr. Paul Gaudio (Yale University) for his kind assistance in the critical editing of this manuscript.