scholarly journals Swab pooling for large-scale RT-qPCR screening of SARS-CoV-2

2020 ◽  
Author(s):  
Ana Paula Christoff ◽  
Giuliano Netto Flores Cruz ◽  
Aline Fernanda Rodrigues Sereia ◽  
Dellyana Rodrigues Boberg ◽  
Daniela Carolina de Bastiani ◽  
...  
Keyword(s):  
Large Scale ◽  
Sample Collection ◽  
Laboratory Capacity ◽  
Positive Individual ◽  
Sample Pooling ◽  
Pooling Strategies ◽  
Low Prevalence ◽  
Pooled Samples ◽  
Sample Dilution ◽  
Large Scale Screening

Pool testing has been proposed as an alternative for large-scale SARS-CoV-2 screening. However, dilution factors proportional to the number of pooled samples have been a source of major concern regarding its diagnostic performance. Further, sample pooling can lead to increased laboratory workload and operational complexity. Therefore, pooling strategies that minimize sample dilution, loss of sensitivity, and laboratory overload are needed to allow reliable and large-scale screenings of SARS-CoV-2. Here, we describe a pooling procedure in which nasopharyngeal swabs are pooled together at the time of sample collection (swab pooling), decreasing laboratory manipulation and minimizing dilution of the viral RNA present in the samples. Paired analysis of pooled and individual samples from 613 patients revealed 94 positive individual tests. Having individual testing as a reference, no false-positives or false-negatives were observed for swab pooling. A Bayesian model estimated a sensitivity of 99% (Cr.I. 96.9% to 100%) and a specificity of 99.8% (Cr.I. 99.4% to 100%) for the swab pooling procedure. Data from additional 18,922 patients screened with swab pooling were included for further quantitative analysis. Mean Cq differences between individual and corresponding pool samples ranged from 0.1 Cq (Cr.I. -0.98 to 1.17) to 2.09 Cq (Cr.I. 1.24 to 2.94). Overall, 19,535 asymptomatic and presymptomatic patients were screened using 4,400 RT-qPCR assays, resulting in 246 positive patients (positivity rate 1.26%). This corresponds to an increase of 4.4 times in laboratory capacity and a reduction of 77% in required tests. Finally, these data demonstrate that swab pooling can significantly minimize sample dilution and sensitivity issues commonly seen in its traditional counterpart. Therefore, swab pooling represents a major alternative for reliable and large-scale screening of SARS-CoV-2 in low prevalence populations.

scholarly journals Swab pooling: A new method for large-scale RT-qPCR screening of SARS-CoV-2 avoiding sample dilution

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246544 ◽  
Author(s):  
Ana Paula Christoff ◽  
Giuliano Netto Flores Cruz ◽  
Aline Fernanda Rodrigues Sereia ◽  
Dellyana Rodrigues Boberg ◽  
Daniela Carolina de Bastiani ◽  
...  
Keyword(s):  
Diagnostic Performance ◽  
Large Scale ◽  
Dilution Effect ◽  
False Negatives ◽  
Laboratory Capacity ◽  
Asymptomatic Patients ◽  
Low Prevalence ◽  
Paired Analysis ◽  
Sample Dilution ◽  
Large Scale Screening

To minimize sample dilution effect on SARS-CoV-2 pool testing, we assessed analytical and diagnostic performance of a new methodology, namely swab pooling. In this method, swabs are pooled at the time of collection, as opposed to pooling of equal volumes from individually collected samples. Paired analysis of pooled and individual samples from 613 patients revealed 94 positive individuals. Having individual testing as reference, no false-positives or false-negatives were observed for swab pooling. In additional 18,922 patients screened with swab pooling (1,344 pools), mean Cq differences between individual and pool samples ranged from 0.1 (Cr.I. -0.98 to 1.17) to 2.09 (Cr.I. 1.24 to 2.94). Overall, 19,535 asymptomatic patients were screened using 4,400 RT-qPCR assays. This corresponds to an increase of 4.4 times in laboratory capacity and a reduction of 77% in required tests. Therefore, swab pooling represents a major alternative for reliable and large-scale screening of SARS-CoV-2 in low prevalence populations.

scholarly journals Pooling of samples to optimize SARS-CoV-2 diagnosis by RT-qPCR: comparative analysis of two protocols.

