Pooled Sample Testing for SARS-CoV-2 Using Rapid RT-PCR COVID-19 Tests

AbstractWe tested an operationally efficient way to pool samples on a rapid, point-of-care PCR device and examined the limit of detection of SARS-CoV-2 for various pool sizes. Pooled testing maintained testing performance similar to individual sample PCR testing, offering the potential for scalable rapid testing at lower cost with less supplies.

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Effectiveness of Sample Pooling Strategies for SARS-CoV-2 Mass Screening by RT-PCR: A Scoping Review

Journal of Laboratory Physicians ◽

10.1055/s-0040-1721159 ◽

2020 ◽

Vol 12 (03) ◽

pp. 212-218 ◽

Cited By ~ 1

Author(s):

Sangeeta Deka ◽

Deepjyoti Kalita

Keyword(s):

Limit Of Detection ◽

Large Population ◽

Pool Size ◽

High Threshold ◽

Conservation Of Resources ◽

Rt Pcr ◽

Sample Pooling ◽

Sample Testing ◽

Low Prevalence ◽

Pooled Sample

AbstractThe ongoing COVID-19 pandemic has hugely impacted the economy of many countries, and there is an acute shortage of diagnostic resources. With the exponential increase in the number of cases and necessity to screen large number of people, there is a steep increase in the demand for diagnostic kits. Pooled-sample testing is a promising strategy to screen a large population rapidly with limited resources. The aim of this work was to compile a cohesive literature review of the effectiveness and accuracy of pooled-sample testing in the detection of SARS-CoV-2 and critically analyze its limitations. Medline, Google Scholar, Embase, and preprint servers (e.g., bioRxiv) were searched for literature on pooled testing for diagnosis of COVID-19, and out of initial 60 articles/reports, nine original articles were retained. Optimal pool size (number of samples in a pool) seemed to be dependent on factors like prevalence or rate of positivity in community. In low-prevalence localities pool size of around 30 seemed to be effective as observed by some authors. All the researchers had found significant reduction in number of tests (depending on pool size, stages, and pooling design), leading to conservation of resources. Pooling can be done with extracted RNA eluate or directly with patient’s sample before extraction. This leads to further reduction in consumables, time and manpower. Risk of false negativity in samples with high-threshold cycle (i.e., low-viral load) value was a concern. Some researchers suggest adding few additional cycles to lower the chances of missing positive cases with low-Ct value. Lower limit of detection (LoD) of RT-PCR kits, that is, sensitivity of kits was another factor to consider. Thus, in a country like India, given the economic benefit and scarcity of resources, pooling strategy can be very effective, especially in low-prevalence areas and in low-risk contacts.

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Development and Evaluation of Novel and Highly Sensitive Single-Tube Nested Real-Time RT-PCR Assays for SARS-CoV-2 Detection

International Journal of Molecular Sciences ◽

10.3390/ijms21165674 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5674

Author(s):

Cyril Chik-Yan Yip ◽

Siddharth Sridhar ◽

Kit-Hang Leung ◽

Anthony Chin-Ki Ng ◽

Kwok-Hung Chan ◽

...

Keyword(s):

Real Time ◽

Limit Of Detection ◽

Rt Pcr ◽

Single Tube ◽

Highly Sensitive ◽

Testing Algorithms ◽

Pcr Assays ◽

Human Coronaviruses ◽

Pooled Sample

Sensitive molecular assays are critical for coronavirus disease 2019 (COVID-19) diagnosis. Here, we designed and evaluated two single-tube nested (STN) real-time RT-PCR assays, targeting SARS-CoV-2 RdRp/Hel and N genes. Both STN assays had a low limit of detection and did not cross react with other human coronaviruses and respiratory viruses. Using 213 initial respiratory specimens from suspected COVID-19 patients, the sensitivity of both the STN COVID-19-RdRp/Hel and the STN COVID-19-N assays was 100% (99/99), while that of the comparator non-nested N assay was 95% (94/99). Among 108 follow-up specimens from confirmed COVID-19 patients who tested negative by the non-nested COVID-19-RdRp/Hel assay, 28 (25.9%) were positive for SARS-CoV-2 by the STN COVID-19-RdRp/Hel or the STN COVID-19-N assay. To evaluate the performance of our novel STN assays in pooled specimens, we created four sample pools, with each pool consisting of one low positive specimen and 49 negative specimens. While the non-nested COVID-19-RdRp/Hel assay was positive in only one of four sample pools (25%), both of the STN assays were positive in two of four samples pools (50%). In conclusion, the STN assays are highly sensitive and specific for SARS-CoV-2 detection. Their boosted sensitivity offers advantages in non-traditional COVID-19 testing algorithms such as saliva screening and pooled sample screening during massive screening.

