Strategies used to scale up testing capacity
- •Diversification of platforms
- •Decentralization away from public health laboratories
- •Conversion of research laboratories into clinical laboratories
- •Maximizing number of samples per plate when supplies were low by adjusting the plate layout
- •Production of viral transport media in house
- •Use of phosphate-buffered saline instead of viral transport media
- •Production of 3D-printed swabs
- •Validation of assays with lower number of targets
- •Multiplexing and automation
- •Applying innovative technologies to COVID-19 diagnostics, such as clustered regularly interspaced short palindromic repeats (CRISPR)-based platforms
- •Pooling method (original specimens vs extracted RNA)
- •Pooling algorithm
- •Size of pool
- •Sensitivity of the pooled test
Size of Pool
|Type of Pooling||Pool Size||Specimen Type||Assay||Number Tested||Results||Reference|
|Simple||5, 10, 15, 5 for large volume analysis||NP||Pathofinder Real Accurate Quadruplex Corona-plus PCR Kit||4475 in 895 pools||Ct ↓ by 2.2,|
Acceptable for Ct 16.7–39.4
|Alcoba-Florez et al.|
|Simple||4||NP MT Nasal||Quest Diagnostics SARS-CoV-2 RNA Qualitative Real-Time RT-PCR||3091||Ct ↓ 1.9/2.38,|
|Borillo et al.|
|Simple||5, 7, 10||NP MT||CDC Assay, Panther Fusion SARS-CoV-2||270, then 7000||Ct ↓ by 2.7–3.6 (10 in pool),|
0.2–1.8 (5, 7 in pool),
Detected all positives with Ct < 36 for all pool sizes
|Das et al.|
|Simple||6||NP Saliva||Roche Cobas SARS-CoV-2||564||Sensitivity: NP 100%,|
Saliva 90%; 25% of samples had Ct > 30
|McMillen et al.|
|Simple||5, 10||NP||TaqPath Covid-19 Multiplex Diagnostic Solution||630||Detected Ct 33 consistently for pool of 5,|
Detected Ct 31 consistently for pool of 10
|More et al.|
|Simple||5, 10||Saliva||Sansure SARS-CoV-2 Nucleic Acid Diagnostic Kit||200||Pools of 5 or 10 acceptable||Pasomsub et al.|
|Simple||2, 4, 8, 16, 32, 64||Nasal and Throat||AgPath ID One-Step RT-PCR, WHO primer/probe, BioRad CFX96||72||10% False negative rate for pool of 32,|
Sensitivity for pool of 16: 96%
|Yelin et al.|
|Simple||6, 9||NP, Nasal + OP||Concentrate pool with Amicon Ultra 0.5 mL Ultracell 30K Filter,|
Z-Path-COVID-19-CE Genesig Real-Time RT-PCR (Primerdesign)
|112||Ct decrease 0.5–3,|
Detected as high as Ct 34
|Sawicki et al.|
|Simple||5, 9||Upper respiratory swab||CDC RT-PCR||20 positives into 60 for pools of 5 and into 39 for pools of 9||For CT ≥ 33, sensitivity 95% for pools of 5% and 87% for pools of 9||Griesemer et al.|
|Simple||5, 10, 20||Saliva||Luna Universal Probe One-Step RTqPCR, Laboratory Developed primer/probe, Biorad CFX 96 q PCR||23 pools of 5, 23 pools of 10,|
31 pools of 20
|Sensitivity: 93% for pools of 5%, 89% for pools of 10%, 85% for pools of 20||Watkins et al.|
|Simple||4, 8||NP||Laboratory-developed assay||320||Sensitivity: 75% for pools of 4,|
62.5% for pools of 8
|Mahmoud et al.|
|Simple||5, 8||NP||Laboratory-developed assay|
TaqPath Master Mix
|133,816||Adjusted pool size with prevalence of 0.5%–6%.|
Spared 76% pf reagents
|Barak et al.|
Sensitivity of Pooled Testing
Coronavirus (COVID-19) update: FDA issues first emergency authorization for sample pooling in diagnostic testing.