2020 ◽  
Author(s):  
Fabiana Volpato ◽  
Daiana Lima-Morales ◽  
Priscila Lamb Wink ◽  
Julia Willig ◽  
Fernanda de-Paris ◽  
...  
Keyword(s):  
Large Scale ◽  
Diagnostic Yield ◽  
Rna Extraction ◽  
Clinical Samples ◽  
The Novel ◽  
Positive Individual ◽  
Sample Pooling ◽  
Novel Coronavirus ◽  
The Individual ◽  
Pooled Samples

RT-qPCR for SARS-CoV-2 is the main diagnostic test used to identify the novel coronavirus. Several countries have used large scale SARS-CoV-2 RT-qPCR testing as one of the important strategies for combating the pandemic. In order to process the massive needs for coronavirus testing, the usual throughput of routine clinical laboratories has reached and often surpassed its limits and new approaches to cope with this challenge must be developed. This study has aimed to evaluate the use pool of samples as a strategy to optimize the diagnostic of SARS-CoV-2 by RT-qPCR in a general population. A total of 220 naso/orofaryngeal swab samples were collected and tested using two different protocols of sample pooling. In the first protocol (Protocol A); 10 clinical samples were pooled before RNA extraction. The second protocol (Protocol B) consisted of pooling the already extracted RNAs from 10 individual samples. Results from Protocol A were identical (100% agreement) with the individual results. However, for results from Protocol B, reduced agreement (91%) was observed in relation to results obtained by individual testing. Inconsistencies observed were related to RT-qPCR results with higher Cycle Thresholds (Ct > 32.73). Furthermore, in pools containing more than one positive individual, the Ct of the pool was equivalent to the lowest Ct among the individual results. These results provide additional evidence in favor of the clinical use of pooled samples for SARS-CoV-2 diagnosis by RT-qPCR and suggest that pooling of samples before RNA extraction is preferrable in terms of diagnostic yield.

scholarly journals Rapid, Reliable and Robust approach for extraction-free RT-PCR based detection of SARS-CoV-2 in clinical setting to expedite large scale screening

2021 ◽  
Author(s):  
Abhilasha Dubey ◽  
Sanjay Upadhyay ◽  
Manjeet Mehta
Keyword(s):  
Large Scale ◽  
Target Genes ◽  
Low Cost ◽  
Diagnostic Methods ◽  
Analysis Method ◽  
Rt Pcr ◽  
Qpcr Method ◽  
Quantitative Results ◽  
Pooled Samples ◽  
Large Scale Screening

Rapid, reliable and robust method for the detection of SARS-CoV-2 is an indispensable need for diagnostics. The development of diagnostic methods will aid to address further waves of the pandemic potentially with rapid surveillance of disease and to allay the fears. To meet this challenge, we have developed a rapid RT-qPCR method for the detection of 3 target genes or confirmatory genes in less than 30 minutes. The assay showed 100% sensitivity and 100% specificity when tested on 120 samples. We compared a conventional extraction based method with extraction-free method, and then further reduced the run time of extraction free method. Additionally, we have validated our rapid RT-qPCR method for the assessment of pooled samples. We hereby propose a most reliable approach for the mass screening of samples with ease of operation at a low cost. Finally we designed a single tube analysis method which provides qualitative as well as quantitative results in minimum time.

scholarly journals Efficient and Practical Sample Pooling for High-Throughput PCR Diagnosis of COVID-19

2020 ◽  
Author(s):  
Haran Shani-Narkiss ◽  
Omri David Gilday ◽  
Nadav Yayon ◽  
Itamar Daniel Landau
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Diagnostic Testing ◽  
Target Frequency ◽  
Public Health Agencies ◽  
Highly Efficient ◽  
Pcr Diagnosis ◽  
Sample Pooling ◽  
Pooling Strategies ◽  
Technical Advances

AbstractIn the global effort to combat the COVID-19 pandemic, governments and public health agencies are striving to rapidly increase the volume and rate of diagnostic testing. The most common form of testing today employs Polymerase Chain Reaction in order to identify the presence of viral RNA in individual patient samples one by one. This process has become one of the most significant bottlenecks to increased testing, especially due to reported shortages in the chemical reagents needed in the PCR reaction.Recent technical advances have enabled High-Throughput PCR, in which multiple samples are pooled into one tube. Such methods can be highly efficient, saving large amounts of time and reagents. However, their efficiency is highly dependent on the frequency of positive samples, which varies significantly across regions and even within regions as testing criterion and conditions change.Here, we present two possible optimized pooling strategies for diagnostic SARS-CoV-2 testing on large scales, both addressing dynamic conditions. In the first, we employ a simple information-theoretic heuristic to derive a highly efficient re-pooling protocol: an estimate of the target frequency determines the initial pool size, and any subsequent pools found positive are re-pooled at half-size and tested again. In the range of very rare target (<0.05), this approach can reduce the number of necessary tests dramatically, for example, achieving a reduction by a factor of 50 for a target frequency of 0.001. The second method is a simpler approach of optimized one-time pooling followed by individual tests on positive pools. We show that this approach is just as efficient for moderate target-product frequencies (0.05<0.2), for example, achieving a two-fold in the number of when the frequency of positive samples is 0.07.These strategies require little investment, and they offer a significant reduction in the amount of materials, equipment and time needed to test large numbers of samples. We show that both these pooling strategies are roughly comparable to the absolute upper-bound efficiency given by Shannon’s source coding theorem. We compare our strategies to the naïve way of testing and to alternative matrix-pooling methods. Most importantly, we offer straightforward, practical pooling instructions for laboratories that perform large scale PCR assays to diagnose SARS-CoV-2 viral particles. These two pooling strategies may offer ways to alleviate the bottleneck currently preventing massive expansion of SARS-CoV-2 testing around the world.