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Development and validation of direct RT-LAMP for SARS-CoV-2

10.1101/2020.04.29.20075747 ◽

2020 ◽

Cited By ~ 5

Author(s):

Abu Naser Mohon ◽

Jana Hundt ◽

Guido van Marle ◽

Kanti Pabbaraju ◽

Byron Berenger ◽

...

Keyword(s):

Sindbis Virus ◽

Limit Of Detection ◽

Point Of Care ◽

Extraction Procedure ◽

Lamp Assay ◽

Cold Chain ◽

Clinical Samples ◽

Lamp Method ◽

Rt Pcr ◽

Percent Agreement

AbstractWe have developed a reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2. The LAMP assay achieves comparable limit of detection as commonly used RT-PCR protocols based on artificial targets, recombinant Sindbis virus, and clinical samples. Clinical validation of single-target (S gene) LAMP (N=120) showed a positive percent agreement (PPA) of 41/42 (97.62%) and negative percent agreement (NPA) of 77/78 (98.72%) compared to reference RT-PCR. Dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 44/48 (91.97%) and NPA 72/72 (100%) when including discrepant samples. The assay can be performed without a formal extraction procedure, with lyophilized reagents which do need cold chain, and is amenable to point-of-care application with visual detection.

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Rapid Isothermal Amplification and Portable Detection System for SARS-CoV-2

10.1101/2020.05.21.108381 ◽

2020 ◽

Cited By ~ 2

Author(s):

A. Ganguli ◽

A. Mostafa ◽

J. Berger ◽

M. Aydin ◽

F. Sun ◽

...

Keyword(s):

Limit Of Detection ◽

Detection System ◽

Point Of Care ◽

Rna Extraction ◽

Clinical Diagnostics ◽

Isothermal Amplification ◽

Sample Collection ◽

Rt Pcr ◽

Point Of Use ◽

Viral Transport Medium

AbstractThe COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay details and primer sequences become widely known, many laboratories could perform diagnostic tests using methods such as RT-PCR or isothermal RT-LAMP amplification. A key advantage of RT-LAMP based approaches compared to RT-PCR is that RT-LAMP is known to be robust in detecting targets from unprocessed samples. In addition, RT-LAMP assays are performed at a constant temperature enabling speed, simplicity, and point-of-use testing. Here, we provide the details of an RT-LAMP isothermal assay for the detection of SARS-CoV-2 virus with performance comparable to currently approved tests using RT-PCR. We characterize the assay by introducing swabs in virus spiked synthetic nasal fluids, moving the swab to viral transport medium (VTM), and using a volume of that VTM for performing the amplification without an RNA extraction kit. The assay has a Limit-of-Detection (LOD) of 50 RNA copies/μL in the VTM solution within 20 minutes, and LOD of 5000 RNA copies/μL in the nasal solution. Additionally, we show the utility of this assay for real-time point-of-use testing by demonstrating detection of SARS-CoV-2 virus in less than 40 minutes using an additively manufactured cartridge and a smartphone-based reader. Finally, we explore the speed and cost advantages by comparing the required resources and workflows with RT-PCR. This work could accelerate the development and availability of SARS-CoV-2 diagnostics by proving alternatives to conventional laboratory benchtop tests.Significance StatementAn important limitation of the current assays for the detection of SARS-CoV-2 stem from their reliance on time- and labor-intensive and laboratory-based protocols for viral isolation, lysis, and removal of inhibiting materials. While RT-PCR remains the gold standard for performing clinical diagnostics to amplify the RNA sequences, there is an urgent need for alternative portable platforms that can provide rapid and accurate diagnosis, potentially at the point-of-use. Here, we present the details of an isothermal amplification-based detection of SARS-CoV-2, including the demonstration of a smartphone-based point-of-care device that can be used at the point of sample collection.

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SalivaAll: Clinical validation of a sensitive test for saliva collected in healthcare and community settings with pooling utility for SARS-CoV-2 mass surveillance.

