Pan-Family Assays for Rapid Viral Screening: Reducing Delays in Public Health Responses During Pandemics
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Abstract
Background
Coronavirus disease 2019 has highlighted deficiencies in the testing capacity of many developed countries during the early stages of pandemics. Here we describe a strategy using pan-family viral assays to improve early accessibility of large-scale nucleic acid testing.
Methods
Coronaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were used as a case study for assessing utility of pan-family viral assays during the early stages of a novel pandemic. Specificity of a pan-coronavirus (Pan-CoV) assay for a novel pathogen was assessed using the frequency of common human coronavirus (HCoV) species in key populations. A reported Pan-CoV assay was assessed to determine sensitivity to 60 reference coronaviruses, including SARS-CoV-2. The resilience of the primer target regions of this assay to mutation was assessed in 8893 high-quality SARS-CoV-2 genomes to predict ongoing utility during pandemic progression.
Results
Because of common HCoV species, a Pan-CoV assay would return false positives for as few as 1% of asymptomatic adults, but up to 30% of immunocompromised patients with respiratory disease. One-half of reported Pan-CoV assays identify SARS-CoV-2 and with small adjustments can accommodate diverse variation observed in animal coronaviruses. The target region of 1 well-established Pan-CoV assay is highly resistant to mutation compared to species-specific SARS-CoV-2 reverse transcriptase-polymerase chain reaction assays.
Conclusions
Despite cross-reactivity with common pathogens, pan-family assays may greatly assist management of emerging pandemics through prioritization of high-resolution testing or isolation measures. Targeting highly conserved genomic regions make pan-family assays robust and resilient to mutation. A strategic stockpile of pan-family assays may improve containment of novel diseases before the availability of species-specific assays.
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SciScore for 10.1101/2020.05.24.20112318: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Sequences for a total of 60 species were identified (Supplementary Table 1), including the 7 species known to infect humans (HCoV-229E, HCoV-NL63, HCoV-HKU1 and HCoV-OC43, SARS-CoV, MERS-CoV and SARS-CoV-2). HCoV-NL63suggested: RRID:CVCL_RW88)Software and Algorithms Sentences Resources Review of common HCoV epidemiolocal characteristics: A search using the terms “HCoV” “epidemiology” and “asymptomatic” was performed in PubMed to … SciScore for 10.1101/2020.05.24.20112318: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Sequences for a total of 60 species were identified (Supplementary Table 1), including the 7 species known to infect humans (HCoV-229E, HCoV-NL63, HCoV-HKU1 and HCoV-OC43, SARS-CoV, MERS-CoV and SARS-CoV-2). HCoV-NL63suggested: RRID:CVCL_RW88)Software and Algorithms Sentences Resources Review of common HCoV epidemiolocal characteristics: A search using the terms “HCoV” “epidemiology” and “asymptomatic” was performed in PubMed to identify studies describing HCoV infection characteristics. PubMedsuggested: (PubMed, RRID:SCR_004846)Validation and modification of a reported Pan-CoV assays: Coronavirus-family reference sequences were identified and downloaded from the NCBI Virus database5 NCBI Virussuggested: (NCBI Virus, RRID:SCR_018253)Pan-coronavirus assay primer targets were derived from four reported assays6-9 and aligned with the SARS-CoV-2 reference genome (NC_045512) to assess consensus between sequences using the Clustal Omega Multiple Sequence Alignment tool10. Clustal Omegasuggested: (Clustal Omega, RRID:SCR_001591)The nucleic acid sequence and predicted protein sequence for each species was aligned and assessed for identity with Pan-CoV primer targets using the software package Geneious Prime 2020.1.2 (https://www.geneious.com). https://www.geneious.comsuggested: (Geneious, RRID:SCR_010519)Analysis was performed with the R Biostrings package12 to identify mismatches in the primer target sites. Biostringssuggested: (Biostrings, RRID:SCR_016949)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:This limitation can be mitigated through multiple strategies, most simply application of this tool as a first-line screen prior to confirmatory analysis with a more specific test. This would allow short-supply specific tests to be reserved as a confirmatory, rather than a primary tool, enabling a higher-throughput screen of large populations at ports-of-entry or for contact tracing during the early stages of pandemic. An alternative strategy, not reliant on the presence of a specific test, would be the addition of a second exclusionary reaction targeting known common species30, though this may not be an effective tool where a novel pathogen is highly genetically similar to a common species and may be overly burdensome for high-throughput population screening. During the initial roll-out of SARS-CoV-2 testing kits by the CDC, several laboratories experienced disruptions due to faulty testing kits31. Pan-family assays, developed prior to an outbreak, would have a relaxed timeframe for test development, ensuring best-practice validation, quality control procedures, regulatory certification and laboratory accreditation can be achieved. Pan-family assays may also have additional ongoing utility as a fallback in the case of quality control issues or disease mutations compromising the accuracy of specific assay detection32. By targeting highly conserved regions and accommodating silent mutations, pan-family assays can be expected to be less prone to mutation induced errors, though r...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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