scholarly journals Toward Community Surveillance: Detecting Intact SARS-CoV-2 Using Exogeneous Oligonucleotide Labels

2021 ◽  
Author(s):  
Thomas R. Carey ◽  
Molly Kozminsky ◽  
Jennifer Hall ◽  
Valerie Vargas-Zapata ◽  
Kristina Geiger ◽  
...  
Keyword(s):  
Gold Standard ◽  
Viral Particles ◽  
Intact Virus ◽  
Sample Pooling ◽  
Pooled Samples ◽  
Sample Dilution ◽  
Testing Efficiency

AbstractThe persistence of the COVID-19 pandemic demands a dramatic increase in testing efficiency. Testing pooled samples for SARS-CoV-2 could meet this need; however, the sensitivity of RT-qPCR, the gold standard, significantly decreases with an increasing number of samples pooled. Here, we introduce DIVER, a method that quantifies intact virus and is robust to sample dilution. DIVER first tags viral particles with exogeneous oligonucleotides, then captures the tagged particles on ACE2-functionalized beads, and finally quantifies the oligonucleotide tags using qPCR. Using spike-presenting liposomes and Spike-pseudotyped lentivirus as SARS-CoV-2 models, we show that DIVER can detect 1×105 liposomes and 100 pfu lentivirus and can successfully identify positive samples in pooling experiments. Overall, DIVER is well-positioned for efficient sample pooling and expanded community surveillance.

scholarly journals Household Representative Sample Strategy for COVID-19 Large-Scale Population Screening

2020 ◽  
Author(s):  
John Takyi-Williams
Keyword(s):  
Continuous Monitoring ◽  
Large Scale ◽  
Group Testing ◽  
Detection Sensitivity ◽  
Disease Group ◽  
Testing Method ◽  
Scale Population ◽  
Virus Exposure ◽  
Pooled Samples ◽  
Sample Dilution

In the advent of COVID-19 pandemic, testing is highly essential to be able to isolate, treat infected persons, and finally curb transmission of this infectious respiratory disease. Group testing has been used previously for various infectious diseases and recently reported for large-scale population testing of COVID-19. However, possible sample dilution as a result of large pool sizes has been reported, limiting testing methods&rsquo; detection sensitivity. Moreover, the need to sample all individuals prior to pooling overburden the limited resources such as test kits. An alternative proposed strategy where test is performed on pooled samples from individuals representing different households is presented here. This strategy intends to improve group testing method through the reduction in the number of samples collected and pooled during large-scale population testing. Moreover, it introduces database system which enables continuous monitoring of the population&rsquo;s virus exposure for better decision making.

scholarly journals Kitchen Range Oven Enabled One-tube RT-LAMP for RNA Detection at Home - A Potential Solution for Large-scale Screening of COVID-19

2020 ◽  
Author(s):  
Yu Lei
Keyword(s):  
Large Scale ◽  
Lamp Reaction ◽  
Sample Collection ◽  
Rna Detection ◽  
Closed Tube ◽  
One Step ◽  
User Friendly ◽  
Target Rna ◽  
Large Scale Screening ◽  
At Home