10.1101/2020.08.26.20182816 ◽

2020 ◽

Cited By ~ 1

Author(s):

Nikhil Shri Sahajpal ◽

Ashis K Mondal ◽

Sudha Ananth ◽

Allan Njau ◽

Pankaj Ahluwalia ◽

...

Keyword(s):

Phase I ◽

Phase Ii ◽

Detection Rate ◽

Limit Of Detection ◽

Rna Extraction ◽

Community Settings ◽

Rt Pcr ◽

Effective Measure ◽

Pooled Testing ◽

Mass Surveillance

Background: The adoption of saliva as a specimen type for SARS-CoV-2 mass surveillance can significantly increase population compliance with decreased exposure risk for healthcare workers. However, studies evaluating the clinical performance of saliva compared to nasopharyngeal swab (NPS) samples have demonstrated conflicting results regardless of the collection being in healthcare or community settings. Further, pooled testing with saliva remains a challenge owing to the ambiguous sensitivity, limit of detection (LoD), and processing challenges. To overcome these limitations, SalivaAll protocol was developed and validated as a cost-effective measure that must be used on saliva collected in health care or community settings with pooling utility for SARS-CoV-2 mass surveillance. Methods: The study evaluated 429 matched NPS and saliva samples collected from 344 individuals in either healthcare or community setting. In phase I (protocol U), 240 matched NPS, and saliva samples were tested for SARS-CoV-2 detection by RT-PCR. In phase II (SalivaAll protocol), 189 matched NPS and saliva samples were tested, with an additional sample homogenization step for saliva before RNA extraction, followed by RT-PCR. Eighty-five saliva samples were evaluated with both protocols (U and SalivaAll). Subsequently, adopting SalivaAll protocol, a five-sample pooling strategy was evaluated for saliva samples based on FDA recommendations. Results: In phase I, 28.3% (68/240) samples tested positive for SARS-CoV-2 from either saliva, NPS, or both. The detection rate was lower in saliva compared to NPS samples (50.0% vs. 89.7%). In phase II, 50.2% (95/189) samples tested positive for SARS-CoV-2 from either saliva, NPS, or both. The detection rate for SARS-CoV-2 was higher in saliva compared to NPS testing (97.8% vs. 78.9%). Of the 85 saliva samples evaluated by both protocols, 57.6% (49) tested positive for SARS-CoV-2 with either protocol U, SalivaAll, or both. The detection rate was 100% for samples tested with SalivaAll, whereas it was 36.7% with protocol U. Also, the LoD with SalivaAll protocol was 20 copies/ml. The pooled testing approach demonstrated a 95% positive and 100% negative percent agreement. Conclusion: This single-site study demonstrated the variability of results reported in the literature for saliva samples, and found that the discrepancies are explained by processing challenges associated with saliva samples. We have optimized a protocol for saliva samples that results in higher sensitivity compared to NPS samples and also breaks the barrier to using pooled saliva testing for SARS-CoV-2.

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Pooled sputum to optimise the efficiency and utility of rapid, point-of-care molecular SARS-CoV-2 testing

BMC Infectious Diseases ◽

10.1186/s12879-021-06316-z ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Alison Burdett ◽

Christofer Toumazou ◽

Rashmita Sahoo ◽

Adam Mujan ◽

Tsz-Kin Hon ◽

...