Summary of Pooling
Need for scale-up of molecular assays for SARS-CoV-2
Strategies to consider for large-scale testing for SARS-CoV-2
Increase in Testing Capacity by Modification of Traditional Laboratory-Developed Tests
Increase in Testing Capacity Using Automated Platforms
|Platform(s) Evaluated||Study Design||Type and Number of Specimens||Comparator Method||Results of Study||Reference|
|Hologic Panther Fusion SARS-CoV-2 Assay (Fusion)|
Hologic Aptima SARS-CoV-2 Assay (Aptima)
BioFire Defense COVID-19 test (Biofire)
|Retrospective and prospective||Nasopharyngeal swab (n = 150)||Consensus results from 3 platforms||94.7%–98.7% PPA, 100% NPA||Smith et al.|
|Hologic Panther Fusion SARS-CoV-2 Assay (Fusion)|
Simplexa COVID-19 Direct (Diasorin) assay GenMark ePlex SARS-CoV-2 (GenMark) assay
|Retrospective and prospective||Nasopharyngeal swab (n = 104)||CDC SARS-CoV-2 assay||96%–100% PPA and NPA||Zhen et al.|
|Hologic Panther Fusion SARS-CoV-2 assay (Fusion) cobas SARS-CoV-2 RT-PCR using cobas 6800 system||Retrospective and prospective||Nasopharyngeal swab (n = 389)||Comparison of 2 platforms and Xpert Xpress SARS-CoV-2 RT-PCR for discrepancy analysis||96.4% agreement in performance||Craney et al.|
|RealTime SARS-CoV-2 assay using m2000 system (Abbott)||Validation and verification||Nasal and nasopharyngeal swab (n = 30)||Comparison to CDC SARS-CoV-2 assay||Sensitivity 93% Specificity 100%||Degli-Angeli et al.|
|RealTime SARS-CoV-2 assay using Alinity m system (Abbott)|
cobas SARS-CoV-2 RT-PCR using cobas 6800 system (Roche)
|Prospective||Nasopharyngeal swab (n = 2129)||Clinical evaluation of performance||100% PPA,96.8% NPA||Kogoj et al.|
|NeuMoDx 96 Molecular System (Ann Arbor, MI)||Retrospective (stored for < 5 d)||Nasopharyngeal swab (n = 159)||Comparison of NeuMoDx to Diasorin Simplexa SARS-CoV-2 direct assay and CDC SARS-CoV-2 assay||100% PPA and NPA||Lima et al.|
|NeuMoDx 96 Molecular System (Ann Arbor, MI)||Multicenter comparison, retrospective||Nasopharyngeal swab (n = 212)||New York SARS-CoV-2 Real-time Reverse Transcriptase (RT)- PCR Diagnostic Panel and RealStar® SARS-CoV-2 RT-PCR Kit 1.0 (Altona Diagnostics, Hamburg, Germany)||99% PPA,91.5% NPA||Mostafa et al.|
|Goal||Key Parameters to be Assessed||Other Factors|
|Is this the right assay for this disease?||Clinical and analytical performance characteristics of assay:|
|Is this the appropriate instrument for this test?|
|Is this the appropriate instrument for my laboratory?|
Alternative Technologies for Diagnosis
Next-Generation Sequencing Large-Scale Surveillance of SARS-CoV-2
- Kirby A.E.
- Welsh R.M.
- Marsh Z.A.
- Yu A.T.
- Vugia D.J.
- Boehm A.B.
- Wolfe M.K.
- White B.J.
- Matzinger S.R.
- Wheeler A.
- Bankers L.
- Andresen K.
- Salatas C.
- Gregory D.A.
- Johnson M.C.
- Trujillo M.
- Kannoly S.
- Smyth D.S.
- Dennehy J.J.
- Sapoval N.
- Ensor K.
- Treangen T.
- Stadler L.B.
- Hopkins L.
Notes from the field: early evidence of the SARS-CoV-2 B.1.1.529 (omicron) variant in community wastewater - United States, november-december 2021.
Limitations of Molecular Assays and Future Strategies for Large-Scale Testing
Clinics Care Points
- •Multiple strategies for pooling of different types specimens provided sensitive and specific SARS-CoV-2 testing.
- •Automated liquid handling, alternative extraction procedures, multiplexing, and rapid commercialization of new testing platforms added to overall testing capacity.
- •Innovation of molecular methods, such as CRISPR-based assays, diversified options for testing and also increased overall testing capacity.
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