Rapid, low-cost, and user-friendly molecular diagnostic methods are prerequisite to address the outbreaks of infectious diseases. Especially during the outbreak of COVID-19 pandemic, there is an urgent need to build the global testing capacity up to 100-fold above what is achievable with current standard approaches. However, current gold standard methods such as RT-PCR and isothermal PCR (e.g., RT-LAMP), which are routinely conducted in laboratories, suffer from limit capacity due to the requirement of special equipment, multiple sample handling steps, and/or the need of skilled personnel. In this study, a kitchen Range Oven enabled RT-LAMP was conducted at a residential home without sacrificing the performance in RNA detection. In addition, one-step, closed-tube RT-LAMP for the detection of target RNA in the oven was accomplished by pre-loading sample collection solution and RT-LAMP reaction reagents into the bottom and cap cavity of PCR tube, respectively. After the addition of target RNA into sample collection solution through either swab swirling or direct pipetting, a flip-and-swing enabled mixing of sample solution and RT-LAMP reaction reagents was conducted first, followed by RT-LAMP detection at a constant temperature in pre-heated oven. The RNA positive sample can be obviously differentiated from the RNA negative sample through both naked-eye based turbidity detection and fluorescence detection under UV light. In conjunction with the user-friendly one-step, closed-tube concept, this study indicates that it is feasible to run RT-LAMP at home using oven with minimum involvement of end-users, thus offering an excellent molecular detection platform, which has the potential to boost the detection capacity for any nucleic acid target. It could be a potential solution for large-scale screening of COVID-19 in short time as the tests can be conducted by residents at home, without the need of well-trained health caregivers and capacity limitation.

scholarly journals Evaluation of efficiency and sensitivity of 1D and 2D sample pooling strategies for diagnostic screening purposes

2020 ◽  
Author(s):  
Jasper Verwilt ◽  
Pieter Mestdagh ◽  
Jo Vandesompele
Keyword(s):  
Quantitative Data ◽  
Large Scale ◽  
Real Life ◽  
Pool Size ◽  
Diagnostic Screening ◽  
Trade Off ◽  
The World ◽  
Sample Pooling ◽  
Pooling Strategies ◽  
Large Scale Simulations

As SARS-CoV-2 continues to spread around the world while the pandemic lasts, testing facilities are forced to massively increment their testing capacities to handle the increasing number of samples. While sample pooling methods have been proposed or are effectively implemented in some labs, no systematic and large-scale simulations have been performed using real-life quantitative data from testing facilities. Here, we use anonymous data from 1632 positive cases to simulate and compare 1D and 2D pooling strategies. We show that the choice of pooling method and pool size is an intricate decision with a prevalence-dependent efficiency-sensitivity trade-off.

scholarly journals Safe and effective pool testing for SARS-CoV-2 detection

2021 ◽  
Author(s):  
Marie Wunsch ◽  
Dominik Aschemeier ◽  
Eva Heger ◽  
Denise Ehrentraut ◽  
Jan Krueger ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Pool Size ◽  
Public Health Issue ◽  
Viral Loads ◽  
Pooling Strategies ◽  
Pooling Strategy ◽  
Pcr Assays ◽  
Low Prevalence ◽  
The Impact

Background / Objectives: The global spread of SARS-CoV-2 is a serious public health issue. Large-scale surveillance screenings are crucial but can exceed diagnostic test capacities. We set out to optimize test conditions and implemented high throughput pool testing of respiratory swabs into SARS-CoV-2 diagnostics. Study design: In preparation for pool testing, we determined the optimal pooling strategy and pool size. In addition, we measured the impact of vortexing prior to sample processing, compared pipette- and swab-pooling method as well as the sensitivity of three different PCR assays. Results: Using optimized strategies for pooling, we systematically pooled 55,690 samples in a period of 44 weeks resulting in a reduction of 47,369 PCR reactions. In a low prevalence setting, we defined a preferable pool size of ten in a two-stage hierarchical pool testing strategy. Vortexing of the swabs increased cellular yield by a factor of 2.34, and sampling at or shortly after symptom onset was associated with higher viral loads. By comparing different pooling strategies, pipette-pooling was more efficient compared to swab-pooling. Conclusions: For implementing pooling strategies into high throughput diagnostics, we recommend to apply a pipette-pooling method, using pool sizes of ten samples, performing sensitivity validation of the PCR assays used, and vortexing swabs prior to analyses. Our data shows, that pool testing for SARS-CoV-2 detection is feasible and highly effective in a low prevalence setting.

Training Aides to Screen Children for Speech and Language Problems

1976 ◽  
Vol 7 (4) ◽  
pp. 236-241 ◽  
Author(s):  
Marisue Pickering ◽  
William R. Dopheide
Keyword(s):  
Rural Areas ◽  
Large Scale ◽  
School Personnel ◽  
School Age Children ◽  
Speech And Language ◽  
Elementary School Age ◽  
Evaluation Procedures ◽  
Language Problems ◽  
Competency Based ◽  
Large Scale Screening

This report deals with an effort to begin the process of effectively identifying children in rural areas with speech and language problems using existing school personnel. A two-day competency-based workshop for the purpose of training aides to conduct a large-scale screening of speech and language problems in elementary-school-age children is described. Training strategies, implementation, and evaluation procedures are discussed.

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