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

High Sensitivity ◽

Pool Size ◽

Pooled Testing ◽

Paired Samples ◽

Absolute Strength ◽

Potential Benefits ◽

First Time ◽

Multiple Patients

Abstract Background As SARS-CoV-2 testing expands, particularly to widespread asymptomatic testing, high sensitivity point-of-care PCR platforms may optimise potential benefits from pooling multiple patients’ samples. Method We tested patients and asymptomatic citizens for SARS-CoV-2, exploring the efficiency and utility of CovidNudge (i) for detection in individuals’ sputum (compared to nasopharyngeal swabs), (ii) for detection in pooled sputum samples, and (iii) by modelling roll out scenarios for pooled sputum testing. Results Across 295 paired samples, we find no difference (p = 0.1236) in signal strength for sputum (mean amplified replicates (MAR) 25.2, standard deviation (SD) 14.2, range 0–60) compared to nasopharyngeal swabs (MAR 27.8, SD 12.4, range 6–56). At 10-sample pool size we find some drop in absolute strength of signal (individual sputum MAR 42.1, SD 11.8, range 13–60 vs. pooled sputum MAR 25.3, SD 14.6, range 1–54; p < 0.0001), but only marginal drop in sensitivity (51/53,96%). We determine a limit of detection of 250 copies/ml for an individual test, rising only four-fold to 1000copies/ml for a 10-sample pool. We find optimal pooled testing efficiency to be a 12–3-1-sample model, yet as prevalence increases, pool size should decrease; at 5% prevalence to maintain a 75% probability of negative first test, 5-sample pools are optimal. Conclusion We describe for the first time the use of sequentially dipped sputum samples for rapid pooled point of care SARS-CoV-2 PCR testing. The potential to screen asymptomatic cohorts rapidly, at the point-of-care, with PCR, offers the potential to quickly identify and isolate positive individuals within a population “bubble”.

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Evaluation of accuracy, exclusivity, limit-of-detection and ease-of-use of LumiraDx™ - Antigen-detecting point-of-care device for SARS-CoV-2

10.1101/2021.03.02.21252430 ◽

2021 ◽

Author(s):

Lisa Johanna Krüger ◽

Julian A.F. Klein ◽

Frank Tobian ◽

Mary Gaeddert ◽

Federica Lainati ◽

...

Keyword(s):

Viral Load ◽

Limit Of Detection ◽

Point Of Care ◽

Scale Up ◽

Cross Reactivity ◽

Ease Of Use ◽

Analytical Sensitivity ◽

Rt Pcr ◽

Clinical Sensitivity ◽

Lateral Flow Assays

Background: Rapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity (≤85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. Materials and Methods: This multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx™ assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally-collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. Results: Study conduct was between November 2nd 2020 and January 21st 2021. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx™, resulting in a sensitivity of 82.2% (95% CI: 75.2%-87.5%). Specificity was 99.3% (CI: 98.3-99.7%). Sensitivity was increased in individuals with viral load ≥ 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI: 86.2%-97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2-56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. Conclusion: The LumiraDx™ assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling.

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Recommendations for sample pooling on the Cepheid GeneXpert® system using the Cepheid Xpert® Xpress SARS-CoV-2 assay

10.1101/2020.05.14.097287 ◽

2020 ◽

Cited By ~ 3

Author(s):

Michael G. Becker ◽

Tracy Taylor ◽

Sandra Kiazyk ◽

Dana R. Cabiles ◽

Adrienne F.A. Meyers ◽

...

Keyword(s):

Public Health ◽

Limit Of Detection ◽

Point Of Care ◽

Test Site ◽

Viral Loads ◽

Pooled Testing ◽

Point Of Care Test ◽

Health Authorities ◽

Public Health Authorities ◽

Sample Pooling

AbstractThe coronavirus disease 2019 (Covid-19) pandemic, caused by SARS-CoV-2, has resulted in a global testing supply shortage. In response, pooled testing has emerged as a promising strategy that can immediately increase testing capacity. Here, we provide support for the adoption of sample pooling with the point-of-care Cepheid Xpert® Xpress SARS-CoV-2 molecular assay. Corroborating previous findings, the Xpert® Xpress SARS-CoV-2 assay limit of detection was comparable to central laboratory reverse-transcription quantitative PCR tests with observed SARS-CoV-2 detection below 100 copies/mL. The Xpert® Xpress assay detected SARS-CoV-2 after samples with minimum viral loads of 461 copies/mL were diluted into six sample pools. Based on these data, we recommend the adoption of pooled testing with the Xpert® Xpress SARS-CoV-2 assay where warranted by population public health needs. The suggested number of samples per pool, or pooling depth, is unique for each point-of-care test site and should be determined by assessing positive test rates. To statistically determine appropriate pooling depth, we have calculated the pooling efficiency for numerous combinations of pool sizes and test rates. This information is included as a supplemental dataset that we encourage public health authorities to use as a guide to make recommendations that will maximize testing capacity and resource conservation.

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Recommendations for sample pooling on the Cepheid GeneXpert® system using the Cepheid Xpert® Xpress SARS-CoV-2 assay

PLoS ONE ◽

10.1371/journal.pone.0241959 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0241959

Author(s):

Michael G. Becker ◽

Tracy Taylor ◽

Sandra Kiazyk ◽

Dana R. Cabiles ◽

Adrienne F. A. Meyers ◽

...

Keyword(s):

Public Health ◽

Limit Of Detection ◽

Point Of Care ◽

Viral Loads ◽

Pooled Testing ◽

Health Authorities ◽

Public Health Authorities ◽

Sample Pooling ◽

Testing Site ◽

The Individual

The coronavirus disease 2019 (Covid-19) pandemic, caused by SARS-CoV-2, has resulted in a global testing supply shortage. In response, pooled testing has emerged as a promising strategy that can immediately increase testing capacity. In pooled sample testing, multiple samples are combined (or pooled) together and tested as a single unit. If the pool is positive, the individual samples can then be individually tested to identify the positive case(s). Here, we provide support for the adoption of sample pooling with the point-of-care Cepheid Xpert® Xpress SARS-CoV-2 molecular assay. Corroborating previous findings, the limit of detection of this assay was comparable to laboratory-developed reverse-transcription quantitative PCR SARS-CoV-2 tests, with observed detection below 100 copies/mL. The Xpert® Xpress assay detected SARS-CoV-2 after samples with minimum viral loads of 461 copies/mL were pooled in groups of six. Based on these data, we recommend the adoption of pooled testing with the Xpert® Xpress SARS-CoV-2 assay where warranted based on public health needs. The suggested number of samples per pool, or the pooling depth, is unique for each point-of-care testing site and can be determined by the positive test rates. To statistically determine appropriate pooling depth, we have calculated the pooling efficiency for numerous combinations of pool sizes and test rates. This information is included as a supplemental dataset that we encourage public health authorities to use as a guide to make recommendations that will maximize testing capacity and resource conservation.

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Evaluation of the QIAstat-Dx Respiratory SARS-CoV-2 Panel, the First Rapid Multiplex PCR Commercial Assay for SARS-CoV-2 Detection

Journal of Clinical Microbiology ◽

10.1128/jcm.00630-20 ◽

2020 ◽

Vol 58 (8) ◽

Cited By ~ 25

Author(s):

Benoit Visseaux ◽

Quentin Le Hingrat ◽

Gilles Collin ◽

Donia Bouzid ◽

Samuel Lebourgeois ◽

...

Keyword(s):

Multiplex Pcr ◽

Limit Of Detection ◽

Clinical Sample ◽

Point Of Care ◽

Sample Pretreatment ◽

Tracheal Aspirate ◽

Clinical Samples ◽

Rt Pcr ◽

Viral Neutralization ◽

Efficient Detection

ABSTRACT In the race to contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), efficient detection and triage of infected patients must rely on rapid and reliable testing. In this work, we performed the first evaluation of the QIAstat-Dx respiratory SARS-CoV-2 panel (QIAstat-SARS) for SARS-CoV-2 detection. This assay is the first rapid multiplex PCR (mPCR) assay, including SARS-CoV-2 detection, and is fully compatible with a non-PCR-trained laboratory or point-of-care (PoC) testing. This evaluation was performed using 69 primary clinical samples (66 nasopharyngeal swabs [NPS], 1 bronchoalveolar lavage fluid sample [BAL], 1 tracheal aspirate sample, and 1 bronchial aspirate sample) comparing SARS-CoV-2 detection with the currently WHO-recommended reverse transcription-PCR (RT-PCR) (WHO-RT-PCR) workflow. Additionally, a comparative limit of detection (LoD) assessment was performed for QIAstat-SARS and WHO-RT-PCR using a quantified clinical sample. Compatibility of sample pretreatment for viral neutralization or viscous samples with the QIAstat-SARS system were also tested. The QIAstat-Dx respiratory SARS-CoV-2 panel demonstrated a sensitivity comparable to that of the WHO-recommended assay with a limit of detection at 1,000 copies/ml. The overall percent agreement between QIAstat-Dx SARS and WHO-RT-PCR on 69 clinical samples was 97% with a sensitivity of 100% (40/40) and specificity at 93% (27/29). No cross-reaction was encountered for any other respiratory viruses or bacteria included in the panel. The QIAstat-SARS rapid multiplex PCR panel provides a highly sensitive, robust, and accurate assay for rapid detection of SARS-CoV-2. This assay allows rapid decisions even in non-PCR-trained laboratory or point-of-care testing, allowing innovative organization.

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